Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Mucosal-associated invariant T (MAIT) cells: an evolutionarily conserved T cell subset

Besides mainstream TCRalphabeta T cells harboring a very diverse repertoire, two subsets display an evolutionarily conserved invariant repertoire. This striking conservation indicates important and unique functions. CD1d-restricted NK-T cells expressing an invariant Valpha14 TCRalpha chain have been implicated in microbial and tumor responses as well as in auto-immunity. In this review, we describe the other subset, which bears the canonical hValpha7.2/mValpha19-Jalpha33 TCRalpha chain paired with a restricted set of Vbeta segments. These invariant T cells are present in mice, humans and cattle. They are preferentially located in the gut lamina propria (LP) of humans and mice and are therefore called mucosal-associated invariant T (MAIT) cells. Selection/expansion of this population requires B lymphocytes expressing MR1, a monomorphic major histocompatibility complex class I-related molecule that is also strikingly conserved in diverse mammalian species. MAIT cells are not present in germ-free mice, indicating that commensal flora is required for their expansion in the gut LP. The nature of the ligand and the putative functions of these MAIT cells are discussed.     

Mechanical remodeling of small-intestine submucosa small-diameter vascular grafts--a preliminary report

Small-intestine submucosa (SIS) is cell-free, 100-mu-thick collagen derived from the small intestine. It has been used as a vascular graft and has the highly desirable ability to be remodeled to become histologically indistinguishable from native adjacent artery. To date there has been limited reporting of its preimplantation and explant mechanical properties as a vascular graft. In this study, compliance, elastic modulus, and burst pressure were measured on preimplant-tested 5- and 8-mm SIS grafts and two 60-day remodeled grafts. Seven prefabricated grafts were implanted in the carotid (n = 7) in dogs, which were sacrificed after 55-63 days. The animals (n = 4) weighed from 22 to 27 kg. One dog received a unilateral carotid graft, and 3 dogs received bilateral carotid grafts. The fabrication technique employed hand-suturing with either nonresorbable or resorbable sutures. None of the grafts had a patency failure. Angiograms taken at 1 month and just before explantation showed uniform flow and no dilation. At the time of explantation, all carotid grafts were found to be encased in fibrous tissue. The grafts made with nonresorbable sutures showed thicker tissue growth at the suture line compared with those made with the resorbable sutures. Along the suture line, the grafts made with resorbable sutures exhibited a more natural color than those sutured with nonresorbable sutures. When the explanted carotid grafts were slit open, the lumen was white, shiny, and glistening. The grafts sutured with nonresorbable sutures exhibited small areas of fibrin and red blood cells when the suture was within the lumen. The resorbable-sutured grafts did not exhibit this response. The mean compliance (percent diameter increase for a pressure rise from 80 to 120 mm Hg) was on average 4.6% (range, 2.9%-8.6%) for the 5-mm preimplant-tested grafts. For the 8-mm preimplant-tested grafts, the increase in diameter for the same pressure rise was 8.7%, on average (range, 7.2% to 9.5%). For comparison, the small-diameter SIS graft at the time of implantation was about one half as compliant as the adjacent dog carotid artery, about 4 times more compliant than a typical vein graft, and more than 10 times more compliant than synthetic vascular grafts. The compliance measured on two 60-day carotid grafts was 10.5% and 7.2%, respectively. This is midway between the original compliance value and the compliance of a typical canine carotid artery (14%), indicating that mechanical remodeling occurred. The modulus of elasticity (E) increased exponentially with increasing pressure according to E = E0e alpha P, where E0 is the zero-pressure modulus and alpha is the exponent that describes the rate of increase in E with pressure; the unit of measure for variables E, E0, and P is g/cm2. The mean value for E0 was 4106 gm/cm2 (range, 1348-5601). The mean value for alpha was 0.0059 (range, 0.0028-0.0125). At 100 mm Hg, the mean value for E was 8.03 x 10(6) dynes/cm2 (range, 4.95-15.7 x 10(6)). For a 60-day SIS graft implant, the elastic modulus at 100 mm Hg decreased from a high value at implant time to twice that of a typical native canine carotid artery. The mean burst pressure for 5.5-mm grafts was 3517 mm Hg (range, 2069-4654). The burst pressure of the remodeled carotid grafts averaged 5660 mm Hg. The burst pressure for a typical carotid artery is about 5000 mm Hg. The results of this preliminary study complement those of previous SIS-vascular-graft studies and add a new factor, namely that the mechanical properties of the remodeled graft approach those of the vessel it replaces.     

What smells? Developing in‐field methods to characterize the chemical composition of wild mammalian scent cues

Olfactory cues play an important role in mammalian biology, but have been challenging to assess in the field. Current methods pose problematic issues with sample storage and transportation, limiting our ability to connect chemical variation in scents with relevant ecological and behavioral contexts. Real‐time, in‐field analysis via portable gas chromatography–mass spectrometry (GC‐MS) has the potential to overcome these issues, but with trade‐offs of reduced sensitivity and compound mass range. We field‐tested the ability of portable GC‐MS to support two representative applications of chemical ecology research with a wild arboreal primate, common marmoset monkeys (Callithrix jacchus). We developed methods to (a) evaluate the chemical composition of marmoset scent marks deposited at feeding sites and (b) characterize the scent profiles of exudates eaten by marmosets. We successfully collected marmoset scent marks across several canopy heights, with the portable GC‐MS detecting known components of marmoset glandular secretions and differentiating these from in‐field controls. Likewise, variation in the chemical profile of scent marks demonstrated a significant correlation with marmoset feeding behavior, indicating these scents’ biological relevance. The portable GC‐MS also delineated species‐specific olfactory signatures of exudates fed on by marmosets. Despite the trade‐offs, portable GC‐MS represents a viable option for characterizing olfactory compounds used by wild mammals, yielding biologically relevant data. While the decision to adopt portable GC‐MS will likely depend on site‐ and project‐specific needs, our ability to conduct two example applications under relatively challenging field conditions bodes well for the versatility of in‐field GC‐MS.     

Short tertatolol treatment does not impair the hormone and metabolic responses to exercise and hypoglycemia in diabetics

Beta-blockers can precipitate hypoglycemia and mask its warning signs. Ten male insulin-dependent, otherwise healthy diabetic patients underwent two submaximal exercise tests and two insulin-induced hypoglycemic events (0.2 u/Kg short-acting insulin IV) after six days administration of placebo followed by tertatolol, a non selective beta-blocker (5 mg once daily). Tertatolol modified neither the exercise-induced changes in blood glucose, lactate and plasma unesterified fatty acid levels, nor those of counter regulatory hormones (glucagon, growth hormone, cortisol), while blood pressure, heart rate and plasma renin activity were significantly reduced, proving that tertatolol had actually been ingested, and was active. During the insulin-induced hypoglycemia, similarly tertatolol did not modify the course of the plasma fuels and hormones. Particularly, hypoglycemia was neither deeper nor more prolonged in the presence than in the absence of tertatolol. Warning symptoms were not affected except for palpitations which were not perceived. These results suggest that tertatolol did not precipitate hypoglycemia following exercise, and did not aggravate insulin-induced hypoglycemia in short term administration, and in otherwise healthy diabetic patients.     

Field immobilization for treatment of an unknown illness in a wild chimpanzee (Pan troglodytes schweinfurthii) at Gombe National Park, Tanzania: findings, challenges, and lessons learned

Infectious diseases are widely presumed to be one of the greatest threats to ape conservation in the wild. Human diseases are of particular concern, and the costs and benefits of human presence in protected areas with apes are regularly debated. While numerous syndromes with fatal outcomes have recently been described, precise identification of pathogens remains difficult. These diagnostic difficulties are compounded by the fact that direct veterinary intervention on wild apes is quite rare. Here we present the unique case of a wild chimpanzee at Gombe National Park that was observed with a severe illness and was subsequently examined and treated in the field. Multiple specimens were collected and tested with the aim of identifying the pathogen responsible for the illness. Our findings represent the first extensive screening of a living wild chimpanzee, yet despite our efforts, the cause and source of illness remain unknown. Nevertheless, our findings represent valuable baseline data for the ape conservation community and for comparison with other recent findings. In addition, we present the case here to demonstrate the planning required and multiple types of expertise necessary to maximize the amount of data obtained from such a rare intervention, and to provide lessons learned for future studies.     

Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis.

Porphyromonas gingivalis, and organism implicated in the etiology and pathogenesis of human periodontal diseases, produces a variety of potent proteolytic enzymes, and it has been suggested that these enzymes play a direct role in the destruction of periodontal tissues. We now report that two cell-associated cysteine proteinases of P. gingivalis W12, with molecular masses of approximately 150 kDa (porphypain-1) and 120 kDa (porphypain-2), as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, have been separated and purified to apparent homogeneity. These proteinases appear to be SDS-stable conformational variants of a 180-kDa enzyme, and they are the largest cysteine proteinases yet purified from P. gingivalis. The purified proteinases hydrolyze fibrinogen, tosyl-Gly-L-Pro-L-Arg p-nitroanilide, and tosyl-Gly-L-Pro-L-Lys p-nitroanilide. While hydrolysis of both synthetic substrates by porphypain-1 and -2 requires activation by reducing agents, is inhibited by EDTA, and is stimulated in the presence of derivatives of glycine, the Arg-amidolytic activity is sensitive to leupeptin and H-D-tyrosyl-L-prolyl-L-arginyl chloromethyl ketone, whereas the Lys-amidolytic activity is sensitive to tosyl-L-lysyl chloromethyl ketone and insensitive to leupeptin. These data suggest that porphypains contain two types of active sites. These cell-associated P. gingivalis proteinases may contribute significantly and directly to periodontal tissue destruction.