Molecular mechanism for recognition of the cargo adapter Rab6GTP by the dynein adapter BicD2.

Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6GTP-positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6GTP by BicD2, using structure prediction and mutagenesis. The binding site of BicD2 spans two regions of Rab6 that undergo structural changes upon the transition from the GDP- to GTP-bound state, and several hydrophobic interface residues are rearranged, explaining the increased affinity of the active GTP-bound state. Mutations of Rab6GTP that abolish binding to BicD2 also result in reduced co-migration of Rab6GTP/BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6GTP/BicD2 interaction for overall motility of the multi-motor complex that contains both kinesin-1 and dynein. Our results provide insights into trafficking of secretory and Golgi-derived vesicles and will help devise therapies for diseases caused by BicD2 mutations, which selectively affect the affinity to Rab6 and other cargoes.

Evaluation of chronic diarrhea in patients newly diagnosed with HIV infection through the FilmArray® gastrointestinal panel.

INTRODUCTION: The treatment and diagnosis of chronic diarrhea in the immunocompromised patient depends on the ability to rapidly detect the etiologic agents. AIMS: Our aim was to evaluate the results of the FilmArray® gastrointestinal panel in patients newly diagnosed with HIV infection that presented with chronic diarrhea. MATERIAL AND METHODS: Utilizing nonprobability consecutive convenience sampling, 24 patients were included that underwent molecular testing for the simultaneous detection of 22 pathogens. RESULTS: In 24 HIV-infected patients with chronic diarrhea, enteropathogen bacteria were detected in 69% of the cases, parasites in 18%, and viruses in 13%. Enteropathogenic Escherichia coli and enteroaggregative Escherichia coli were the main bacteria identified, Giardia lamblia was found in 25%, and norovirus was the most frequent viral agent. The median number of infectious agents per patient was three (range of 0 to 7). The biologic agents not identified through the FilmArray® method were tuberculosis and fungi. CONCLUSIONS: Several infectious agents were simultaneously detected through the FilmArray® gastrointestinal panel in patients with HIV infection and chronic diarrhea.

Successful treatment using gentamicin liposomes of Salmonella dublin infections in mice.

Gentamicin entrapped within stable multilamellar liposomes was used to treat mice after they were infected per os with Salmonella dublin. Of 10 mice, 8 survived after a single intravenous (i.v.) injection of 2 mg of gentamicin liposomes per kg compared with 0 of 10 treated with the same amount of free gentamicin. All mice survived after treatment with a single i.v. or intraperitoneal injection of 20 mg of gentamicin liposomes per kg, whereas that dose of free drug was completely ineffective and caused neuromuscular paralysis when injected rapidly i.v. In mice treated with gentamicin liposomes, there was a steady decrease in the number of salmonellae in spleens for 2 weeks after treatment. High concentrations of gentamicin were present in the spleen for at least 10 days after treatment. Although gentamicin was not detected in the mesenteric lymph nodes of mice treated with gentamicin liposomes, bacterial counts in the nodes also decreased over time. Small numbers of bacteria remained viable in the mesenteric lymph nodes and Peyer's patches but not in the spleens of mice treated with 20 to 80 mg/kg. Mice treated with doses of gentamicin liposomes as high as 80 mg/kg showed only a transient increase in blood urea nitrogen and no rise in serum creatinine. These results confirm that gentamicin in liposomes is less toxic in mice than is free gentamicin and is extremely effective therapy for disseminated Salmonella infections in mice.

A further study of the neuromuscular effects of vesamicol (AH5183) and of its enantiomer specificity.

1. The effects of vesamicol (2-(4-phenylpiperidino) cyclohexanol), an inhibitor of acetylcholine storage, and its two optical isomers have been studied on neuromuscular transmission in rat and frog muscle, and on nerve conduction in frog nerve. 2. Racemic vesamicol produced a pre-block augmentation of twitch tension that also occurred in directly-stimulated muscle. This effect is thus at least partially due to an increase in muscle contractility. 3. (-)-Vesamicol was approximately 20 times more potent than (+)-vesamicol in blocking twitches elicited at 1 Hz. This degree of stereoselectivity is similar to that measured for inhibition of acetylcholine uptake by isolated synaptic vesicles. Both enantiomers were equally weak in reducing nerve action potential amplitude in frog nerve. 4. Further studies with the active isomer, (-)-vesamicol, showed that, like that produced by racemic vesamicol, the neuromuscular block was highly frequency-dependent. The block was not reversed by choline or neostigmine, but was partially reversed by 4- or 3,4-aminopyridine. 5. Preliminary electrophysiological studies showed that vesamicol reduced miniature endplate potential amplitude in rapidly-stimulated frog nerve-muscle preparations. Addition of lanthanum ions increased the frequency of miniature endplate potentials and led to the appearance of apparently normal-sized potentials amongst those of reduced amplitude. 6. The results show the close agreement between pharmacological and biochemical observations indicating the suitability of the rat diaphragm as a test model for substances of this nature. The degree of reversibility of the vesamicol-induced neuromuscular block by aminopyridines was unexpected, and it is suggested that in the presence of a drug which greatly increases release, a pool of acetylcholine is capable of being released which is not normally releasable after block of storage by vesamicol. It is also considered possible that the results from the intracellular recording studies may be explained in these terms.