Our objective was to study the effect of tobramycin solution for inhalation (TSI; TOBI(R), Chiron Corp.) on lung function decline rate in 400 young persons with cystic fibrosis (CF) and mild lung disease. Effects on hospitalization, antibiotic use, school days missed, and nutritional status also were determined. This was an open-label, randomized (stratified by sex and age group, i.e., 6-10 and 11-15 years), parallel-group, multicenter study. Routine subject management (control group) was compared to routine management plus 28 days of twice-daily TSI inhalation, followed by 28 days off the drug (TSI group) for 56 weeks. Primary efficacy endpoints included rate of lung function decline (as measured by forced expiratory volume in 1 sec; FEV1), hospitalization, and concomitant antibiotic use. Safety was assessed by analysis of treatment-emergent adverse events. Only 184 of 400 planned subjects were recruited and randomized (93 to the TSI group, and 91 to the control group). Enrollment was ended after 2 years because of difficult recruitment. An interim safety review showed a 2.42-fold risk of respiratory hospitalization for control group subjects (P=0.020), and the study was terminated. Sixty-three subjects (34.2%) completed the entire study (30 in the TSI group, or 32.3%; and 33 in the control group, or 36.3%). Significantly fewer TSI subjects were hospitalized for worsening of respiratory symptoms (11.0% vs. 25.6%; P=0.011), and fewer TSI subjects were hospitalized overall (16.5% vs. 27.8%; P=0.065). Fewer TSI subjects received antibiotics other than the study drug (78.0% vs. 95.6%), and significantly fewer received oral antibiotics (76.9% vs. 91.1%; P=0.009). No other safety or adverse event differences were observed. In conclusion, significant reductions in respiratory hospitalizations, concomitant antibiotic use, and a trend towards improvement in percent predicted forced expiratory flow (FEF25-75) provide evidence of a clinical benefit of TSI use in young persons with CF and mild lung disease. An effect on lung function decline rate could not be evaluated as planned, due to inadequate enrollment and early study termination. (C) 2004 Wiley-Liss, Inc.

Chromosome inheritance must be amazingly efficient to ensure that each of the 100 trillion (1014) cells in the human body contains the full complement of 46 chromosomes required for normal viability and development. Mitotic chromosome inheritance requires successful completion of three basic functions—replication, sister chromatid cohesion and separation, and attachment to and movement along the spindle. Chromosomes contain many origins of DNA replication and sites of cohesion, which helps to ensure that these tasks are accomplished even when individual sites fail to function. However, in many organisms spindle attachment occurs at a single, localized region of each chromosome, the centromere (Figure 1A), whose chromosome location does not appear to change from one division to the next. What specifies the site of centromere function, and how does the centromere assemble the components necessary for spindle attachment and movement? Despite the essential role of centromeres in cell division, answers to these questions have baffled biologists for over a century. The best-characterized centromere is found in the yeast Saccharomyces cerevisiae. In this unicellular eukaryote, centromere function is conferred by a small (125 bp) DNA sequence whose overall organization and sequence composition are highly conserved among the 16 different centromeres. The apparent dependence on primary DNA sequence is reminiscent of transcriptional regulation—specific sequences bind specific proteins, which in this case mediate chromosome movement rather than gene expression. In contrast, Schizosaccharomyces pombe and Drosophila melanogaster do not appear to contain such a “magic sequence” within their more complex and larger centromeres and may instead rely on higher order structure and epigenetic regulation rather than primary DNA sequence (reviewed in6Karpen G.H Allshire R Trends Genet. 1997; 13: 489-496Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 13Wiens G.R Sorger P.K Cell. 1998; 93 (this issue): 313-316Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar [this issue of Cell). Studies of human centromeres over the last decade have led to controversy concerning the role of a prominent candidate DNA sequence, the centromere-associated alpha satellite, also known as alphoid DNA. Here, we evaluate recent, exciting advances that address the following question—is alpha satellite necessary and sufficient for centromere function? We discuss growing evidence that centromere behavior in humans and other eukaryotes is paradoxical—centromeres are stably propagated at the same chromosomal location through replication and division, but also display remarkable plasticity (activation and inactivation). Finally, we explore the implications of epigenetic regulation of centromere activity to chromosome function and evolution. There is ample evidence implicating a role for alpha satellite in centromere function. The first supportive evidence is “guilt by association”; alpha satellite is the only known human DNA that is exclusively localized to the centromeric regions of all normal human chromosomes (Figure 1A). The arrays are composed of tandemly arranged 171 bp monomers organized into higher-order repeats and range in size from 200 to 9000 kb (Figure 1B). Secondly, the human X and Y centromeres have been localized using chromosome rearrangements found in natural populations or created artificially by fragmenting chromosomes with cloned telomeric DNA. In one study, two derivative chromosomes deleted for either most of the Y long or short arms shared only 70 kb of overlapping sequences that are predominantly composed of alpha satellite. All of the chromosome rearrangements analyzed to date retain at least 140 kb of alpha satellite, further implicating this sequence as having a role in centromere function (4Heller R Brown K.E Burgtorf C Brown W.R Proc. Natl. Acad. Sci. USA. 1996; 93: 7125-7130Crossref PubMed Scopus (124) Google Scholarreferences therein). If guilt by association were sufficient, we would conclude that alpha satellite is the “magic sequence” that determines human centromere identity and function, analogous to the conserved elements present in the S. cerevisiae centromere. However, structural analyses are currently not detailed enough to exclude the importance of nonalphoid DNA. Long-range restriction mapping suggests that the alpha satellite arrays are mostly homogeneous, but some interspersed sequences are present. These include LINEs, Alu repeats, and other satellites (7Lee C Wevrick R Fisher R.B Ferguson-Smith M.A Lin C.C Hum. Genet. 1997; 100: 291-304Crossref PubMed Scopus (188) Google Scholar), which are predominantly present in other parts of the genome that never associate with kinetochores, suggesting that they are not by themselves sufficient for centromere function. However, complete sequencing of alpha satellite arrays may identify functionally important interspersed sequences. Critical centromeric sequences may be recognized by proteins located in the kinetochore inner plate, where DNA interfaces with the kinetochore (Figure 1A). CENP-A and CENP-C are only present at functional centromeres, and have been localized to the inner plate by coimmunofluorescence and immunoelectron microscopy, respectively (12Warburton P.E Cooke C.A Bourassa S Vafa O Sullivan B.A Stetten G Gimelli G Warburton D Tyler-Smith C Sullivan K.F Poirier G.G Earnshaw W.C Curr. Biol. 1997; 7: 901-904Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholarreferences therein). CENP-A is a highly divergent H3-like histone that may form a specialized chromatin structure in the kinetochore, and CENP-C contains a novel DNA-binding motif. 10Vafa O Sullivan K.F Curr. Biol. 1997; 7: 897Abstract Full Text Full Text PDF PubMed Google Scholar used chromatin immunoprecipitation to isolate the DNA associated with CENP-A in vivo and found a 10- to 20-fold enrichment of alpha satellite in comparison to its representation in control genomic libraries. These results demonstrate that alpha satellite is present in the kinetochore inner plate and associates with critical centromere proteins; therefore, alpha satellite is at the right place to determine centromere identity and function. If alpha satellite specifies the site of kinetochore formation, then it should be possible to create functional centromeres from cloned alphoid DNA, as demonstrated previously for S. pombe and S. cerevisiae centromeric DNAs. 3Harrington J.J Van Bokkelen G Mays R.W Gustashaw K Willard H.F Nat. Genet. 1997; 15: 345-355Crossref PubMed Scopus (527) Google Scholar devised an elegant in vitro concatamerization strategy to construct synthetic alpha satellite arrays up to 1 Mb in size, starting from a 2.7 kb alphoid clone. These arrays were joined to a selectable marker and lipofected into human cells along with telomeric and total genomic DNA. Some of the recovered clones contained 6–10 Mb minichromosomes bearing synthetic alpha satellite arrays, which are significantly larger than the starting material. Of four minichromosomes that were further characterized, at least two contained de novo centromeres that bound CENP-C and CENP-E. Using a different approach, 5Ikeno M Grimes B Okazaki T Nakano M Saitoh K Hoshino H McGill N.I Cooke H Masumoto H Nat. Biotech., in press. 1998; Google Scholar retrofitted a YAC containing ∼100 kb of alpha satellite with nested human telomeres and introduced it into human cells. Most of the recovered clones contained small minichromosomes (∼1–5 Mb) derived from the YAC DNA. Further analyses of three minichromosomes indicated that they had functional centromeres and were composed of ∼30 copies of the original YAC. FISH with chromosome paint or satellite probes suggested that they had not acquired large amounts of additional host sequences; however, nonalphoid DNA could be present in the original YAC and be required for centromere formation. What can we conclude from these reconstitution experiments? The production of minichromosomes from synthetic alphoid arrays suggests that alpha satellite may be sufficient for centromere function. In addition, not all alpha satellite clones were competent to produce artificial minichromosomes, suggesting that the type of substrate may be important. However, in both studies all de novo chromosomes contained substantial rearrangements of the introduced arrays. Rearrangements may be required to incorporate nonalphoid genomic sequences necessary for de novo centromere function or to achieve a minimum overall size necessary for a noncentromeric component of human chromosome function. Furthermore, the kinetics of de novo centromere formation could not be determined, and it is possible that a rare activation step is required in addition to the presence of alpha satellite. Nevertheless, these studies constitute important breakthroughs that will help answer many extant questions about the substrate “rules” for de novo centromere formation in humans. It is exciting that the relative importance of alpha satellite can now be assessed directly by comparing the frequency of de novo minichromosome assembly with different, defined DNA substrates, such as alphoid repeats versus other repeats versus nonrepetitive DNA. The localization of alpha satellite to kinetochores and the successes in generating de novo centromeres from alphoid clones suggest that alpha satellite encodes human centromere function. However, colocalization experiments indicate that only a subset of the alpha satellite array at each centromere is associated with CENP-A, CENP-C, and the kinetochore inner plate (12Warburton P.E Cooke C.A Bourassa S Vafa O Sullivan B.A Stetten G Gimelli G Warburton D Tyler-Smith C Sullivan K.F Poirier G.G Earnshaw W.C Curr. Biol. 1997; 7: 901-904Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), suggesting that identical alphoid sequences can differ in their ability to bind critical kinetochore proteins. In addition, spatially separated alpha satellite arrays on a single chromosome can differ in function. Normally chromosomes that contain two active centromeres (dicentrics) are unstable because they frequently attach to opposite spindle poles and produce chromatin bridges in anaphase. However, numerous stable dicentric chromosomes have been described in which one of the two alpha satellite blocks is inactivated and no longer binds critical kinetochore proteins (Figure 2) (9Sullivan B.A Schwartz S Hum. Mol. Genet. 1995; 4: 2189-2197Crossref PubMed Scopus (180) Google Scholarreferences therein). Similarly, several groups have analyzed the centromere function of arrays of alphoid DNA constructs integrated into human, simian, or hamster chromosomes. For example, 11Warburton P.E Cooke H.J Chromosoma. 1997; 106: 149-159Crossref PubMed Scopus (33) Google Scholarreferences therein demonstrated that arrays of human alphoid DNA integrated into hamster chromosomes did not bind CENP-C or CENP-E, and did not associate with spindle microtubules during mitosis. Anaphase bridges were observed, but they were caused by delayed sister chromatid separation in the region of the amplified arrays rather than centromere activity of the integrated alphoid DNA. Thus, alphoid DNA in stable dicentrics or integrated into ectopic sites is not sufficient for kinetochore formation. Centromeres can also function without alphoid DNA. At least 17 different human marker chromosomes have been described that lack detectable amounts of alphoid DNA by FISH. Somehow, these chromosomes have compensated for loss of the normal centromere by forming a “neocentromere” on nonalphoid sequences (Figure 2), indicated by the presence of CENP-A, -C, or -E. du 1du Sart D Cancilla M.R Earle E Mao J.I Saffery R Tainton K.M Kalitsis P Martyn J Barry A.E Choo K.H Nat. Genet. 1997; 16: 144-153Crossref PubMed Scopus (266) Google Scholarreferences therein recently localized one neocentromere to an 80 kb region of chromosome 10 euchromatin. Extensive restriction analyses indicated that the structure of this region had not changed upon neocentromere acquisition, suggesting that the same sequences can have two different functional states. Thus, alphoid DNA is not necessary for (neo)centromere function. To our knowledge, human neocentromere activation has not been observed in chromosome rearrangements induced experimentally by irradiation or telomere-associated fragmentation, suggesting that it requires a rare activation step. Although a role for unidentified sequences in centromere function cannot be ruled out, the available evidence suggests that alpha satellite plays an important role in human centromere function. Kinetochores contain alpha satellite DNA, and transformation with alphoid clones can produce functional artificial chromosomes. However, neocentromere activation and centromere inactivation demonstrate that alpha satellite is not absolutely necessary or sufficient for centromere function. These observations present a paradox—how can centromeres consistently and efficiently form at the same sites, without an absolute requirement for specific primary DNA sequences, and also exhibit remarkable plasticity? One possibility is that centromere activity is normally specified by the primary sequence of alphoid DNA, and rare regulatory mechanisms or independent pathways are responsible for inactivation and neocentromere activation. However, inactive or active states are efficiently maintained despite the presence or absence of alphoid DNA, respectively, suggesting that these events reflect the normal mechanism for human centromere function. We prefer a more parsimonious model, which requires the fewest elements to account for both centromere plasticity and stability. Human centromere function could be determined by an epigenetic system that prefers specific substrates. In this model (Figure 3), de novo centromere formation requires an activation step that “marks” or “imprints” the DNA. Initial activation may be biased towards some characteristic of alpha satellite, such as high A+T composition, repetitiveness, or secondary structure, but other sequences can also be activated, perhaps at a lower frequency (neocentromere formation). Once marked, any sequence can self-propagate the mark and function as a centromere in the next cell division (centromere and neocentromere propagation/maintenance). Centromere inactivation would occur by random reversal of the activation/marking or propagation steps. Epigenetic specification of centromere identity may be a universal feature of eukaryotic regional centromeres. Recent studies have indicated that epigenetic mechanisms influence centromere function in S. pombe and Drosophila (reviewed in6Karpen G.H Allshire R Trends Genet. 1997; 13: 489-496Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 13Wiens G.R Sorger P.K Cell. 1998; 93 (this issue): 313-316Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Evolutionary comparisons suggest that primary sequences are not important for centromere activity. Centromere-associated satellite DNAs show no obvious sequence conservation among fungi, plants, insects, mammals, and other vertebrates, and differ considerably even in closely related species. The sequences tend to be A+T-rich, have monomer subunits of nucleosome size, and possess a natural DNA curvature, but these are general characteristics of many satellite sequences. These observations can be explained if centromere function is specified by an epigenetic mechanism that prefers the overall DNA composition or repetitiveness of satellite DNA. How could the epigenetic mark be specified and maintained during DNA replication? Active S. pombe centromeres contain underacetylated histones. Aberrant centromeric acetylation patterns are correlated with abnormal centromere function; these states are epigenetically maintained for many cell divisions (2Ekwall K Olsson T Turner B.M Cranston G Allshire R.C Cell. 1997; 91: 1021-1032Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Another attractive paradigm is suggested by the fact that late replication of human alpha satellite coincides with the G2 peak of CENP-A expression, and misexpression of CENP-A throughout S phase results in nonspecific localization of CENP-A (8Shelby R.D Vafa O Sullivan K.F J. Cell Biol. 1997; 136: 501-513Crossref PubMed Scopus (249) Google Scholar). Perhaps a chromatin duplication mechanism acts in late S phase to copy the centromere-specific pattern of acetylation or CENP-A incorporation (reviewed in6Karpen G.H Allshire R Trends Genet. 1997; 13: 489-496Abstract Full Text PDF PubMed Scopus (376) Google Scholar). At first glance, there are significant advantages for the individual cell and organism to contain centromeres that are dependent entirely on primary DNA sequence; given the importance of centromeres to cell and organismal viability, there should be no room for gain or loss of centromere function. Then why would centromeres utilize epigenetic mechanisms of regulation? Perhaps because it is adaptive and advantageous during evolution. Epigenetic mechanisms could help buffer organisms from frequent and possibly detrimental changes to the DNA sequence. In addition to frequent base changes, satellite arrays readily expand and contract, changing the relative representation of neighbors (Figure 4A). A primary sequence-independent, self-propagation mechanism would tolerate changes in DNA sequence, because the centromere would simply follow the rule “love the one you're with…” Alternatively, an epigenetic mechanism may even be adaptive. For example, if centromere function per se results in recombination suppression, the resulting accumulation of ephemeral satellites in the centromeric region would then favor evolution of sequence-independent mechanisms of centromere specification. Centromere plasticity could also facilitate chromosome evolution. Related species frequently differ in the arrangement and association of chromosome arms, even when the DNA sequences are nearly identical. Chromosome parts are exchanged by fusion and fission events—the requirement for one, and only one, centromere would render many of the resulting dicentric and acentric chromosome rearrangements useless unless centromeres could be inactivated and neocentromeres activated (Figure 4B). Thus, the ability to move the centromere from one DNA sequence to another, by spreading, hopping, or activation (Figure 4A and Figure 4B) may expedite chromosome evolution. Do centromeres move within a chromosome, in evolutionary or even cellular timescales? Perhaps our perception of the absolute stability of centromere location is false. Most centromeres have been localized by light microscopy, which does not provide the resolution necessary to answer this question. Functional centromeric DNAs need to be identified and mapped precisely within populations, and within extensive phylogenies that contain both closely related and distant species that are accessible to experimental manipulation. Lessons learned from centromere studies may be relevant to understanding other chromosomal functions, such as homolog pairing, long-distance regulation of gene expression, and replication. For example, replication origins in S. cerevisiae are competent to function when cloned and reintegrated into chromosomes; this does not appear to be true for mammalian origins. Perhaps other chromosomal functions in higher eukaryotes have, like centromeres, been resistant to comprehension because these biological processes are determined by epigenetic mechanisms; if so, new tools and ways of thinking need to be developed before we can completely understand chromosome behavior and inheritance. The determinants of centromere identity and function are being investigated intensively in humans and other eukaryotes, and in the coming years we expect to gain a deeper understanding of this essential and complex biological function.

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere, but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centromere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.

The DNA elements responsible for centromere activity in a metazoan have been localized using the Drosophila minichromosome Dp1187. Deleted minichromosomes were generated by irradiation mutagenesis, and their molecular structures were determined by pulsed-field Southern blot analysis. Analyses of the transmission behavior of Dp1187 derivatives localized sequences necessary for chromosome inheritance within the centric heterochromatin. The essential core of the centromere is contained within a 220 kb region that includes significant amounts of complex DNA. Completely normal inheritance also requires approximately 200 kb on either side of the essential core. This flanking DNA predominantly contains highly repeated sequences, and the amount required for normal transmission differs among division types and between the sexes. We propose that the essential core is the site of kinetochore formation and that flanking DNA provides two functions: sister chromatid cohesion and indirect assistance in kinetochore formation or function.

Group B Streptococcus (GBS) causes an impairment of diaphragmatic pressure generation (Pdi) in 2-wk-old piglets, whereas 4-wk-old piglets are unaffected. In this study, we examined the effect on 4-wk-old piglets of a higher dose of GBS than previously utilized. We sought to determine whether an eicosanoid product of arachidonic acid metabolism accounted for the decrease in Pdi during GBS infusion and whether thromboxane A2 (TxA2) is the putative eicosanoid mediator of decreased Pdi during GBS infusion. Measuring Pdi during phrenic nerve stimulation, we studied four groups of anesthetized spontaneously breathing 4-wk-old piglets. Group 1 (GBS) was infused with live GBS, which caused a decrease in Pdi by 1 h at 20-, 30-, 50-, and 100-Hz stimulation frequencies. Group 2 [GBS + indomethacin (Indo)] was pretreated with Indo before GBS infusion. In the GBS + Indo group, Pdi did not decrease throughout 4 h of GBS infusion. Because Indo proved to be protective of Pdi during GBS infusion, we examined the role of TxA2, the only eicosanoid present at 1 h in the serum of GBS-infused piglets. Group 3 was infused with the TxA2 analogue U-46619 only for 1 h. Group 4 was treated with the TxA2-receptor antagonist SQ-29548 before and concomitant with GBS infusion for 1 h; the SQ-29548 was then discontinued, and GBS was continued for 1 h more. In the U-46619-infused group, Pdi decreased at 1 h, and in the SQ-29548-treated group, Pdi did not decrease during GBS infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

PURPOSE:Self-assessment of sexual maturity by healthy male adolescents has been found to correlate closely with physician ratings. Data are lacking, however, in adolescents with cystic fibrosis (CF), where an altered body image may affect self-assessment. Delayed sexual maturation occurs in many patients with CF, particularly those with severe disease. We hypothesized that self-assessment of sexual maturation by adolescents with CF would agree with physician ratings. METHODS:Using Tanner's standard photographs for pubic hair (PH) and genital (G) development, we compared self-assessment of sexual maturation to physician rating in 34 adolescent male patients with CF and 27 healthy male controls (C). RESULTS:The two groups did not differ in age. All subjects were initially examined and Tanner-staged by a physician (SRB), and instructed in self-assessment using the Tanner photographs; they then performed a self-assessment. Scores by physician and subjects were assessed for inter-observer agreement by Kappa analysis. For the CF group, 29 of 34 PH assessments and 21 of 34 G assessments demonstrated exact inter-observer agreement between physician and subject ratings. The Kappa coefficient, kappa, (weighted for the degree of closeness between two observers) was 0.946 for PH and 0.840 for G and the C group, kappa was 0.905 for PH and 0.737 for G. Repeat analysis combining stages 3 and 4 PH and G development yielded higher inter-observer agreement in the CF group (33 of 34 PH assessments and 26 of 34 G assessments) and in the C group (24 of 27 PH assessments and 18 of 27 G assessments). CONCLUSIONS:Self-assessment is a valid method to assess sexual maturity in clinical evaluation and as a research tool in the study of patients with CF.

In this study, we demonstrate a role for extracentromeric sequences in chromosome inheritance. Genetic analyses indicate that transmission of the Drosophila minichromosome Dp1187 is sensitive to the dosage of nod+, a kinesin-like gene required for the meiotic transmission of achiasmate chromosomes. Minichromosome deletions displayed increased loss rates in females heterozygous for a loss-of-function allele of nod (nod/+). We have analyzed the structures of nod-sensitive deletions and conclude that multiple regions of Dp1187 interact genetically with nod+ to promote normal chromosome transmission. Most nod+ interactions are observed with regions that are not essential for centromere function. We propose that normal chromosome transmission requires forces generated outside the kinetochore, perhaps to maintain tension on kinetochore microtubules and stabilize the attachment of achiasmate chromosomes to the metaphase spindle.

ABSTRACT: Recent studies indicate that diaphragmatic pressure generation (Pdi) is impaired by bacterial infection. However, group B streptococcus (GBS) had no effect on Pdi when infused into 4-wk-old piglets. As responsiveness to GBS is age-dependent, we therefore studied the acute effect of GBS infusion on Pdi, using a younger, 2-wk-old piglet model. Using trans-Pdi with phrenic nerve stimulation, we studied the effect of continuous GBS infusion in seven anesthetized, spontaneously breathing 2-wk-old piglets. Pdi was measured under baseline conditions (50% O2/ 50% N2) and at 1, 2, and 4 h of GBS infusion. GBS was infused at a rate which caused a doubling of the pulmonary artery pressure but which avoided hypotension or acidosis—both of which can decrease Pdi. In addition, the piglets were kept hyperoxic [PaO2 > 13.3 kPa (100 torr)], and no piglet with hypercapnia [PaCO2 > 8.7 kPa (65 torr)] was included, as hypoxia and hypercapnia can also cause respiratory muscle dysfunction. For the GBS group, diaphragmatic contractility declined significantly by 1 h at 30-, 50-, and 100-Hz stimulation frequency and, by 2 h, was significantly decreased at all frequencies. We conclude that 2-wk-old piglets, in contrast to 4-wk-old piglets, demonstrate a decline in Pdi during GBS infusion. These data demonstrate an age-related response to GBS in the piglet. This finding implies that a decline in Pdi during acute GBS infusion is not due to a secondary effect from shock (i.e. acidosis, hypercapnia, or hypoxemia) or due to the catabolic effects of a chronic infection, but is due to a direct effect of sepsis on diaphragmatic muscle function.

Recent studies indicate that diaphragmatic contractility is adversely affected by bacterial infection. Using transdiaphragmatic pressure (Pdi) with phrenic nerve stimulation, the effect of continuous Group B Streptococcus (GBS) infusion on diaphragmatic force output was studied in seven anesthetized, spontaneously breathing 1-month old piglets. Pdi was measured under baseline condition (50% O2/50% N2) and at 1, 2, and 4 h of GBS infusion. The GBS was infused at a level that caused a doubling of the pulmonary artery pressure and a 32% decrease in cardiac output but which avoided hypotension or acidosis--both of which can decrease diaphragmatic contractility. In addition, the piglets were kept hyperoxic (PaO2 greater than 100) and no piglet with hypercapnia (PaCO2 greater than 65) was studied, as hypoxia and hypercapnia also can cause respiratory muscle dysfunction. Pdi in response to phrenic nerve stimulation did not change during GBS infusion. We conclude that GBS infusion, in the absence of hypotension, hypercapnia, hypoxia, or acidosis, has no effect on diaphragmatic force generation in the piglet.

Inspiratory resistive loaded (IRL) breathing results in hypoventilation and diaphragmatic fatigue in the piglet. We studied the effects of 6 h of IRL on ten 1-mo-old piglets. The load was adjusted to increase spontaneously generated transdiaphragmatic pressure five to six times baseline. Six 1-mo-old piglets acted as controls and were identically instrumented but were not subjected to IRL. Measurements of ventilation, blood gases and pH, diaphragmatic electromyogram, force-frequency curve, blood flow, and end-expiratory lung volume were obtained hourly. Diaphragmatic muscle samples were obtained after 6 h for determination of ATP, phosphocreatine, lactate, and glycogen levels. No changes occurred in the control animals. IRL resulted in a significant decrease in ventilation, an increase in diaphragmatic EMG, onset of abdominal expiratory muscle activity, and a fall in end-expiratory lung volume by 1 h. The force-frequency curve adjusted for lung volume change fell by 20% at all frequencies of stimulation at 1 h and by 40% at 6 h. Blood flow to the costal and crural diaphragm increased by 51 and 141%, respectively. No differences were noted in ATP, phosphocreatine, lactate, or glycogen between control and IRL animals. It is concluded that submaximal spontaneous contractions of the piglet diaphragm over a 6-h period cause a substantial decrease in its maximal force-generating capacity that is not related to substrate depletion.

Acquisition of category-level information can be based on experience with category members (induced) as well as on direct presentation of prototypical values (given). To investigate the effects of these two types of information, a relational coding model of categorization was developed in which classification is based on a mixture of exemplar and prototype information. In two experiments, subjects learned about two ill-defined categories. Stimuli were geometric shapes varying along four binary-valued dimensions. For three groups of subjects, training consisted of (a) experience with exemplars only, (b) learning prototype values followed by exemplar experience, or (c) learning prototype values concurrently with exemplar experience. Following training, all subjects received classification tests on prototype values as well as on old and new exemplars. By varying the relative use of prototype and exemplar information, the mixture model accurately accounted for category judgements in all three groups. For subjects directly presented with prototype values, classification was based on a mixture of similarity to prototypes and to stored exemplars. In contrast, subjects who only received experience with exemplars appeared to base their category judgements solely on similarity to stored exemplars, even though they could accurately judge the prototype values. The two components of the mixture model are related to subjects' classification strategies and the nature of abstracted, category-level information.