Membrane insertion of the Escherichia coli MalF protein in cells with impaired secretion machinery

The MalF protein is an integral membrane protein of Escherichia coli containing eight membrane-spanning stretches and a large periplasmic domain of approximately 180 amino acids. We have asked whether this protein is dependent for its membrane insertion on the bacterial secretion machinery specified by the sec genes. Using azide to inhibit the SecA protein and sec mutants to reduce the functioning of the machinery, we have studied the membrane assembly of MalF and beta-galactosidase and alkaline phosphatase fusions to MalF. In no case did we see an effect of reducing sec gene function on the insertion of MalF or fusion proteins. Selection for mutants that would cause internalization of a MalF-beta-galactosidase hybrid protein yielded no mutations in sec genes. Our results suggest that MalF can assemble in the membrane independently of the bacterial secretion machinery.     

Cryptic species,cryptic endosymbionts,and geographical variation in chemical defences in the bryozoan Bugula neritina

Molecular markers often offer the only means to discriminate between species and to elucidate the specificity of many community interactions, both of which are key to the understanding of ecological patterns. Western Atlantic populations of the bryozoan Bugula neritina vary in the palatability of their larvae to predators: individuals south of Cape Hatteras produce chemical deterrents to fish predators that are absent in more northern individuals. We use mitochondrial cytochrome oxidase c subunit I (COI) sequences to show that the differences in palatability between populations correlate with the geographical distributions of two cryptic species within B. neritina. Furthermore, these cryptic species differ in their associations with bacteria that may confer chemical resistance to predation. Small subunit rRNA primers specific to a subset of gamma-proteobacteria amplified only the bacterium Endobugula sertula from the southern cryptic species. Endobugula sertula produces a family of chemical compounds (bryostatins) that may deter predators of its animal host. In contrast, the same primers amplified an array of gamma-proteobacteria from the unprotected northern cryptic bryozoan species, but never E. sertula. In combination, these findings suggest that the geographical variation in palatability observed in the larvae of B. neritina is not the result of local adaptation of a single species to regions of differing predation pressure, but rather results from the comparison of cryptic species that differ in the presence or absence of a bacterium that may provide protection against predators. The ability to identify the cryptic Bugula species and their differing relationships with bacterial associates provides an example of the important role molecular techniques may play in addressing ecological questions.     

Stress wave velocities in bovine patellar tendon.

The velocity of longitudinal stress waves in an elastic body is given by the square root of the ratio of its elastic modulus to its density. In tendinous and ligamentous tissue, the elastic modulus increases with strain and with strain rate. Therefore, it was postulated that stress wave velocity would also increase with increasing strain and strain rate. The purpose of this study was to determine the velocity of stress waves in tendinous tissue as a function of strain and to compare these values to those predicted using the elastic modulus derived from quasi-static testing. Five bovine patellar tendons were harvested and potted as bone-tendon-bone specimens. Quasi-static mechanical properties were determined in tension at a deformation rate of 100 mm/s. Impact loading was employed to determine wave velocity at various strain levels, achieved by preloading the tendon. Following impact, there was a measurable delay in force transmission across the specimen and this delay decreased with increasing tendon strain. The wave velocities at tendon strains of 0.0075, 0.015, and 0.0225 were determined to be 260 +/- 52 m/s, 360 +/- 71 m/s, and 461 +/- 94 m/s, respectively. These velocities were significantly (p < 0.01) faster than those predicted using elastic moduli derived from the quasi-static tests by 52, 45, and 41 percent, respectively. This study has documented that stress wave velocity in patellar tendon increases with increasing strain and is underestimated with a modulus estimated from quasi-static testing.     

ALGICIDAL BACTERIA ACTIVE AGAINST GYMNODINIUM BREVE (DINOPHYCEAE). I. BACTERIAL ISOLATION AND CHARACTERIZATION OF KILLING ACTIVITY1,3

Interactions between bacteria and species of harmful and/or toxic algae are potentially important factors affecting both the population dynamics and the toxicity of these algae. Recent reports of bacteria lethal to certain harmful algal bloom (HAB) species, coupled with a rapidly evolving interest in attempting to minimize the adverse effects of HABs through various prevention, control, and mitigation strategies, have focused attention on defining the role of algicidal bacteria in bloom termination. The aim of the present study was to determine whether algicidal bacteria active against Gymnodinium breve Davis, a dinoflagellate responsible for frequent and protracted red tides in the Gulf of Mexico, are present in the waters of the west Florida shelf. To date, we have isolated two bacterial strains from this region lethal to G. breve and have begun to characterize the algicidal activity of one of these strains, 41-DBG2. This bacterium, a yellow-pigmented, gram-negative rod, was isolated from waters containing no detectable G. breve cells, suggesting that such bacteria are part of the ambient microbial community and are not restricted to areas of high G. breve abundance. Strain 41-DBG2 produced a dissolved algicidal compound(s) that was released into the growth medium, and the algicide was effective against the four Gulf of Mexico G. breve isolates tested as well as a closely related HAB species that also occurs in this region, Gymnodinium mikimotoi Miyake et Kominami ex Oda. Nonetheless, data showing that a nontoxic isolate of Gymnodinium sanguineum Hirasaka from Florida Bay was not affected indicate that the algicidal activity of this bacterium does exhibit a degree of taxonomic specificity. Our efforts are currently being directed at resolving several critical issues, including the identity of the algicide(s), the mechanisms regulating its production and ability to discriminate between target algal species, and how the growth rate of 41-DBG2 is affected by the presence of G. breve cells. We have also proposed a conceptual model for interactions between algicidal bacteria and their target species to serve as a testable framework for ensuing field studies.     

Fluorescence microscopy of mycobacteria in pleural effusions

The diagnostic advantage of fluorescence microscopy (FM) of Papanicolaou-stained cytological specimens obtained by bronchoscopy has been described previously. This study was designed to evaluate the method's diagnostic benefit in cytological preparations of pleural effusions in cases of active pulmonary tuberculosis. In contrast to bronchial material there is no advantage in cytological evaluation of pleural effusions by FM.     

The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity

Leptin is required for normal energy and glucose homeostasis. The hypothalamic arcuate nucleus (ARH) has been proposed as an important site of leptin action. To assess the physiological significance of leptin signaling in the ARH, we used mice homozygous for a FLPe-reactivatable, leptin receptor null allele (Lepr(neo/neo) mice). Similar to Lepr(db/db) mice, these mice are obese, hyperglycemic, hyperinsulinemic, infertile, and hypoactive. To selectively restore leptin signaling in the ARH, we generated an adeno-associated virus expressing FLPe-recombinase, which was delivered unilaterally into the hypothalamus using stereotaxic injections. We found that unilateral restoration of leptin signaling in the ARH of Lepr(neo/neo) mice leads to a modest decrease in body weight and food intake. In contrast, unilateral reactivation markedly improved hyperinsulinemia and normalized blood glucose levels and locomotor activity. These data demonstrate that leptin signaling in the ARH is sufficient for mediating leptin's effects on glucose homeostasis and locomotor activity.     

Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.     

Referral and experience with genetic testing among women with early onset breast cancer

The purpose of this study was to determine whether physicians refer women with early onset breast cancer for genetic testing for BRCA1 and BRCA2, and how women respond to being offered testing and use the results. A web-based survey was distributed to 1221 women with early onset breast cancer. The survey included 158 questions divided into the following sections: demographics, family history of cancer, medical history, treatment history, and experience with genetic testing. Of 551 women diagnosed since 1993 who responded to the survey (45.1%), less than half (45%, n = 246) had ever discussed genetic testing with their physician and/or been referred to see a genetic counselor. Women with a family history of cancer (53%) and Ashkenazi Jewish women (81%) were more likely to have been referred. Of those who had discussed testing, 60% had undergone or were interested in testing. Overall 92 women were tested and 19 (20.6%) of these tested positive for a deleterious BRCA1 or BRCA2 mutation. Fourteen (74%) who tested positive subsequently underwent prophylactic surgery. Satisfaction with counseling and the decision to be tested was high. Among women who were not offered testing, the fact that the test had not been offered by their physician (89%), and fear of discrimination (83%) were the two most frequently cited factors for lack of interest in testing. A substantial number of women are not being referred to genetic counseling and/or testing after a diagnosis of early onset breast cancer. Among those who were tested, there was high interest in prophylactic surgery after confirmation of a BRCA1/2 mutation.