Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis

The “cysteine string protein” (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a “J” domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the “tall cells” of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.     

Progesterone effects on three regional gastrointestinal tissues.

Esophageal, antral, and colonic tissues excised from progesterone pretreated males were demonstrated to exhibit a significantly reduced contractile activity compared to the corresponding regions excised from non-treated male control animals. Progesterone blood levels (measured with RIA) of the treated males were found to fall within the range reported for non-pregnant cyclic females.     

Local recombination and mutation effects on molecular evolution in Drosophila.

I studied the cause of the significant difference in the synonymous-substitution pattern found in the achaete-scute complex genes in two Drosophila lineages, higher codon bias in Drosophila yakuba, and lower bias in D. melanogaster. Besides these genes, the functionally unrelated yellow gene showed the same substitution pattern, suggesting a region-dependent phenomenon in the X-chromosome telomere. Because the numbers of A/T --> G/C substitutions were not significantly different from those of G/C --> A/T in the yellow noncoding regions of these species, a AT/GC mutational bias could not completely account for the synonymous-substitution biases. In contrast, we did find an approximately 14-fold difference in recombination rates in the X-chromosome telomere regions between the two species, suggesting that the reduction of recombination rates in this region resulted in the reduction of the efficacy of selection in D. melanogaster. In addition, the D. orena yellow showed a 5% increase in the G + C content at silent sites in the coding and noncoding regions since the divergence from D. erecta. This pattern was significantly different from those at the orena Adh and Amy loci. These results suggest that local changes in recombination rates and mutational pressures are contributing to the irregular synonymous-substitution patterns in Drosophila.     

Local and systemic effects of intranodally injected CpG-C immunostimulatory-oligodeoxyribonucleotides in macaques

Immunostimulatory CpG-C oligodeoxyribonucleotides (ISS-ODNs) represent a promising strategy to enhance vaccine efficacy. We have shown that the CpG-C ISS-ODN C274 stimulates macaque blood dendritic cells (DCs) and B cells and augments SIV-specific IFN-gamma responses in vitro. To further explore the potential of C274 for future vaccine studies, we assessed the in vivo effects of locally administered C274 (in naive and healthy infected macaques). Costimulatory molecules were marginally increased on DCs and B cells within cells isolated from C274-injected lymph nodes (LNs). However, cells from C274-injected LNs exhibited heightened responsiveness to in vitro culture. This was particularly apparent at the level of CD80 (less so CD86) expression by CD123(+) plasmacytoid DCs and was further boosted in the presence of additional C274 in vitro. Notably, cells from C274-injected LNs secreted significantly elevated levels of several cytokines and chemokines upon in vitro culture. This was more pronounced when cells were exposed to additional stimuli in vitro, producing IFN-alpha, IL-3, IL-6, IL-12, TNF-alpha, CCL2, CCL3, CCL5, and CXCL8. Following C274 administration in the absence of additional SIV Ag, endogenous IFN-gamma secretion was elevated in LN cells of infected animals, but SIV-specific responses were unchanged. Endogenous and SIV-specific responses decreased in blood, before the SIV-specific responses rebounded by 2 wk after C274 treatment. Elevated IFN-alpha, CCL2, and CCL5 were also detected in the plasma after C274 injection. Thus, locally administered C274 has local and systemic activities, supporting the potential for CpG-C ISS-ODNs to boost immune function to enhance anti-HIV vaccine immunogenicity.     

Local and systemic effects of microbiome‐derived metabolites

Commensal microbes form distinct ecosystems within their mammalian hosts, collectively termed microbiomes. These indigenous microbial communities broadly expand the genomic and functional repertoire of their host and contribute to the formation of a “meta‐organism.” Importantly, microbiomes exert numerous biochemical reactions synthesizing or modifying multiple bioactive small molecules termed metabolites, which impact their host''s physiology in a variety of contexts. Identifying and understanding molecular mechanisms of metabolite–host interactions, and how their disrupted signaling can contribute to diseases, may enable their therapeutic application, a modality termed “postbiotic” therapy. In this review, we highlight key examples of effects of bioactive microbe‐associated metabolites on local, systemic, and immune environments, and discuss how these may impact mammalian physiology and associated disorders. We outline the challenges and perspectives in understanding the potential activity and function of this plethora of microbially associated small molecules as well as possibilities to harness them toward the promotion of personalized precision therapeutic interventions.     

A targeted gene knockout in Drosophila

We previously described a method for targeted homologous recombination at the yellow gene of Drosophila melanogaster. Because only a single gene was targeted, further work was required to show whether the method could be extended to become generally useful for gene modification in Drosophila. We have now used this method to produce a knockout of the autosomal pugilist gene by homologous recombination between the endogenous locus and a 2.5-kb DNA fragment. This was accomplished solely by tracking the altered genetic linkage of an arbitrary marker gene as the targeting DNA moved from chromosome X or 2 to chromosome 3. The results indicate that this method of homologous recombination is likely to be generally useful for Drosophila gene targeting.     

Local and systemic effects on inflammation during Eimeria nieschulzi infection

Rats infected with Eimeria nieschulzi, a coccidium that inhabits intestinal epithelium, have a lower basal inflammatory state in their intestinal mucosa eight days postinoculation as reflected by a drop in mucosal peroxidase activity and a decrease in the number of granulocytes in the lamina propria. The reduction of systemic inflammation in infected rats was assessed from a reduction in the formation of granulation tissue around a sterile cotton string implanted under the abdominal skin of the hosts. This reduced inflammatory response, both locally and systemically, occurs during the development of gamonts by the parasite and the release of oocysts from the host. These results plus the presence of normal or slightly elevated numbers of granulocytes in peripheral blood lead to the conclusion that the parasite does not affect hematopoiesis but interferes with some phase in the directed migration of leukocytes to specific sites.     

pH-induced microtubule-dependent redistribution of late endosomes in neuronal and epithelial cells

The interaction between late endocytic structures and microtubules in polarized cells was studied using a procedure previously shown to cause microtubule-dependent redistribution of lysosomes in fibroblasts and macrophages (Heuser, J. 1989. J. Cell Biol. 108:855-864). In cultured rat hippocampal neurons, low cytoplasmic pH caused cation-independent mannose-6-phosphate receptor-enriched structures to move out of the cell body and into the processes. In filter grown MDCK cells lowering the cytosolic pH to approximately 6.5 caused late endosomes to move to the base of the cell and this process was shown to be microtubule dependent. Alkalinization caused a shift in distribution towards the apical pole of the cell. The results are consistent with low pH causing the redistribution of late endosomes towards the plus ends of the microtubules. In MDCK cells the microtubules orientated vertically in the cell may play a role in this process. The shape changes that accompanied the redistribution of the late endosomes in MDCK cells were examined by electron microscopy. On low pH treatment fragmentation of the late endosomes was observed whereas after microtubule depolymerization individual late endosomal structures appeared to fuse together. The late endosomes of the MDCK cell appear to be highly pleomorphic and dependent on microtubules for their form and distribution in the cell.     

Different effects on zinc redistribution if ethanol is consumed before or immediately after birth

AimsThe effect of ethanol consumption, either during pregnancy and/or lactation, on the altered metabolism of zinc (Zn) is not well-defined. Therefore, this study was performed to analyse the effect of chronic ethanol exposure on Zn redistribution in dams and offspring during either gestation and/or lactation.MethodsWe have used three groups of Wistar rat dams: control (CD), ethanol (ED), and pair-fed dams (PD). Some of the newborns were cross-fostered to dams at birth and we formed five experimental groups of offspring: control (CO); those exposed to ethanol during gestation only (GO); those exposed to ethanol during lactation only (LO); those exposed to ethanol during both periods (EO); and pair-fed groups (PO). Zn levels were measured by flame atomic absorption spectrophotometry.ResultsZinc distribution is altered in ED with respect to CD, presenting significantly higher Zn values in the brain and spleen, and lower levels in the liver. However, total organs Zn levels are similar between dams. Ethanol-treated offspring (GO, LO, EO) consumed significantly less Zn than the CO. However, LO and EO showed significantly higher Zn serum levels. Zn distribution was altered in ethanol-treated offspring. GO and LO showed lower Zn levels in liver than CO; GO presents the lowest Zn liver levels. These levels were significantly lower than EO and PO. Ethanol-treated pups present significantly higher spleen and testes values than CO and PO. Total organ Zn levels were significantly lower in GO.ConclusionsMaternal adaptation resulted in organ Zn retention in order to meet the demands of pup’s growth in the face of a lower diet intake. However, there was a redistribution of Zn in organ contents. Therefore, the ethanol route administration (via placenta and/or milk) affects Zn redistribution in pups in a different way.     

In vivo effects of zinc deficiency on calmodulin concentrations in selected rat tissues

Two groups of male Sprague-Dawley rats, one fed zinc-deficient diet, ad libitum, the other, pair-fed with the same diet, but given supplemental zinc in the drinking water (8 mg Zn++/ml) were studied. After ten weeks of diet, rats were exsanguinated and zinc and calmodulin concentrations in brain and testis were measured. Mean zinc concentration in testis was significantly decreased in rats fed zinc-deficient diet without supplemental Zn++, but mean zinc concentration in brain was not different. Similarly, mean calmodulin concentration in testis was decreased in rats fed zinc-deficient diet without supplemental Zn++ whereas mean calmodulin concentration in brain was not different. Distribution studies of zinc and calmodulin showed that both zinc and calmodulin were released more freely into soluble fractions of testis in rats fed zinc-deficient diet without supplemental Zn++. These results indicate, for the first time in in vivo studies, that zinc influences the calmodulin content of testis.     

Local redistribution of fungicides on leaves by water

Local redistribution of various fungicides applied as large or small droplets to detached broad-bean leaflets was examined by laboratory bio-assay with Botrytis fabae and by leaf-printing techniques. The results showed that fungicidal particulate and soluble material will spread in water from the initial deposits and that the protected areas on leaves inoculated after washing were often much greater than on unwashed leaves. The extent of redistribution was related to the inherent toxicity, physical form, duration of weathering and tenacity of the fungicides. The effects of formulating copper oxychloride and zineb fungicides with surface-active agents and adhesives seemed due to differences in the tenacity of the deposits. Results indicated that similar types of redistribution would occur in the field, but the possible importance of the electrokinetic theory of redistribution was not proven.     

The prion protein and neuronal zinc homeostasis

Although the prion protein (PrP) is known to be the causative agent of the neurodegenerative transmissible spongiform encephalopathies, its normal cellular function remains elusive. Octapeptide repeats in the N terminus of PrP bind metal ions and are required for the endocytosis of PrP upon exposure of cells to copper or zinc. As the concentration of zinc in the extracellular spaces of the brain is higher than that for copper, we put forward the hypothesis that PrP is involved in neuronal zinc homeostasis; PrP might be involved in transport of zinc into the cell or might act as a zinc sensor. In prion disease, when the protein undergoes a conformational change to the infectious form, this function of PrP in zinc homeostasis might be compromised.     

Adrenergic and calcium-mediated subcellular redistribution of protein kinase C in primary neuronal cultures

Incubation of primary neuronal cultures prepared from the brains of neonatal rats with 50 microM epinephrine resulted in the transient redistribution of protein kinase C from the cytosol to the particulate fraction. This effect occurred after 1 and 5 min of incubation and resulted in a decrease in cytosolic protein kinase C activity with a corresponding increase in particulate protein kinase C of approximately 30% and 15%, respectively. The epinephrine-stimulated translocation of protein kinase C was blocked by 1 microM prazosin indicating the involvement of alpha 1-adrenergic receptors. Further, inclusion of 0.1 microM Ca2+ in the homogenization buffer was found to significantly enhance the binding of protein kinase C to cellular membranes prepared from neuronal cultures. These results indicate that alpha 1-adrenergic receptors in neuronal brain cell cultures are linked to the activation of protein kinase C and that the mobilization of Ca2+ may enhance this effect.     

Local and systemic effects of phosphorus and nitrogen on nodulation and nodule function in Alnus incana

Phosphorus (P) and nitrogen (N) effects on nodulation, nitrogenase activity and plant growth were studied in the root-hair-infected actinorhizal plant Alnus incana (L.) Moench. A split-root experiment, as well as a short-term experiment with entire root systems and a broader range of P concentrations, showed that P effects were specific on nodulation and not a general stimulation via a plant growth effect. These results indicate that nodule initiation and nodule growth have a high P demand. The split-root assay, comprising seven combinations of two N and two P levels, showed that P could counteract systemic N inhibition of nodulation, but did not counteract N inhibition of nitrogenase activity.