The pro-domain of the zebrafish Nodal-related protein Cyclops regulates its signaling activities

Nodal proteins are secreted signaling factors of the transforming growth factor beta (TGFbeta) family with essential roles in embryonic development in vertebrates. Mutations affecting the Nodal factors have severe consequences in mammals and fish. Furthermore, increased Nodal levels have been associated with melanoma tumor progression. Like other TGFbeta-related proteins, Nodal factors consist of a pro-domain and a mature domain. The pro-domain of mouse Nodal protein stabilizes its precursor. However, the mechanisms by which the pro-domains exert their activities are unknown. Here, we characterize the zebrafish Nodal-related factor Cyclops (Cyc) and find unexpected functions for the pro-domain in regulating Cyc activity. We identified a lysosome-targeting region in the Cyc pro-domain that destabilizes the precursor and restricts Cyc activity, revealing the molecular basis for the short-range signaling activities of Cyc. We show that both the pro- and mature-domains of Cyc regulate its stability. We also characterize a mutation in the pro-domain of human NODAL (hNODAL) that underlies congenital heterotaxia. Heterologous expression of mutant hNODAL increases expression of Nodal-response genes. Our studies reveal unexpected roles for the pro-domain of the Nodal factors and provide a possible mechanism for familial heterotaxia.     

Alteration of superoxide- and nitric oxide-mediated antimicrobial function of macrophages by in vivo cocaine exposure

Cocaine is a popular drug of abuse and despite impressive advances in the understanding of its physiological, pharmacological, and toxic effects, its mechanism of immunosuppression at the cellular level is not well understood. In this paper we report the role of effector molecules like superoxide and nitric oxide in the antibacterial function of macrophages exposed to acute and chronic doses of cocaine in vivo. Bacterial killing by acute cocaine-exposed macrophages (ACE-Mphis) increased significantly, with a concomitant rise in respiratory burst and generation of superoxide and nitric oxide, compared with control macrophages. In contrast, chronic cocaine-exposed macrophages (CCE-Mphis) exhibited limited antimicrobial activity, which correlated closely with diminished respiratory burst and reduced production of superoxide and nitric oxide. Further, a killing assay was carried out in the presence of N(G)-methyl-L-arginine acetate, an inhibitor of iNOS, to evaluate the role of nitric oxide in the killing process. The results obtained indicate that while about 30% killing of input bacteria by control and ACE-Mphis was attributable to NO-mediated killing, only about 6% killing from NO was found with CCE-Mphis. The findings indicate that acute exposure to cocaine possibly caused upregulation of enzymes responsible for the generation of ROI (reactive oxygen intermediates) and RNI (reactive nitrogen intermediates), leading to enhanced antimicrobial function. On the other hand, chronic exposure to cocaine impaired the oxygen-dependent microbicidal capacity of macrophages, possibly through impaired expression of enzymes responsible for ROI and RNI formation. Proinflammatory cytokines may play a key role in cocaine-mediated immunosuppression, since exposure of macrophages to cocaine impairs the ability of the cells to produce these cytokines.     

7-[1H-Indol-2-yl]-2,3-dihydro-isoindol-1-ones as dual Aurora-A/VEGF-R2 kinase inhibitors: design, synthesis, and biological activity

A novel series of 7-[1H-indol-2-yl]-2,3-dihydro-isoindol-1-ones designed to be inhibitors of VEGF-R2 kinase was synthesized and found to potently inhibit VEGF-R2 and Aurora-A kinases. The structure-based design, synthesis, and initial SAR of the series are discussed.     

Efficiency of RAPD,ISSR and ITS markers in detecting genetic variability among Salacia species sampled from the Western Ghats of Karnataka

Diversity and phylogenetic relationship between four closely related Salacia species, i.e., Salacia chinensis, Salacia macrosperma, Salacia fruticosa and Salacia oblonga, collected from the Western Ghats of Karnataka, India, was assessed. Ten each of RAPD and ISSR primers generated a total of 76 and 68 loci, generating polymorphisms of 92.21 and 89.71%, respectively. Maximum likelihood analysis of the ITS sequences revealed three clades. Dendrogram analyses of RAPD and ISSR revealed two and four clusters, respectively. Overall polymorphism revealed by RAPD was 41.45?±?10%, ISSR was 33.58?±?6.52%, and ITS was 25.50?±?17.25%. Molecular variance revealed significant variance within and among the Salacia species. Tajima’s D neutrality test and Fu’s Fs were negative for all four species, implying presences of rare alleles and population expansion. Comparative study of RAPD, ISSR and ITS for Salacia species has given an insight into the efficiency of each technique in detecting diversity within and among the population sampled in the Western Ghats of Karnataka.

     

Amino acid deprivation and central carbon metabolism regulate the production of outer membrane vesicles and tubes by Francisella

Francisella tularensis is a highly virulent Gram‐negative bacterial pathogen that causes the zoonotic disease tularemia. F. novicida, a model tularemia strain, produces spherical outer membrane vesicles (OMV), as well as novel tubular vesicles and extensions of the cell surface. These OMV and tubes (OMV/T) are produced in a regulated manner and contain known virulence factors. Mechanisms by which bacterial vesicles are produced and regulated are not well understood. We performed a genetic screen in F. novicida to decipher the molecular basis for regulated OMV/T formation, and identified both hypo‐ and hyper‐vesiculating mutants. Mutations in fumA and tktA, involved in central carbon metabolism, and in FTN_0908 and FTN_1037, of unknown function, resulted in severe defects in OMV/T production. Cysteine deprivation was identified as the signal that triggers OMV/T formation in F. novicida during growth in rich medium. We also found that fully virulent F. tularensis produces OMV/T in a similarly regulated manner. Further analysis revealed that OMV/T production is responsive to deprivation of essential amino acids in addition to cysteine, and that the hypo‐vesiculating mutants are defective in responding to this signal. Thus, amino acid starvation, such as encountered by Francisella during host cell invasion, regulates the production of membrane‐derived structures.     

A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells

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Identification of new HLA class I region genes by sample sequencing

 Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region. We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases. Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes. Cosmids were sonnicated to produce fragments of 0.5 – 1 kilobases, subcloned, and sequenced using an automated sequencer. Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm. A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 – 8 gene family. These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens. Received: 10 December 1996 / Revised: 7 January 1997     

Dexamethasone induced alterations in enzymatic and nonenzymatic antioxidant status in heart and kidney of rats

This study was designed to investigate the alterations in thiobarbituric acid reactants (TBA-reactants) and enzymatic and nonenzymatic antioxidant levels induced by dexamethasone (Dex) in heart and kidney and to find out whether these alterations induced by Dex and its hypertensive effect had any role in the maintenance of hypertension in this model. Administration of dexamethasone induced severe loss of body weight, significant increase in heart and kidney weights and also marked electrocardiographic changes. The protein content in heart and kidney increased significantly during Dex administration and returned to near normalcy after withdrawal. Total activity of lactate dehydrogenase showed a significant increase in heart till day 8 of treatment, whereas in serum, it exhibited a significant decrease. The activity of CK in heart showed an increase till day 8 of treatment and approached normalcy thereafter. In serum, CK exhibited a decrease till day 8, remaining insignificant thereafter. CKMB in heart showed an insignificant increase initially, reaching normal levels on Dex withdrawal, whereas in serum, it showed a significant decrease throughout the experimental period. Mean arterial pressure (MAP) and heart rate increased significantly, while a significant elevation in the ST segment was noticed during administration as well as after withdrawal of dex. The TBA-reactants levels were found to increase in heart and kidney during days 12 and 16 of administration with Dex and even after withdrawal of Dex, the levels were insignificantly elevated. The level of glutathione in heart and kidney increased from day 4 onwards and reached normalcy during the later stages of treatment and after withdrawal of Dex. The total sulfhydryl groups exhibited a significant increase in both heart and kidney throughout the experiment. The antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase exhibited a significant decrease in heart during Dex administration whereas, in kidney, they exhibited a significant increase during treatment and after withdrawal of Dex. Thus, Dex induced rise in mean arterial pressure, significant alterations in electrocardiographic parameters and also marked alterations in enzymatic and nonenzymatic antioxidant levels and in the TBA-reactants level in heart and kidney.     

Strong reproductive isolation and narrow genomic tracts of differentiation among three woodpecker species in secondary contact

Hybrid zones allow the measurement of gene flow across the genome, producing insight into the genomic architecture of speciation. Such analysis is particularly powerful when applied to multiple pairs of hybridizing species, as patterns of genomic differentiation can then be related to age of the hybridizing species, providing a view into the build‐up of differentiation over time. We examined 33 809 single nucleotide polymorphisms (SNPs) in three hybridizing woodpecker species: Red‐breasted, Red‐naped and Yellow‐bellied sapsuckers (Sphyrapicus ruber, Sphyrapicus nuchalis and Sphyrapicus varius), two of which (ruber and nuchalis) are much more closely related than each is to the third (varius). To identify positions of SNPs on chromosomes, we developed a localization method based on comparative genomics. We found narrow clines, bimodal distributions of hybrid indices and genomic regions with decreased rates of introgression. These results suggest moderately strong reproductive isolation among species and selection against specific hybrid genotypes. We found 19 small regions of strong differentiation between species, partly shared among species pairs, but no large regions of differentiation. An association analysis revealed a single strong‐effect candidate locus associated with plumage, possibly explaining mismatch among the three species in genomic relatedness and plumage similarity. Our comparative analysis of species pairs of different age and their hybrid zones showed that moderately strong reproductive isolation can occur with little genomic differentiation, but that reproductive isolation is incomplete even with much greater genomic differentiation, implying there are long periods of time when hybridization is possible if diverging populations are in geographic contact.