Purification and preliminary characterization of permethrinase from a pyrethroid-transforming strain of Bacillus cereus.

Bacillus cereus SM3 was isolated on a mineral salts medium with Tween 80 as the primary carbon source. It was able to hydrolyze second- and third-generation pyrethroids, thereby generating noninsecticidal products. The enzyme responsible for this hydrolytic reaction was named permethrinase for this study. This is the first instance in which pyrethroid detoxification has been achieved with a cell-free microbial enzyme system. Permethrinase was purified by ion-exchange chromatography and gel filtration chromatography. The molecular mass of native permethrinase was 61 +/- 3 kDa, as estimated by Sephadex G-100 gel filtration. This novel microbial esterase seems to be a carboxylesterase. Permethrinase activity had an optimum pH of 7.5 and a temperature optimum of 37 degrees C. No cofactors or coenzymes were required for permethrinase activity. The enzyme may be a serine esterase, as it seems to be sensitive to the organophosphorus compound tetraethylpyrophosphate at concentrations in the micromolar range. Addition of dithiothreitol afforded permethrinase protection against the inhibitory effects of the sulfydryl agents p-chloromercuribenzoate and N-ethylmaleimide. The enzyme was stable over a range of temperatures. Cell extracts of strain SM3 also contained another esterase, which was active towards beta-naphthylacetate, but this enzyme was distinct from permethrinase.     

Effects of Preservatives on Viability of Cryptosporidium parvum Oocysts

Potassium dichromate and formalin reduced the viability of Cryptosporidium parvum oocysts as assessed by inclusion or exclusion of 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) and excystation. Some formalin-treated oocysts containing dead sporozoites excluded PI; that this fluorogenic assay relies not solely upon exclusion of PI but also upon highlighting of sporozoite nuclei by DAPI is reiterated.     

Solid state NMR study of [epsilon-13C]Lys-bacteriorhodopsin: Schiff base photoisomerization.

Previous solid state 13C-NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration of the retinal-lysine Schiff base linkage from the [14-13C]retinal chemical shift (1-3). Here we verify the interpretation of the [14-13C]-retinal data using the [epsilon-13C]lysine 216 resonance. The epsilon-Lys-216 chemical shifts in bR555 (48 ppm) and bR568 (53 ppm) are consistent with a C = N isomerization from syn in bR555 to anti in bR568. The M photointermediate was trapped at pH 10.0 and low temperatures by illumination of samples containing either 0.5 M guanidine-HCl or 0.1 M NaCl. In both preparations, the [epsilon-13C]Lys-216 resonance of M is 6 ppm downfield from that of bR568. This shift is attributed to deprotonation of the Schiff base nitrogen and is consistent with the idea that the M intermediate contains a C = N anti chromophore. M is the only intermediate trapped in the presence of 0.5 M guanidine-HCl, whereas a second species, X, is trapped in the presence of 0.1 M NaCl. The [epsilon-13C]Lys-216 resonance of X is coincident with the signal for bR568, indicating that X is either C = N anti and protonated or C = N syn and deprotonated.     

ANNUAL DIFFERENCES IN FEMALE REPRODUCTIVE SUCCESS AFFECT SPATIAL AND COHORT-SPECIFIC GENOTYPIC HETEROGENEITY IN PAINTED TURTLES

Long-term ecological data were used to evaluate the relative importance of movements, breeding structure, and reproductive ecological factors to the degree of spatial and age-specific variation in genetic characteristics of painted turtles (Chrysemys picta) on the E. S. George Reserve in southeastern Michigan. Estimates of the degree of spatial genetic structuring were based on the proportion of total genotypic variance partitioned within and between subpopulations (inferred from hierarchical F-statistics based on variation at 18 protein loci), and in terms of gene correlations (co-ancestry among individuals derived from reproductive data on full-sib families of females nesting at specific nesting areas). Little variation in allele frequency was observed among turtles from different marshes (F_mt = 0.003), though significant variation was observed among turtles from different nesting areas associated with each marsh (F_nm = 0.046). Gene correlations among individuals within nesting areas varied greatly over years (0.032-0.171; mean = 0.069) and were negatively correlated to the proportion of females that successfully nested during each year. General concordance between independent estimates of genotypic correlations (i.e., F_nm derived from protein electrophoretic variation vs. mean co-ancestry) suggests that allozyme data, when collected over spatial scales consistent with species behavioral characteristics and reproductive ecology, may accurately reflect the apportionment of gene diversity within and among subpopulations. The magnitude and patterning of allelic variation among nesting areas and individuals appears to be primarily a function of gametic correlations among members of full-sib families, irrespective of the degree of gene flow or female nesting-site fidelity. Comparisons of genetic characteristics among 11 cohorts (1974-1984) revealed that heterozygosity (H) and inbreeding coefficients (F) varied greatly. Cohort estimates of H and F were correlated to female nesting success and to estimates of co-ancestry for the same years. Results clearly reflect the concomitant importance of ecological factors (principally the proportion of the female population that successfully produce offspring during each year) in determining the magnitude and patterning of gene correlations within and among groups, and to the genotypic composition of offspring born during each year.     

Cardiovascular responses to sustained maximal isometric contractions of the finger flexors

This study investigated cardiovascular responses to 2 min sustained submaximal (20% MVC) and maximal (100% MVC) voluntary isometric contractions of the finger flexors in healthy young women. Cardiovascular variables investigated were: heart rate (f _c), mean arterial pressure ( _a), and stroke volume (SV). Doppler echocardiography was used to estimate SV from measures of aortic diameter (AD) and time-velocity integrals. Preliminary studies indicated that AD did not change significantly after 2 min sustained 100% MVC. Therefore, pre-exercise AD values were used to calculate SV before, during and after exercise. During the 2-min 100% MVC period, f _c and _aincreased significantly during the first 30 s of contraction. f _c then remained constant during the remainder of the 2-min contraction period, while _acontinued to rise. SV did not change significantly during the 100% MVC task but increased significantly during recovery from sustained 100% MVC. The data suggest that the magnitude of cardiovascular responses to isometric exercise is dependent on the specific task performed, and that there is a different pattern of response for f _c, _a, and SV during 20% and 100% MVC tasks. Unlike f _c and _a, SV did not change significantly during isometric exercise, but increased significantly after sustained 100% MVC.     

The effects of intensity of exercise on excess postexercise oxygen consumption and energy expenditure in moderately trained men and women

This experiment investigated the effects of intensity of exercise on excess postexercise oxygen consumption (EPOC) in eight trained men and eight women. Three exercise intensities were employed 40%, 50%, and 70% of the predetermined maximal oxygen consumption (VO_2max). All ventilation measured was undertaken with a standard, calibrated, open circuit spirometry system. No differences in the 40%, 50% and 70% VO_2max trials were observed among resting levels of oxygen consumption (V0₂) for either the men or the women. The men had significantly higher resting VO₂ values being 0.31 (SEM 0.01) 1·min^–1 than did the women, 0.26 (SEM 0.01) 1·min^–1 (P < 0.05). The results indicated that there were highly significant EPOC for both the men and the women during the 3-h postexercise period when compared with resting levels and that these were dependent upon the exercise intensity employed. The duration of EPOC differed between the men and the women but increased with exercise intensity: for the men 40% – 31.2 min; 50% – 42.1 min; and 70% – 47.6 min and for the women, 40% – 26.9 min; 50% – 35.6 min; and 70% – 39.1 min. The highest EPOC, in terms of both time and energy utilised was at 70% VO_2max. The regression equation for the men, where y=O₂ in litres, and x=exercise intensity as a percentage of maximum was y=0.380x + 1.9 (r ²=0.968) and for the women is y=0.374x–0.857 (r ²=0.825). These findings would indicate that the men and the women had to exercise at the same percentage of their VO_2max to achieve the maximal benefits in terms of energy expenditure and hence body mass loss. However, it was shown that a significant EPOC can be achieved at moderate to low exercise intensities but without the same body mass loss and energy expenditure.     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.     

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland.     

Autonomous replication in human cells of multimers of specific human and bacterial DNA sequences.

Using modules of a specific 2,712-bp human DNA sequence and a specific 2,557-bp Escherichia coli DNA sequence, we created plasmids containing between 1 and 12 modules of single or chimeric sequence composition and tested them in human cells for their autonomous replication ability. We found that replication efficiency per generation increased with successive addition of human modules, to essentially 100% by six copies. Although a single copy of the bacterial module had negligible replication ability, the replication efficiency per generation of 12 bacterial modules was 66%. Chimeras composed of human and bacterial modules displayed intermediate replication levels. We also used two-dimensional gel electrophoresis to physically map where replication initiated on a half human-half E. coli plasmid. Our results suggest that autonomous replication in human cells is stimulated by simple sequence features which occur frequently in human DNA but are more rare in bacterial DNA.