Methanogenesis is a key biological process contributing to both global climate change and renewable energy via methane. Methylotrophic methanogenesis is an important, but often underappreciated, component of this process. Recently, there has been an increasing interest in methylotrophic methanogenesis from quaternary amines such as glycine betaine, choline, and tetramethylammonium (QMA). QMA was the original quaternary amine identified as a direct substrate for methanogens and the components of its catabolic pathway were isolated from Methanococcoides methylutens NaT1. Loss of strain NaT1 and the absence of genomic sequencing leaves into question the identity of the genes responsible. We previously isolated a related strain of Methanococcoides methylutens called strain Q3c, which is capable of growth on the quaternary amines QMA and choline. We conducted proteomic analysis of strain Q3c in which we identified potential targets for QMA and choline metabolism, involving the methyltransferase, MtqB, and its cognate corrinoid protein, MtqC, because they were highly produced in QMA-grown and choline-grown cells differentially compared to trimethylamine grown cells and the 82.8 and 96.4%, identity of their N-termini, respectively, to MtqBC identified previously in NaT1. Surprisingly, MtqB was identified as a nonpyrrolysine monomethylamine methyltransferase (MtmB) homolog. This was validated via enzymatic assay and molecular co-docking of MtqB with QMA and choline along with its cognate partner MtqC, representing the first ever function for a non-Pyl MtmB demonstrated. The phylogeny of MtqB and MtqC showed unique clustering of these proteins compared to other homologs, suggesting these proteins may be important in our continued study of the evolution of Pyl- and non-Pyl methyltransferase systems.

The MttB superfamily member MtgB catalyzes the methylation of a cognate corrinoid protein with glycine betaine, and representatives have been described from both bacteria and archaea. Here, we focused on MtgB from Desulfitobacterium hafniense, a protein for which a crystal structure had been previously obtained. We employed different programs to predict the binding of glycine betaine and identified a consensus binding site. The modelled binding site consisted of two aromatic residues, Y97 and F356, which are both proposed to interact with the quaternary amine portion of glycine betaine via pi:cation interactions. Additionally, two basic residues, H348 and R312, were proposed to interact with the carboxylate group. We carried out site-directed substitutions and subsequently tested the necessity of these residues for glycine betaine:cob(I)alamin methyltransferase activity. These experiments supported a role in catalysis for each residue, presumably in the placement of glycine betaine at the proper position for nucleophilic attack by the Co(I) ion of cobalamin. Subsequently, the structure of the glycine betaine-bound enzyme was obtained, confirming the interaction of these residues with glycine betaine. Other MttB superfamily members with specificity for different quaternary amines were modeled and compared with the glycine betaine-bound structure of MtgB. The nitrogen of each quaternary amine was brought within an average value of 1.8 Å to each other, suggesting that members of the superfamily bring their methyl groups into nearly the same space within the TIM barrel prior to methyl group transfer to cob(I)alamin.

Gut microbiota and human physiology are closely linked, and our microbiota can influence disease through immunological and metabolic activity. Quaternary amines, such as choline and carnitine, are abundant in the human gut and are found in red meat, beef, eggs, seafood, wheat, and beets. Canonically, choline and carnitine are broken down into the pro-atherogenic metabolite, trimethylamine (TMA), by various microbes in the human gut. A glycyl radical enzyme, CutC, is involved in the breakdown of choline to TMA. Carnitine is broken down to TMA via a gamma-butyrobetaine intermediate. TMA from the human gut increases levels of TMA N-oxide in blood and promotes atherosclerosis. Citrobacter amalonaticus CJ25, a gut strain isolated and characterized in our lab, has been shown to grow on choline or carnitine as the sole carbon-energy source without generating TMA. Because the genome lacks canonical enzymes involved in the degradation of choline and no TMA was produced in both choline and carnitine growth conditions, we analyzed the choline and carnitine metabolism using a combined metabolomic and proteomic approach. CJ25 metabolizes choline and carnitine into glycine betaine (GB) via pathways involving novel enzyme homologs, as indicated by proteomic analysis. The proteomics showed putative dehydrogenases that could be oxidizing choline and carnitine to GB. These non-atherogenic pathways involving novel enzyme homologs that we identify in CJ25 may also exist in other gut microbiota, which could amplify the effects of these pathways significantly, possibly reducing the risk of atherosclerotic cardiovascular disease in individuals harboring these microbiota.IMPORTANCEThe human gut microbiome has been shown to contribute to atherosclerotic cardiovascular disease with adverse health effects throughout the world. Gut microbes canonically metabolize quaternary amines into proatherogenic TMA. In this study, a gut bacterium, CJ25, metabolizes choline and carnitine to a non-atherogenic product, glycine betaine, potentially using novel dehydrogenase homologs for their oxidation. Notably, the ability of CJ25 to metabolize choline and carnitine in a non-atherogenic manner establishes its potential as a beneficial human gut bacterium. Additionally, enzymes identified in CJ25 for choline and carnitine breakdown may be present in other gut microbes, which could amplify the effects of these pathways and reduce the risk of atherosclerotic cardiovascular disease more universally.

Gut microbiota metabolism can have profound effects on human health. Choline, a quaternary amine (QA) highly abundant in our diet, is canonically cleaved by a glycyl radical enzyme, choline trimethylamine lyase (CutC), and its SAM-dependent radical activator, CutD. CutC cleaves choline to form trimethylamine (TMA) and acetaldehyde. TMA is oxidized to TMAO by FMO3 in the liver, which plays a role in causing atherosclerosis. We hypothesized that alternative pathways for choline degradation occur within gut microbes and that certain gut microbiota can anaerobically respire or ferment QAs, such as choline. Based on this prediction we established QA-supplemented enrichment cultures using fecal material from healthy volunteers as the inocula. We have isolated, from a choline-supplemented enrichment of a human fecal sample, a strain of Citrobacter amalonaticus, that we have designated CJ25. This strain is capable of anaerobically utilizing choline as its sole carbon and energy source. Its genome does not contain the cutCD genes or genes encoding any COG5598 methyltransferases. We have confirmed the degradation of choline and production of acetate by the organism during growth of the strain. However, we used multiple analytical methods to confirm that no TMA accumulated in the medium during growth. Hence, strain CJ25 is a unique bacterium that degrades choline without the production of the proatherogenic metabolite TMA.

Recent studies indicate that environmentally abundant quaternary amines (QAs) are a primary source for methanogenesis, yet the catabolic enzymes are unknown. We hypothesized that the methanogenic archaeon Methanolobus vulcani B1d metabolizes glycine betaine (GB) through a corrinoid-dependent GB:coenzyme M (CoM) methyl transfer pathway. The draft genome sequence of M. vulcani B1d revealed a gene encoding a predicted non-pyrrolysine MttB homolog (MV8460) with high sequence similarity to the GB methyltransferase encoded by Desulfitobacterium hafniense Y51. MV8460 catalyzes GB-dependent methylation of free cob(I)alamin indicating it is an authentic MtgB enzyme. Proteomic analysis revealed that MV8460 and a corrinoid binding protein (MV8465) were highly abundant when M. vulcani B1d was grown on GB relative to growth on trimethylamine. The abundance of a corrinoid reductive activation enzyme (MV10335) and a methylcorrinoid:CoM methyltransferase (MV10360) were significantly higher in GB-grown B1d lysates compared to other homologs. The GB:CoM pathway was fully reconstituted in vitro using recombinant MV8460, MV8465, MV10335, and MV10360. Demonstration of the complete GB:CoM pathway expands the knowledge of direct QA-dependent methylotrophy and establishes a model to identify additional ecologically relevant anaerobic quaternary amine pathways.

PERSPECTIVES IN BIOLOGY AND MEDICINE Volume 30 ¦ Number 3 ¦ Spring 1987 THE ACTUAL EXTENT OF MASTECTOMY: A KEY TO SURVIVAL DONALDJ. FERGUSON* During the past 3 decades, surgery for breast cancer has been gradually attenuated by a turn of thought that began in the United Kingdom and has spread worldwide, holding that efforts to excise all the local and regional disease do not improve results. It is now widely taught that "dissemination rates are unaffected by the type of primary treatment" [1], "operable breast cancer is a systemic disease" [2], and "local treatment does not influence survival" [3]. One basis for these beliefs is the appearance of disseminated cancer many years after treatment that is locally effective. Another reason is the similarity in rates of survival after nominally different types of mastectomy. If, on the other hand, it may be observed by a more careful scrutiny of the evidence that the actual extent of mastectomy can affect survival and that a significant proportion of mastectomy patients are cured—in the sense that they live a normal life span without recurrent cancer—the quoted generalizations are seen to apply only to a certain fraction of operable patients. Members of this doomed subgroup cannot be individually recognized at the time of mastectomy and therefore cannot safely be treated differently from the others. It may, of course, be postulated that all cancer patients, or all human beings for that matter, have dormant cancer cells that never proliferate, but this theory has no apparent usefulness. The purpose of this essay is to review the evidence for and against mastectomy, by showing that the present, generally disparaging opinion *Department of Surgery, University of Chicago, 5841 Maryland Avenue, Chicago, Illinois 60637.© 1987 by The University of Chicago. All rights reserved. 003 1-5982/87/3003-054 1$01 .00 Perspectives in Biology and Medicine, 30,3 ¦ Spring 1987 | 311 TABLE 1 Ten-year Survival in Randomized Comparisons of the Extent of Mastectomy Project Types of Mastectomy Survival Local Recurrence P (%) Cases (N) 1st series 2d series Total vs. I—III extendedI— III TotalII vs. radicalII—III Total vs. radical Partial vs. radical Partial vs. radical Partial vs. radical Radical vs. extended Total vs. radical Total vs. radical Total vs. radical Quadrant vs. radical I—III I—III I I II II I—III I—III I I II II II II I (<2 cm) I (<2 cm) 44 48 46 49 52 61 57 52 59 68 35 49 53 56 54 58 39 38 31 35 79 78 NS <.05 NS <.002 <.05 .40 .50 NS 21 24 Not NSgiven 23 15 34 16 33 8 63 26 24 14 10 8 16 Not .90given NS Not given <.003 <.001 <.001 <.05 NS NS NS NS 199 183 113 91 242 256 112 108 122 130 70 80 746 697 365 362 294 292 159 148 352 349 312 Nodes Found (N) Adjuvants Internal Axillary Mammary Randomization X-ray Rads EndoDrugcrineMethodExclusions (9i ) Not described Not described Not described Not described 12 15 15 15 15 15 2-4 Not described Not described 4300 0 3300 3300 4500 0 3800 3000 3800 3000 3800 3000 Yes Yes Yes Yes 6000 0 4000 0 6000 0 Yes Yes Yes Yes Case40 no. 45 OK3 deaths OK OK OK OK Not given Not given 15 0.3 2 5.7 11 Kaae [9, 10] Brinkley[ll, 12] Bruce [5, 13] Atkins [6, 7] Lacour [14, 15] Fisher [16, 17] Lythgoe [3, 18] Veronesi [19, 20] 313 is not well founded and by demonstrating that not only the extent but the detailed care applied to the resection determines survival. To relate extent to survival, we need a randomized comparison of defined variations in resection with adequate follow-up. Results up to 5 years have been shown to mislead in part because of the often slow progression of breast cancer (4 vs. 5, 6, 6 vs. 7). Patients who remain well for 15—20 years after radical mastectomy have little further risk of recurrence , and their average life expectancy is then the same as that of a matched control population [8]. There is only one randomized study carried to 20 years [7]. If we compromise by accepting 10 years, only seven other projects meet these minimum requirements. We can briefly examine all of this evidence; the studies and their references are listed in table 1. Descriptions of the Surgery Names of the various types of mastectomy are listed in table 1 . The actual resections performed are found to vary within study groups as well as between studies that are nominally of the same procedure. Insofar as such variations may affect survival, they obviously impair the validity of any conclusions that are drawn, and they are therefore described here in more detail. The smallest operation was partial mastectomy. In project 4, it was called "extended tylectomy" and defined as "wide excision of the lump together with the surrounding breast tissue within 3 cm of palpable or visible growth" [6]. Deep and peripheral tumors obviously could not have such a margin. Patients with medial tumors were excluded from the study, perhaps for this reason. Another variation of partial mastectomy called "quadrantectomy" was studied in project 8, in which only patients with no palpable nodes and cancers less than 2 cm in diameter, as measured by the pathologist after excision, were included; 45 percent of the tumors were less than 1 cm in diameter [19]. The involved segment of the breast was removed, including skin and fascia, with at least 2 cm of tissue surrounding the cancer. Through the same or a separate incision, the axillary nodes (numbers not given) were dissected out, with removal of part of the minor pectoral muscle. Cosmetic results were thought to be good in "more than 70 percent of the cases." Total (simple) mastectomy was evaluated in projects 1, 2, 3, 6, and 7. In project 2, 79 percent of total mastectomies included axillary lymph nodes, and, in 42 percent of these patients, there were nodal metastases. Probably the most prominent nodes were removed, and this may have altered survival [21]. In project 6, total mastectomy included an average of 15 nodes, the same number as this group found in their radical 314 I DonaldJ. Ferguson ¦ Mastectomy and Survival mastectomies [22]. In their hands, these nominally different procedures are in an important respect exactly alike. That the results are also similar might have been anticipated. Total mastectomy with lymphadenectomy does not differ in basic concept from modified radical mastectomy, the most-used operation in recent years, for which there are no 10-year randomized comparisons. Radical mastectomy, included in all the projects except the first, has this name because of the myectomy. Resection of the sternal part of pectoralis major and the pectoralis minor continues to be advocated by some surgeons because it increases the margin of normal tissue beneath the cancer, resects lymphatic vessels traversing the muscle, and facilitates complete nodal dissection [23]. The posterior margin of total mastectomies , or modified radical resections, is commonly within 0—10 mm of the tumor or biopsy cavity. (In 126 patients, our pathologist noted the margin in 39, of which 31 were SlO mm.) In the radical mastectomies of project 7, myectomy was optional. In its absence, the distinction from the total mastectomies of the comparison group is diminished. The radical mastectomies of project 2 could be supplemented ad lib. by removal of internal mammary nodes. These variations were not enumerated or distinguished in the survival data. The radical mastectomies of project 6, performed by various surgeons in 34 institutions, removed on the average about half as many nodes as can be obtained [8]. In 20 percent of patients undergoing radical mastectomy in project 2, healing was delayed more than 2 months. The percentage of patients who had local first recurrences after radical operations was high in all these studies (table 1). In our institution among all patients eligible for randomization at stages I and II and treated by radical surgery from 1960 to 1978, the local recurrence rate is 3 percent (17/513). The incidence of local recurrence , like the incidence of delayed healing and the count of lymph nodes removed, is an indication of the care and thoroughness with which the operation was done. Extended radical mastectomy, performed as described by Urban [24], aims to remove both of the primary lymph node drainage areas en bloc with the breast and intervening tissues. About 40 percent of patients at combined clinical stages I and II have axillary nodal metastases and roughly 20 percent have internal mammary involvement. The good reasons there are for removing axillary nodes therefore should apply also to the internal mammary nodes, but these reasons f^e not applied, probably because complete removal of the latter is surgically difficult and, if not done properly, dangerous. To obtain practically all of the internal mammary nodes, it is necessary to resect part of the sternum, costal cartilages 2-5, and pleura and to dissect out the vessels and adjacent tissues above the first interspace. The resulting defect in the chest wall is at least 4.5 cm in width. It must be closed, preferably with Cortex®, Perspectives in Biology and Medicine, 30, 3 ¦ Spring 1987 \ 315 under strong tension to prevent pain, deformity, and paradoxical respiratory motion. There is no visible or palpable postoperative defect apparent to the patient when this has been done properly. The need for removing pleura en bloc is clearly shown in figure 7 of McDonald, Haagensen, and Stout [25], a photomicrograph showing metastases adjacent to this membrane. Apparently, few surgeons are trained to handle this resection expertly: most of the extended operations in studies 1 and 5 were not en bloc; after the radical mastectomy was finished, some nodes were dissected extrapleurally from the intercostal spaces [14, 26]. About half the extended radical operations in project 5 were said to be en bloc, a procedure pictured as a narrow parasternal excision filled in by a strip of denervated and devascularized muscle that could not hold any tension [27]. The average number of internal mammary nodes removed in our experience with extended radical mastectomy [28] has been six. No number is given for project 1. The averages for the various groups of project 5 were two to four. Veronesi [29] published his part in project 5 separately, showing results that cannot be numerically reconciled with data in the parent study. We have tabulated only the latter. Neither of the two studies nominally about extended radical mastectomy used the Urban operation, and their resections were evidently not complete. The Quality of Surgery—Effect on Survival By a combination ofchance and design, in project 4, survival following radical mastectomy done by trainees was compared with that following nominally the same procedure performed by specialists in breast surgery (table 1; compare the two paired groups at stage I). (See fig. 1.) The difference in survival favored the specialists by 16 percent at 10 years, P < .0007 [7]. Although the quality of partial mastectomy in the second group was presumably also improved, survival remained equally poor. None of the other studies dealt overtly with quality of surgery, and yet, on the basis of the evidence cited of incompleteness of both radical and extended operations, this is probably the factor that explains their small differences in survival results, exactly as it did in the first group of stage I patients in the Guy's trial. The quality of surgery in our institution, where fully trained surgeons have always scrubbed in, underwent change when a blood bank and well-developed anesthesia service allowed the duration of radical mastectomy to increase from an average of 1.6 hours from 1927 through 1944 to 3.7 hours from 1945 through 1978. While survival of patients at pathologic stage I remained the same (74 and 73 percent) at 10 years in the two eras, survival at pathologic stages II and III, in which the better care in dissection now possible would likely make the most difference, 316 I DonaldJ. Ferguson ¦ Mastectomy and Survival ----- 1st Series ------ 2nd Series Significance P<.0007 —i--------1--------1--------1--------r--------1--------r--------1-------1--------r-------1 ? t~ 84 96 108 120 U2 14a 156 168 100 19? 204 216 2:8 Months Fig. 1.—Survival of patients at stage I in project 4 after radical mastectomy performed by surgical trainees (first series) compared to that following the same operation performed by specialists in breast surgery (second series). Reprinted from [7] with permission. increased from 29 to 47 percent and from 8 to 26 percent, respectively [8]. It may also be pertinent to the quality of mastectomy that survival of black patients at 5 years at each stage is 4 to 14 percent lower than that of Caucasians in a widely based sample from this country [30]. Black patients , mostly in a lower socioeconomic group, are more often operated on by trainees with only nominal supervision. In our institution, survival in the two races is the same [28]. Survival rates would be the ultimate test of the quality of resection if they could be reliably compared between one project and another. Such comparisons are not appropriate because of uncontrolled variations in patient selection and clinical staging, both of which are partly subjective, and because of unique restrictions to entry such as found in project 8. Nevertheless it can be noted that survivals are unusually low after radical mastectomy in projects 6 and 7 and the first series of project 4 compared to other experience in project 4, project 2, and elsewhere [8]. Survival and Recurrences: The End Results The objective of the Guy's Hospital trial, project 4, was, according to Atkins, to test "the hypothesis that radical mastectomy was no more effective in preserving life in cases ofearly cancer ofthe breast than such restricted surgery as simple removal of the lump" [6]. The results at 10 Perspectives in Biology and Medicine, 30, 3 ¦ Spring 1987 \ 317 ____ Radical ------ W.E. Significance P<002 40 Months 80 96 104 Fig. 2.—Survival of patients at clinical stage I up to 10 years after radical mastectomy, compared to that after partial mastectomy (Wide Excision, W.E.) in project 4. Reprinted from [7] with permission. and 20 years show that the smaller procedure failed for both clinical stages I and II (table 1, figs. 2 and 3). Adjuvant radiotherapy, given to both treatment groups, has been criticized as being inadequate for the smaller procedure. To prove equal to radical mastectomy, it would have to reduce mammary and axillary recurrences to zero without causing appreciable morbidity, an unlikely possibility [21]. Whether or not radiotherapy was at the optimum dosage in project 4, the basic question pertained to the extent of surgery, and it was given a clear answer. The Bruce trial, project 3, also significantly favors radical mastectomy over a smaller operation for patients at stage I, though not for those at stages II and III. Only half the patients had been followed for 10 years at last report. No significant differences were found in the other studies, except that local recurrence was higher in project 5 after the smaller operation (table 1). In this study at 5 years, patients at stage II with medial-central cancers had a significantly higher survival after extended, compared to radical, mastectomy; this comparison of subgroups was not reported at 10 years. (We also observed higher survival for medial-central tumors, 62 percent of the total in our series, after extended radical mastectomy 3 18 I DonaldJ. Ferguson ¦ Mastectomy and Survival ------ Radical ----- WE Significance P< .05 —?-------1-------1-------1-------t------t-------1-------1-------1-------1-------1-------1-------1-------1-------1-------1-------1 48 60 72 84 96 108 120 132 144 156 168 180 192 204 216 228 240 FiG. 3.—Survival of patients at clinical stage II up to 20 years after radical mastectomy, compared to that after partial mastectomy (Wide Excision, W.E.) in project 4. Reprinted from [7] with permission. at 5 years [28] and will report on this randomization, in which the survival difference continues, at 10 years.) The nonsignificant differences in survival in the table also are mainly in favor of the larger procedure. Postoperative Radiotherapy Radiotherapy was used to supplement only the smaller of the two procedures in projects 1, 3, 6 (stage II), 7, and 8. Assuming that it may have affected survival, both the extent of surgery and the radiation are without controls in these protocols. Another group of total mastectomies in project 6 who received radiotherapy is not listed in the table because it is not relevant to the surgical comparison; their survival was 6 percent higher than that Ofcontrols who had total mastectomy without radiation, a difference not statistically significant. No indication of late (8 + years) toxicity of radiotherapy was given in these studies. (Serious complications have been observed at 8 to 40 years in 1 1 percent of patients who had adjuvant radiation after radical mastectomy [31], and, to date, there has been a 4 percent mortality directly attributable to radiation in this series.) The basic problem with any adjuvant is that some cancer cells are Perspectives in Biology and Medicine, 30, 3 ¦ Spring 1987 \ 3 19 resistant to it. The chance that adjuvant chemotherapy, endocrine treatment , or radiation can eliminate all remaining cancer cells after mastectomy is therefore better if there are fewer of them and should be maximal following the most complete operations. No controlled trial of any adjuvant has been carried out in groups of patients each of whom had a demonstrably careful and complete operation. The Changing Faces of the Data It is instructive to compare sequential reports on the same projects. The Guy's trial (partial vs. radical mastectomy) has often been cited as evidence that smaller operations are "just as good," viz. Atkins 1972 (referring to all the data): "No significant difference between the results of the two operations can be detected at any time up to 10 years after treatment." But later: "in stage 2 cases radical mastectomy gives significantly better survival ... at 10 years but not at 5 years" [6]. Hayward , in 1977, stated, "There was no significant difference between the two operations in 5- and 10-year survival" (referring to patients in both stages). And, after tylectomy at stage I, "In almost all cases, local recurrence can be treated successfully by further surgery or radiotherapy and it does not appear to affect distant recurrence or survival" [32]. Hayward stated in 1980, ". . . there is no firm evidence that local control of disease can be achieved by any treatment other than mastectomy" [33]. Hayward , in 1981, stated, "If the primary complex is treated inadequately, the tumour recurs locally, the distant metastatic rate increases and survival is diminished" [34]. (Halsted may have wafted this thought from the empyrean.) The most recent report, published in a book mainly about radiotherapy—not in our library or bookstore and not indexed by Medline—documents significantly higher survival at 10-20 years for both stages I and II after radical mastectomy [7]. It is as if this result, not what was expected and wanted, although honestly reported, was laid to rest. In 1986, this project continues to be referred to as supporting the avoidance of mastectomy [35]. The Bruce trial similarly was, for many years, cited as evidence that the radical procedure was no better (viz. Bruce 1971). There is "no statistically significant difference in survival data." (The 5-year survival for radical mastectomy was 76 percent, for total mastectomy 66 percent) [13]. Hamilton stated in 1974, "There is no significant difference in survival between patients randomly allocated to simple mastectomy plus radical radiotherapy or standard radical mastectomy" at 5 years [36]. Hamilton statec' in 1977 that the difference was 3 percent at 10 years [37]. Three years later, the latest report [5] gives radical mastectomy a 9.5 percent advantage at 10 years, P < .05 (table 1). This paper contains a fairly complete statistical analysis. 320 I DonaldJ. Ferguson ¦ Mastectomy and Survival Earlier Diagnosis, Smaller Cancers, and Cosmesis Preclinical or minimal breast cancer, usually discovered by mammography , includes noninvasive proliferations and microscopic cancers, or those up to 5 mm in diameter. Survival after total mastectomy has been close to 100 percent, and there is no need for more surgery. Partial mastectomy is not adequate because such tumors are commonly multicentric . In project 8, for example, there have already been a number of recurrences and "new" cancers in the breasts so treated. Cancers over 5 mm in diameter have appreciable risk of adenopathy, and the safest treatment is a complete operation. Given the clear choice that exists between preservation of mammary tissues and a reduced risk of recurrent cancer, the great majority of patients chooses the latter. Differences in appearance or function in postmastectomy patients who have had total, radical, or extended radical mastectomy are not recognized by most patients, nurses, or surgical residents who follow them in the clinic, assuming that the procedures have been properly performed. Replacement of muscle by shifting the origin of the latissimus dorsi at the time of mastectomy without alteration of the incision leaves the patient ready to receive a prosthesis under local anesthesia. Thus complete surgery need not be more deforming than total mastectomy (or modified radical mastectomy), and the patient who receives only the latter is subjected to increased risk for no good reason. Conclusions Analysis of the eight randomized comparisons of different types of mastectomy that have been carried to at least 10 years reveals some basic deficiencies. In four of the studies, radiotherapy was added only to the smaller procedure, thus clouding the surgical comparison. Nevertheless, in three of the four, the nonirradiated patients with the larger operations had the higher survival. Numbers of lymph nodes removed and examined were not given in six projects. In the other two, they were about half the number expected with careful surgery. In one study, the small number of nodes obtained was the same for radical as for simple or partial mastectomy, indicating that there was not much difference in the procedures. The only two studies of "extended radical" operations documented inadequate margins of resection. The two studies with significant differences in survival both favored the larger operation. Survival following mastectomies performed by surgical trainees was inferior to that following more thorough and careful operations performed by surgical specialists. The available data support the reasonable assumption that survival of breast cancer patients is proportional both to the nominal extent of the mastectomy and to the competence with which it is carried out. Perspectives in Biology and Medicine, 30, 3 ¦ Spring 1987 \ 32 1 REFERENCES 1.Forrest, A. P. M. Conservative management of breast cancer: a review of British controlled trials. Ann. R. Coll. Surg. Engl. 62:41-43, 1980. 2.Fisher, B. Laboratory and clinical research in breast cancer—a personal adventure. Cancer Res. 40:3863-3874, 1980. 3.Lvthgoe, P. J., and Palmer, M. K. Manchester regional breast study—5 and 10 year results. Br. J. Surg. 69:693-696, 1982. 4.Hamilton, T.; Langlands, A. O.; and Prescott, R. P. The treatment of operable cancer of the breast. Br. J. Surg. 61:758-761, 1974. 5.Langlands, A. O.; Prescott, R. J.; and Hamilton, T. A clinical trial in the management of operable cancer ofthe breast. Br.J. Surg. 67:170-174, 1980. 6.Atkins, H.; Hayward, J. L.; Klugman, D. 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COG5598 comprises a large number of proteins related to MttB, the trimethylamine: corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptors such as nitrate or fumarate, producing dimethylglycine and CO2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obviousmeans to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I) alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine: corrinoid methyltransferase described, and a designation of MtgB is proposed. In addition, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin: tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. They are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.

Two novel strains of methanogens were isolated from an estuarine sediment with the capability to utilize quaternary amines. Based on the 16S rRNA analysis, strain B1d shared 99 % sequence identity with Methanolobus vulcani PL-12/M T and strain Q3c shared 99 % identity with Methanococcoides sp. PM1 and PM2, but our current isolates display clearly different capabilities of growth on quaternary amines and were isolated based on these capabilities. Strain Q3c was capable of growth on tetramethylammonium and choline, while strain B1d was capable of growth on glycine betaine. Ml. vulcani PL-12/M T was incapable of growth on glycine betaine, indicating an obvious distinction between strains B1d and PL-12/M T . Strain Q3c now represents the only known tetramethylammonium-utilizing methanogen in isolation. Strain B1d is the first quaternary amine-utilizing methanogen from the genus Methanolobus . This study suggests that quaternary amines may serve as ready precursors of biological methane production in marine environments.

The family Methanosarcinaceae has an expanded repertoire of growth substrates relative to most other methanogenic archaea. Various methylamines, methylated thiols, and methanol can serve as precursors to both methane and carbon dioxide. These compounds are mobilized into metabolism by methyltransferases that use the growth substrate to methylate a cognate corrinoid protein, which in turn is used as a substrate by a second methyltransferase to methylate Coenzyme M (CoM), forming methyl-SCoM, the precursor to both methane and carbon dioxide. Orthologs of the methyltransferases, as well as the small corrinoid proteins, are found in many archaeal and bacterial genomes. Some of these are homologs of the methylamine methyltransferases predicted to require pyrrolysine, an atypical genetically encoded amino acid, for synthesis. As a resource for the study of these sizable families of proteins, we describe here techniques our laboratories have used for the study of methanogen corrinoid-dependent methyltransferases, focusing especially on isolation and assay techniques useful for various activities of components of the methylamine- and methylthiol-dependent CoM methyltransferase systems.

Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO 2 oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO 2 interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the α 2 ε 2 component from the α 8 β 8 γ 8 δ 8 ε 8 CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H 2 O/OH - bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the ε-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.

This study focused on the interpretation of scanned cephalometric images after the digitization of the original analog film, with a variety of standard scanner settings. The purpose was to determine the minimum allowable resolution, grayscale, and color settings of these digital images, without compromising the precision of orthodontic landmark identification. Forty-nine orthodontic residents and faculty at Saint Louis University identified 13 landmarks in 3 separate trials on either an original lateral cephalogram or 1 of 6 digitized radiographic images. They also subjectively assessed the quality of the image. Ability to identify landmarks did not differ significantly among the groups tested. Conversely, opinions of image quality differed significantly among the groups. Participants found the quality of the grayscale images without color to be poorer than that of the original film but could detect differences only at resolution extremes. Prediction of ability to identify landmarks was substantially greater for higher resolution images. In conclusion, the scanner settings used in the digitization of a cephalometric film did not matter significantly when standard settings were used. In addition, subjective opinion of image quality was predictive of how precisely landmarks were identified when the resolution was relatively high.

ABSTRACT Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM). Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time. A single copy of mttB , the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene, mtbB1 . However, two other nearly identical copies of mtbB1 , designated mtbB2 and mtbB3 , were also found in the genome. A 6.8-kb transcript was detected with probes to mttB and mtbB1 , as well as to mtbC and mttC , encoding the cognate corrinoid proteins for DMA:CoM and TMA:CoM methyl transfer, respectively, and with probes to mttP , encoding a putative membrane protein which might function as a methylamine permease. These results indicate that these genes, found on the chromosome in the order mtbC , mttB , mttC , mttP , and mtbB1 , form a single transcriptional unit. A transcriptional start site was detected 303 or 304 bp upstream of the translational start of mtbC . The MMA, DMA, and TMA methyltransferases are not homologs; however, like the MMA methyltransferase gene, the genes encoding the DMA and TMA methyltransferases each contain a single in-frame amber codon. Each of the three DMA methyltransferase gene copies from Methanosarcina barkeri contained an amber codon at the same position, followed by a downstream UAA or UGA codon. The C-terminal residues of DMA methyltransferase purified from TMA-grown cells matched the residues predicted for the gene products of mtbB1 , mtbB2 , or mtbB3 if termination occurred at the UAA or UGA codon rather than the in-frame amber codon. The mttB gene from Methanosarcina thermophila contained a UAG codon at the same position as the M. barkeri mttB gene. The UAG codon is also present in mttB transcripts. Thus, the genes encoding the three types of methyltransferases that initiate methanogenesis from methylamine contain in-frame amber codons that are suppressed during expression of the characterized methyltransferases.

Reconstitution of trimethylamine-dependent coenzyme M (CoM) methylation was achieved with three purified polypeptides. Two of these polypeptides copurified as a trimethylamine methyl transfer (TMA-MT) activity detected by stimulation of the TMA:CoM methyl transfer reaction in cell extracts. The purified TMA-MT fraction stimulated the rate of methyl-CoM formation sevenfold, up to 1.7 micromol/min/mg of TMA-MT protein. The TMA-MT polypeptides had molecular masses of 52 and 26 kDa. Gel permeation of the TMA-MT fraction demonstrated that the 52-kDa polypeptide eluted with an apparent molecular mass of 280 kDa. The 26-kDa protein eluted primarily as a monomer, but some 26-kDa polypeptides also eluted with the 280-kDa peak, indicating that the two proteins weakly associate. The two polypeptides could be completely separated using gel permeation in the presence of sodium dodecyl sulfate. The corrinoid remained associated with the 26-kDa polypeptide at a molar ratio of 1.1 corrin/26-kDa polypeptide. This polypeptide was therefore designated the TMA corrinoid protein, or TCP. The TMA-MT polypeptides, when supplemented with purified methylcorrinoid:CoM methyltransferase (MT2), could effect the demethylation of TMA with the subsequent methylation of CoM and the production of dimethylamine at specific activities of up to 600 nmol/min/mg of TMA-MT protein. Neither dimethylamine nor monomethylamine served as the substrate, and the activity required Ti(III) citrate and methyl viologen. TMA-MT could interact with either isozyme of MT2 but had the greatest affinity for the A isozyme. These results suggest that TCP is uniquely involved in TMA-dependent methanogenesis, that this corrinoid protein is methylated by the substrate and demethylated by either isozyme of MT2, and that the predominant isozyme of MT2 found in TMA-grown cells is the favored participant in the TMA:CoM methyl transfer reaction.

An immunochemical approach was employed as a direct test for functional activities of isozymes of methylcobamide:coenzyme M methyltransferase (MT2-M and MT2-A) in the metabolic pathways of methane formation from: methanol, acetate, monomethylamine, dimethylamine, and trimethylamine. Specific removal of the MT2 isozymes from buffer soluble cell extracts of Methano-sarcina barkeri was accomplished by use of immobilized, affinity-purified, ovine polyclonal antibodies. Extracts of methanol-grown cells depleted of MT2-M lost entirely the ability to carry out conversion of methanol to 2-(methylthio)ethanesulfonate (methyl-CoM). Methanol:CoM methyl transfer activity was completely restored by addition of purified MT2-M, but no activity was recovered by addition of MT2-A In contrast, the activity of trimethylamine-grown cell extracts to convert monomethylamine and dimethylamine to methyl-CoM was lost almost entirely by immunosorptive removal of MT2-A. Addition of purified MT2-A, but not MT2-M, to the MT2-A-depleted extract fully reconstituted methyl-CoM formation from both mono- and dimethylamine. Interestingly, in extracts resolved of MT2-A, trimethylamine-dependent methylation of coenzyme M was observed at approximately 20% of the rate of controls not treated with antibody. Furthermore, both isozymes were effective in full restoration of trimethylamine conversion. Tests indicated that neither of the two MT2 isozymes are involved in methane formation from acetate. The results establish that MT2-A plays a specific role in metabolism of methylated amine substrates, whereas, MT2-M functions in methane formation from trimethylamine and methanol.