Patricia Hannah Clarke was a distinguished British biochemist and microbiologist who won an international reputation for her work on microbial evolution. After completing the Natural Sciences Tripos at the University of Cambridge at the beginning of World War II, she chose to work for the Armaments Research Department, before moving into microbiological research on bacterial toxoids. She was appointed to an assistant lectureship in biochemistry at University College London in 1953, eventually becoming Professor of Microbial Biochemistry in 1974. Her pioneering work on the directed evolution of bacterial metabolic capability led to her election to Fellowship of the Royal Society in 1976. Patricia gave dedicated service to the scientific community through her many years of committee work with the Royal Society, the Biochemical Society and the Society for General Microbiology. She was a passionate advocate of the importance of equal opportunities for women in education and scientific careers.

Sir Kenneth Murray—Ken to his friends—was held in high esteem and affection by all who knew him. In a remarkable career, which began after he left school at the age of 16 years, he played a prominent part, through his elegant and meticulous research, in the evolution of biotechnology; he trained and inspired generations of students entering this bright new discipline; he developed the means of diagnosing, preventing and treating a feared disease, thereby saving many thousands of lives; he was one of a small coterie that founded the first Europe-based biotechnology company; and he used the profits that this yielded to enrich his university and promote, through the charity that he founded, the careers of promising young scientists. He remained, throughout, modest, unassuming and ever willing to give of his time and knowledge to all who asked.

Salivary fluid, the collective product of numerous major and minor salivary glands, contains a range of secretory proteins that play key defensive, digestive, and gustatory roles in the oral cavity. To understand the distinct protein "signature" contributed by individual salivary glands to salivary secretions, we studied a family of proteins shown by in vitro mRNA translation to be abundantly expressed in mouse sublingual glands. Molecular cloning, Southern blotting, and restriction fragment length polymorphism analyses showed these to represent one known and two novel members of the common salivary protein (CSP-1)/Demilune cell and parotid protein (Dcpp) salivary protein family, the genes for which are closely linked in the T-complex region of mouse chromosome 17. Bioinformatic analysis identified a putative human CSP-1/Dcpp ortholog, HRPE773, expressed predominantly in human salivary tissue, that shows 31% amino acid identity and 45% amino acid similarity to the mouse Dcpp query sequence. The corresponding human gene displays a similar structure to the mouse Dcpp genes and is located on human chromosome 16 in a region known to be syntenic with the T-complex region of mouse chromosome 17. The predicted mouse and human proteins both display classical NH2-terminal signal sequences, putative jacalin-related lectin domains, and potential N-linked glycosylation sites, suggesting secretion via sublingual saliva into the oral cavity where they may display antimicrobial activity or provide a defensive coating to enamel. Identification of a human CSP-1/Dcpp ortholog therefore provides a key tool for investigation of salivary protein function in human oral health and disease.

A Type II restriction endonuciease, Mmel, has been purified from the obligate methylotroph, Methylophilus methylotrophus. The enzyme was shown to have the non-pallndromic recognition sequence 5' T C C Pu A C (N)2o " 3' 3' A G G Py T G (N) 1 8 " 5' and to cleave (as Indicated) on the 3' side, generating a two nucleotide 3' projection. Determination of the recognition sequence was achieved U3lng two new computer programs; RECOG, which predicts recognition sequences from the pattern of restriction fragments obtained from DNAs of known sequence, and GELSIM, which generates graphical simulations of DNA band patterns obtained by gel electrophoresis of restriction digests of sequenced DNA molecules. INTRODUCTION Type II restriction endonucleases that cut DNA molecules at, or near, defined nucleotide sequences, have now been obtained from a wide variety of bacterial species (1) and have become essential tools in molecular biology. The sequence-speclflcity of an endonuciease can often be determined by analysis of the ends generated by the action of the enzyme. It is also possible to define a target sequence by analysis of the pattern of cleavage of DNA molecules of known nucleotide sequence (2,3,1),5). Here we describe the isolation of a new restriction enzyme, Mmel, from the Gram-negative bacterium Methylophilus methylotrophus, an obligate methylotroph and source of commercial single cell protein (SCP). A computer program, RECOG, which includes an algorithm not used in similar programs (2, 3,4,5), was developed to determine the recognition sequence of the enzyme using accurate mapping data on a 3equenced DNA, pBR322 (6). (The pBR322 sequence revision, adding one base pair to the TcR gene (7), has a negligible effect on the analyses described below, so the original coordinates are used throughout.) Mmel restriction generates a complex © IRL Press Limited, Oxford, England. 5255 at Y le U niersity on Jne 6, 2015 http://narrdjournals.org/ D ow nladed from Nucleic Acids Research mixture of partial and complete digest products; therefore, another computer procedure, GELSIM, was written to simulate graphically the migration of DNA fragments during agarose gel electrophoresis, simplifying analysis of Mnel gel patterns. Computer methods described below are generally applicable to the problem of characterizing newly isolated restriction enzymes, especially preparations too crude to be analysed biochemically. MATERIALS AND METHODS Methylotroph culture conditions M.methylotrophus is an obligate methylotroph used commercially as a 3ource of single cell protein (8). Cells were grown aeroblcally at 37°C in medium containing 0.9J (v/v) methanol, and (per litre), NaH2P0i| d.'tg), K2HPOi4 (1.9g), (NHi))2SOi) (1.8g), MgSOi, (0.2g) with frace elements CuSOn-SHjO (0.02mg), MnSOi4.4H2O (0.1mg), FeCl3 (0.98mg), ZnSOn-TH^ (0.1mg) and CaCO3 (1.8mg). Substrate DNAs and enzymes Plasmid pBR322 and M13mp7 RF DNAa were prepared by a scaled-up alkallne/SDS Iysl3 method followed by purification on a CsCl/ethidlum bromide gradient (9, 10). The E.coll strain GMH8 (11) (dam-3 dcm-6 gal ara lac thr leu thl tonA tax) was transformed with pBR322 and was used as a source of "dam*"" plasmid DNA. Phage Lambda (cj_857, Sam7) DNA was prepared from a suitable lysogen by a standard technique (12). SV10, PhiX17H RF and H13 RF DNAs were gifts from (respectively) B.K. Ely, G.E. Blair and J.G.G. Schoenmakers. Restriction enzymes P3_tI, Sau3A, TaqI, EcoRI and BamHI were from BRL Inc. and used as recommended. Symbols for degenerate nucleotldes The programs described here were developed before any standard symbols for degenerate nucleotlde positions were accepted. Therefore, as well as the commonly used symbols X purlne, Y pyrimidine, N A, C, G or T, new ones were devised mnemonically to describe degeneracies found in many recognition sequences. These are: 1 A or C ("ACe"); 8 A or T ("ATe") and their "arithmetic complements" 9 G o r T ; 2 G o r C ( 1 + 9 2 + 8 10). Under this system, for example, AccI recognizes GT19AC and Hael recognizes

The leading region of the conjugal bacterial plasmid ColIb-P9 contains three dispersed repeats of a 328 bp sequence homologous to Frpo, a sequence from plasmid F that acts as a promoter in single-stranded DNA. One of these sequences, ssi3, inactive in the double-stranded form, promoted in vitro transcription exclusively from the single strand that is transferred during conjugation. Promoter activity was dependent on the presence of RNA polymerase holoenzyme containing sigma 70. Transcription initiated from the position predicted from folding the single-stranded DNA to form a pseudo double-stranded hairpin structure containing recognizable -35 and -10 promoter elements. Footprinting of RNA polymerase holoenzyme on single-stranded ssi3 DNA was consistent with this suggestion. Mutagenesis of the putative -35 region inactivated the promoter, but random mutations in the -10 region had little effect. The putative -10 region is a poor match to the consensus sequence and contains mismatched bases. Elimination of these mismatches invariably destroyed single-strand promoter activity. These observations reveal the crucial contribution of the unpaired bases in the -10 region in potentiating the formation of the productive open complex with RNA polymerase.

The ArdA antirestriction protein of the IncB plasmid R16 selectively inhibited the restriction activity of EcoKI, leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The results are consistent with the hypothesis that ArdA functions in bacterial conjugation to allow an unmodified plasmid to evade restriction in the recipient bacterium and yet acquire cognate modification.

The inwardly rectifying potassium ion channel Kir2.2 has recently been demonstrated to have nuclear and plasma membrane subcellular localization. Nuclear expression of Kir2.2 is controversial, as a functional role for Kir2.0 potassium channels in the nucleus has not been investigated. However, in this report we have demonstrated Kir2.2 nuclear localization in sections of rat hindbrain and dorsal root ganglia tissue, using two anti- Kir2.2 polyclonal antisera with different epitope specificities. These data confirm nuclear localization and are suggestive of new functions of Kir2.0 potassium ion channels in the nucleus.

The leading region of a plasmid is the first sector to enter the recipient cell in bacterial conjugation. This sector of IncI1 plasmid ColIb-P9 includes genes that are transcribed in a transient pulse early in the conjugatively infected cell to promote establishment of the immigrant plasmid. Evidence is presented that the burst of gene expression is regulated by a process which is independent of a repressor but dependent on the orientation of the genes on the unique plasmid strand transferred in conjugation. The nucleotide sequence of 11.7 kb of the leading region was determined and found to contain 10 ORFs; all are orientated such that the template strand for transcription corresponds to the transferred strand. The leading region contains three dispersed repeats of a sequence homologous to a novel promoter in ssDNA described by H. Masai & K. Arai (1997, Cell 89, 897-907). It is proposed that the repeats are promoters that form in the transferring strand of ColIb to support transient transcription of genes transferred early in conjugation.

Inwardly rectifying potassium (Kir) channels are expressed in a wide range of excitable and non-excitable cells, where they set the resting membrane potential. Because Kirs are open in the range of the resting potential, their modulation is an important factor in the regulation of cellular excitability. In the noradrenergic neurons of locus coeruleus (LC), the neuropeptide substance P (SP) has been shown to suppress Kir activity.1,2 In this report we demonstrate, by immunohistochemistry with site-directed polyclonal antibodies, the co-localization of Kir2.2 and substance P receptor (SPR) proteins in single LC neurons and associated oligodendroglia. In addition, we show a nuclear localization in LC neurons for Kir2.2 protein. This was supported by a nuclear signal with Kir2.2 antibodies obtained in Chinese hamster ovary (CHO) cells transiently transfected with Kir2.2. Expression of Kir2.2 protein could not be detected in untransfected cells. The Kir2.2 antibody detected a single 62 kD band on Western blots containing rat brain nuclear and plasma membrane protein fractions. This band was blocked by incubation with the 10 μg/ml Kir2.2 antigenic peptide (data not shown). Similarly Kir2.2 immunostaining on rat brain tissue sections was also blocked by incubation with the Kir2.2 antigenic peptide (data not shown). Adjacent coronal cerebellar rat brain tissue sections (10 μm deep, paraffin-embedded) were immunostained with the affinity purified SPR antibody (1:500), or the affinity purified Kir2.2 antibody (12 μg/ml). The large diameter (∼40 μm) of LC neurons made it possible to identify cells present in serial tissue sections. Strong nuclear and plasma membrane signals in neurons were observed for Kir2.2, with a clear nuclear signal also in oligodendroglia (FIG. 1A). SPR was strongly expressed on the plasma membranes of neurons and oligodendroglia, as well as neural processes within the LC (FIG. 1B). In order to establish co-expression of Kir2.2 and SPR, the Macromedia XRES program was used to overlay the two adjacent tissue sections. This overlay demonstrated a co-localization of SPR and Kir2.2 proteins in single neurons and oligodendroglia of the LC (FIG. 1C). This is consistent with previous

The ardA gene of the enterobacterial plasmid CoIIbP-9 acts to alleviate restriction of DNA by type I systems, while psiB inhibits induction of the bacterial SOS response. Both genes are transferred early in a round of bacterial conjugation as part of the plasmid leading region. We report here that ardA and psiB are transcribed transiently after their conjugative transport into the recipient cell. Transcript levels, monitored by competitive reverse transcription-polymerase chain reaction (RT-PCR) amplification of RNA templates, started to increase about 5 min after the initiation of conjugation in a cell population and probably before the first round of plasmid transfer was completed. Genetic evidence is given that the expression of ardA and psiB is activated when the genes enter the recipient cell on the transferring plasmid strand. It is proposed that these and other leading region genes function to promote the establishment of the immigrant plasmid in the new host and are expressed by transcription from promoters active only in single-stranded DNA.

Intercellular Channels (GAP JUN): Intercellular Gap Junctional Channels Formed By Connexin Proteins [ECC]. Intracellular Ligand-Gated Channels [ILG]: The Intracellular Ligand-Gated Channel Group-Key Facts [ECC]. Native Ca2+ Channels Gated By the Arachidonic Acid Metabolite Leukotreine C4. Ion Channels Associated with Ca2+ Channels Sensitive to Inositol 1,3,4,5-Tetrakisphophate (Insp4) [ECC]. Caffeine-Sensitive C2+. Release Channels (Ryanodine Receptors, Ryr) [ECC] Candidate Native Intracellular-Ligand-Gated Ca2+-Store Repletion Channels [ECC]. Inositol 1,4,5-Trisphosphate-Sensitive Ca2+-Release Channels (Insp3r) [ECC]. Subject Indexes.

Rats at day 15.5 of gestation were dosed intraperitoneally with 300 mg.kg-1 of clofibrate for three consecutive days at 24-hr intervals and were culled 24 hr after the final injection. This regime produced maximal induction of the cytochrome P4504A (CYP4A) mRNAs in the maternal liver and kidney and in 18.5-day fetal tissues. The maternal hepatic and renal CYP4A mRNA levels had risen 12- and 2-fold, respectively, above the constitutive levels seen in untreated pregnant rats at an equivalent stage of gestation. Clofibrate was capable of traversing the placenta and modulating the fetal CYP4A mRNA expression as demonstrated by a 3-fold elevation in the mRNA levels in those fetuses explanted from drug-induced mothers, compared with those fetuses removed from untreated mothers. The CYP4A mRNAs were demonstrated in the fetal liver via dot-blot and Northern blot analyses. In addition, low levels of CYP4A mRNA expression were detected in the induced placenta via Northern blot analysis. Western blot analysis revealed that the CYP4A protein levels increased in the maternal liver and in the kidney and fetal livers after exposure to clofibrate. Peroxisome proliferation, a phenomenon associated with induction of CYP4A1 expression in rodents, was demonstrated in both maternal and fetal livers, with the use of light and electron microscopy.

Conference Abstract| November 01 1996 THE ROLE OF POTASSIUM CHANNELS IN T LYMPHOCYTES S. Badley; S. Badley 1Centre for Mechanisms of Human Toxicity, University of Leicester, P.O Box 138, Lancaster Road, Leicester, LE1 9HN Search for other works by this author on: This Site PubMed Google Scholar C. Dart; C. Dart 1Centre for Mechanisms of Human Toxicity, University of Leicester, P.O Box 138, Lancaster Road, Leicester, LE1 9HN Search for other works by this author on: This Site PubMed Google Scholar E. Conley; E. Conley 1Centre for Mechanisms of Human Toxicity, University of Leicester, P.O Box 138, Lancaster Road, Leicester, LE1 9HN Search for other works by this author on: This Site PubMed Google Scholar W. Brammar W. Brammar 1Centre for Mechanisms of Human Toxicity, University of Leicester, P.O Box 138, Lancaster Road, Leicester, LE1 9HN Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (4): 509S. https://doi.org/10.1042/bst024509sc Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation S. Badley, C. Dart, E. Conley, W. Brammar; THE ROLE OF POTASSIUM CHANNELS IN T LYMPHOCYTES. Biochem Soc Trans 1 November 1996; 24 (4): 509S. doi: https://doi.org/10.1042/bst024509sc Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.

Lactating mothers of 7.5-day neonatal rats were injected intraperitoneally with 500 mg kg−1 clofibrate for 3 consecutive days at 24-hour intervals; 24 hours after the final injection, the maternal cytochrome P450 4A (CYP4A) mRNA levels had risen 14- and 2.5-fold above the constitutive levels of expression seen in the liver and kidney, respectively. Lactational transfer of clofibrate to the suckling 10.5-day litter was demonstrated by the 15- and 5-fold elevation observed in the neonatal hepatic and renal CYP4A mRNAs, respectively, following suckling from drug-induced mothers. A significant decrease in the relative liver weights of these neonatal pups was seen following clofibrate exposure via maternal milk, in total contrast to the normally observed increase in liver/body weight ratios of rats treated with clofibrate. Western blot analysis using a polyclonal goat anti-rat CYP4A1 antibody also demonstrated a rise in the CYP4A protein levels in both the mothers and their litters following maternal clofibrate treatment.

Conference Article| August 01 1995 Distribution of cGMP-gated cation channel expression in bovine and porcine cardiovascular tissue Charlotte F. Ratcliffe; Charlotte F. Ratcliffe 1CMHT, University of Leicester, Hodgkin Building, PO Box 138, Lancaster Road, Leicester, LE1 9HN Search for other works by this author on: This Site PubMed Google Scholar Edward C. Conley; Edward C. Conley 1CMHT, University of Leicester, Hodgkin Building, PO Box 138, Lancaster Road, Leicester, LE1 9HN Search for other works by this author on: This Site PubMed Google Scholar William J. Brammar William J. Brammar 1CMHT, University of Leicester, Hodgkin Building, PO Box 138, Lancaster Road, Leicester, LE1 9HN Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (3): 441S. https://doi.org/10.1042/bst023441s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation Charlotte F. Ratcliffe, Edward C. Conley, William J. Brammar; Distribution of cGMP-gated cation channel expression in bovine and porcine cardiovascular tissue. Biochem Soc Trans 1 August 1995; 23 (3): 441S. doi: https://doi.org/10.1042/bst023441s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CNGC, cyclic nucleotidegated channel, BAEC, bovine aorta endothelial cell, PCASM, porcine coronary artery smooth muscle This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.