Construction of recombination-deficient strains of Streptococcus gordonii by disruption of the recA gene.

Degenerate oligonucleotide primers were used in a polymerase chain reaction (PCR) to amplify a region of the recA sequence of Streptococcus gordonii Challis. The resulting PCR fragment was cloned into the suicide vector pAM6199 and introduced into strain Challis, giving rise to recombination-deficient strains in which the recA gene was specifically inactivated.     

Temperature preferences and tolerances of three fish species inhabiting hyperthermal ponds on mangrove islands

The fish species Cyprinidon artifrons, Floridichthys carpio, and Gambusia yucatana inhabit shallow mangrove ponds off the coast of Belize. Portions of these ponds experience a diurnal temperature change from 26 °C at night to 40 °C and above during midday. Repeated field observations indicate Cyprinidon prefer the warmer (and much larger) portions of the ponds whereas the other two species stay in the cooler areas.The hypothesis that temperature is serving as a cue for partitioning within the ponds was supported by laboratory thermal gradient tests in which Cyprinidon preferred temperatures clearly higher than the other two species.The critical thermal maximum (CTM) was determined for the three species using members that had been acclimated to either a daily cycling temperature similar to that for the ponds, or to the mean of the 24-hour cycle (30 °C). Cyprinidon acclimated to the cycling temperature had a CTM of 45.5 °C, which apparently sets a new record for fish CTM. Acclimation to a constant 30 °C lowered the CTM to 43.7 °C. Floridichthys and Gambusia acclimated to the cycled temperature had CTMs of 43.9 and 43.3 °C respectively, and 42.5 and 42.6 °C for those acclimated to 30 °C.All three species appear to have the ability to tolerate the high temperatures throughout the ponds but only Cyprinidon utilize the whole pond during the day. This may help to explain the large populations of Cyprinodon found in these mangrove ponds compared to the other species.     

An in situ study of rock bass (Ambloplites rupestris) physiology: effect of season and mercury contamination

Selected physiological and biochemical variables were examined in rock bass, Ambloplites rupestris, which were collected on five different sampling dates from an area of chronic mercury contamination and a reference site on the South River, Virginia.The onset of spawning represented the most significant seasonal influence in the physiological profile of the fish, with elevations in hematocrit, hemoglobin, plasma protein, and plasma glucose. Sex-related differences in plasma calcium, liver glycogen and liver ascorbic acid were also unique to the period. Female rock bass had significantly higher levels of liver glutathione than did males on all but one of the sampling dates, although the cause of this difference is not clear.Rock bass from the mercury contaminated site had an average muscle mercury concentration of 1.37 mg Hg g^–1, and an average liver mercury concentration of 2.86 mg Hg g^–1. These levels were approximately an order of magnitude greater than those found in the tissues of the reference fish which averaged 0.165 and 0.101 mg Hg g^–1 in muscle and liver respectively. In July 1987, mercury concentrations in the liver of both reference and contaminated fish increased significantly, possibly the result of greater uptake of the metal through increased feeding or changes in the mercury level of selected prey items. Rock bass collected from the two sites in July also had significantly different levels of liver glutathione: reference fish exhibited an elevation and contaminated fish a depression. When fish from the two sampling stations received a 96-hr exposure to 150 µg HgCl₂ in the laboratory, both groups exhibited elevated liver mercury and decreased liver glutathione. Mercury levels in the gall bladders of the exposed fish were also elevated, suggesting that glutathione may have been lost through excretion with the metal in the bile.On the whole, physiological differences between the two groups of rock bass were limited, indicating that exposure to the mercury is not having a significant impact on the rock bass from the contaminated area. This is further supported by field examination of the fish and comparison of condition indices from rock bass previously taken from the same two stations.Those factors which significantly altered the physiology of the rock bass were unique to certain times of the year, indicating that the most appropriate sampling approach in future studies is one which examines a number of variables over a range of environmental conditions.     

Formation of crystalloid inclusions in the small intestine of neonatal pigs: an immunocytochemical study using colloidal gold

Summary Enterocytes of the small intestine in 1-day-old suckling piglets contain numerous vesicles in the apical cytoplasm and a large granule located beneath the nucleus. Within the next 3 days, these granules transform into electron-dense crystalloid inclusions. These membrane-bound inclusions are up to 10 m in length and 1–2 m in diameter, and they are composed of electron-dense lamellae 3.9 nm apart. Postembedding immunocytochemistry, using rabbit anti-porcine IgG and goat anti-rabbit IgG conjugated to 10 nm colloidal gold, revealed that both the granules and the crystalloid inclusions contained a high concentration of maternal IgG. Although the IgG content of the crystalloid inclusions was detected on epoxy-embedded sections, the use of LR White resin resulted in a much higher density of labelling. Quantification of the labelling density showed that the concentration of IgG in the crystalloid inclusions was approximately ten times higher than that in the lumenal colostrum and approximately three times higher than that in the granules. These observations showed that there are at least three compartments involved in the accretion of IgG in the small intestine of neonatal piglets: smaller apical endocytotic vesicles, large subnuclear granules and crystalloid inclusions. The role of these compartments in maternal immunoglobulin absorption and in the acquisition of passive immunity has yet to be explored.     

Reliability and stability of mothers' reports about their pregnancies with twins.

The objective of this study was to determine if mothers' retrospective reports about events in their pregnancies with twins are reliable and stable. Six hundred and twenty-four mothers completed psychiatric interviews about their twins. These interviews also contained questions about the mothers' pregnancies, the perinatal period, and the child's early development. The mothers reported first on one twin and then on the other with interviews spaced from 3 days to 2 weeks apart. Thus mothers reported on the same pregnancy twice. Of these mothers, 47 were re-interviewed 6 to 18 months later by raters blind to the results of the initial interview. The twin design allowed us to compare the short-term reliability of the 624 mothers' reports of the same pregnancy. The re-interview of the 47 mothers enabled us to compare the stability of reports over a longer time period. Agreement between the reports was measured with the kappa statistic. Kappas were good to excellent for the short-term reports of pregnancy for each twin for the 624 mothers. Kappas were equally high for the 47 mothers that were re-interviewed 6 to 18 months later. Mothers show good reliability and stability of reporting about events during pregnancy.     

Cleavage of Nuclear DNA into Oligonucleosomal Fragments during Cell Death Induced by Fungal Infection or by Abiotic Treatments

It is often claimed that programmed cell death (pcd) exists in plants and that a form of pcd known as the hypersensitive response is triggered as a defense mechanism by microbial pathogens. However, in contrast to animals, no feature in plants universally identifies or defines pcd. We have looked for a hallmark of pcd in animal cells, namely, DNA cleavage, in plant cells killed by infection with incompatible fungi or by abiotic means. We found that cell death triggered in intact leaves of two resistant cowpea cultivars by the cowpea rust fungus is accompanied by the cleavage of nuclear DNA into oligonucleosomal fragments (DNA laddering). Terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labeling of leaf sections showed that fungus-induced DNA cleavage occurred only in haustorium-containing cells and was detectable early in the degeneration process. Such cytologically detectable DNA cleavage was also observed in vascular tissue of infected and uninfected plants, but no DNA laddering was detected in the latter. DNA laddering was triggered by [greater than or equal to]100 mM KCN, regardless of cowpea cultivar, but not by physical cell disruption or by concentrations of H2O2, NaN3, CuSO4, or ZnCl2 that killed cowpea cells at a rate similar to that of ladder-inducing KCN concentrations. These and other results suggest that the hypersensitive response to microbial pathogens may involve a pcd with some of the characteristics of animal apoptosis and that DNA cleavage is a potential indicator of pcd in plants.     

Critical evaluation of the VSC model for tip growth

The vesicle supply centre (VSC) model (Bartnicki-Garcia et al., 1989) for hyphal tip growth is powerful because it can model diverse developmental morphologies and predicts cellular organization based in current cell biology. It predicts that tip growth results from the random distribution of cell surface synthesizing vesicles from a point in the tip, the VSC, which determines their pattern of impact and fusion at the plasma membrane. We derive equations for tip-high gradients of vesicle fusions, generated by mechanisms not related to a supply centre, which create typical hyphal morphologies. These equations direct the conceptual basis for tip growth to vesicle fusion gradients, presumably mediated by a putative membrane skeleton associated with the plasma membrane. We also show that the organization and behaviour of motile organelles in growing hyphal tips of the oomycete,Saprolegnia ferax, argue against the presence of an apparatus capable of generating the distribution of vesicles postulated by the VSC model. We conclude that the VSC model is unlikely to describe the mechanistic basis of tip growth inS. ferax, and therefore, at best, it is not universally applicable.     

Mycobacterium kansasii infection in squirrel monkeys (Saimiri sciureus sciureus)

Abstract: This report documents asymptomatic infections of Mycobacterium kansasii in four of five tuberculin positive squirrel monkeys (Saimiri sciureus sciureus). The mycobacterial DNA amplified by polymerase chain reaction (PCR) from a bronchial lymph node had no affinity for the species specific probes of M. tuberculosis, M. avium, and M. intracellular, thus allowing the presumptive diagnosis of an atypical mycobacterial infection. Infection by Mycobacterium kansasii was confirmed by culture of bronchial lymph nodes from three monkeys. The source of the infection was never identified.     

Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols).

A physical map of the chromosome of Treponema pallidum subsp. pallidum (Nichols), the causative agent of syphilis, was constructed from restriction fragments produced by NotI, SfiI, and SrfI. These rare-cutting restriction endonucleases cleaved the T. pallidum genome into 16, 8, and 15 fragments, respectively. Summation of the physical lengths of the fragments indicates that the chromosome of T. pallidum subsp. pallidum is approximately 1,030 to 1,080 kbp in size. The physical map was constructed by hybridizing a variety of probes to Southern blots of single and double digests of T. pallidum genomic DNA separated by contour-clamped homogeneous electric field electrophoresis. Probes included cosmid clones constructed from T. pallidum subsp. pallidum genomic DNA, restriction fragments excised from gels, and selected genes. Physical mapping confirmed that the chromosome of T. pallidum subsp. pallidum is circular, as the SfiI and SrfI maps formed complete circles. A total of 13 genes, including those encoding five membrane lipoproteins (tpn47, tpn41, tpn29-35, tpn17, and tpn15), a putative outer membrane porin (tpn50), the flagellar sheath and hook proteins (flaA and flgE), the cytoplasmic filament protein (cfpA), 16S rRNA (rrnA), a major sigma factor (rpoD), and a homolog of cysteinyl-tRNA synthetase (cysS), have been localized in the physical map as a first step toward studying the genetic organization of this noncultivable pathogen.     

The mechanism of inhibition of ureogenesis by acidosis

In perfused rat liver a decrease of cytosol pH, determined with pH-sensitive microelectrodes7 from 7.2 to 6.85 is associated with a 50% fall in ureogenesis from ammonium chloride. In isolated rat hepatocytes the fall in ureogenesis due to acidosis is associated with decrease in the mitochondrial and cytosolic concentration of citrulline. Limitation of carbamoyl phosphate synthesis and thus citrulline supply could be responsible for the inhibition of ureogenesis observed.