An antibody against secretogranin I (chromogranin B) is packaged into secretory granules

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.     

Cyanobacteria of Rocks and Soils of the Orinoco Lowlands and the Guayana Uplands,Venezuela

The occurrence of 23 cyanobacterial species, belonging to 9 different genera and 5 cyanobacterial lichen species of 5 different genera on exposed, open rock surfaces of inselbergs and on soil in savannas of the Orinoco lowlands and the Guayana uplands is described. Their distribution patterns and frequency within the different habitats are given. The filamentous procaryotic blue-green algae/cyanobacteria Stigonema ocellatum and Scytonema crassum, together with the unicellular cyanobacterium Gloeocapsa sanguinea were the most frequent species on rocks, whereas the filamentous cyanobacterium, Schizothrix telephoroides, dominated in cyanobacterial mats on the savanna soil. All species showed intensively coloured sheaths, either brown or yellow in the case of Stigonema ocellatum and Scytonema crassum, or red in Gloeocapsa sanguinea and Schizothrix telephoroides. In addition, a number of cyanobacterial lichens occurred.     

The novel flightless-I gene brings together two gene families, actin- binding proteins related to gelsolin and leucine-rich-repeat proteins involved in Ras signal transduction

The Drosophila melanogaster gene flightless-I, involved in gastrulation and muscle degeneration, has Caenorhabditis elegans and human homologues. In these highly conserved genes, two previously known gene families have been brought together, families encoding the actin- binding proteins related to gelsolin and the leucine-rich-repeat (LRR) group of proteins involved in protein-protein interactions. Both these gene families exhibit characteristics of molecular changes involving replication slippage and exon shuffling. Phylogenetic analyses of 19 amino acid sequences of 6 related protein types indicate that actin- associated proteins related to gelsolin are monophyletic to a common ancestor and include flightless proteins. Conversely, comparison of 24 amino acid sequences of LRR proteins including the flightless proteins indicates that flightless proteins are members of a structurally related subgroup. Included in the flightless cluster are human and mouse rsp-1 proteins involved in suppressing v-Ras transformation of cells and the membrane-associated yeast (Saccharomyces cerevisae) adenylate cyclase whose analogous LRRs are required for interaction with Ras proteins. There is a strong possibility that ligands for this group could be related and that flightless may have a similar role in Ras signal transduction. It is hypothesized that an ancestral monomeric gelsolin precursor protein has undergone at least four independent gene reorganization events to account for the structural diversity of the extant family of gelsolin-related proteins and that gene duplication and exon shuffling events occurred prior to or at the beginning of multicellular life, resulting in the evolution of some members of the family soon after the appearance of actin-type proteins.      

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Two cellular single-strand-specific DNA-binding proteins interact with two regions of the bovine papillomavirus type 1 genome, including the origin of DNA replication.

We have identified and purified to near homogeneity two specific single-stranded DNA-binding factors (SPSF I and II) with molecular masses of 42 and 39 kDa, respectively, from calf thymus. Gel retention analysis and competition experiments demonstrate that the ubiquitous proteins SPSF I and II specifically interact with single-stranded DNA derived from the minimal in vitro origin of replication of bovine papillomavirus type 1 and a region of the viral genome proposed to be involved in plasmid maintenance. Bovine papillomavirus type 1 proteins do not interfere with DNA binding of SPSF I and II. The exact location of the binding domains of SPSF I and II on the DNA has been determined by methylation interference and T4 DNA polymerase footprinting. A potential cellular binding site for SPSF I and II is the major promoter (P2) of the human c-myc gene.     

Ion distribution in roots of barley seedlings measured by electron probe x-ray microanalysis

The distribution of ions, particularly K and Na, was studied in roots of barley seedlings grown on various ionic solutions. Analyses were made by means of electron probe x-ray microanalysis using frozen, fractured bulk specimens. By this technique, it was demonstrated that there can be variability in the ratio K/Na measured in the vacuoles of cortical cells, with this ratio often being lower in epidermal cells of the root than in the inner cortex. A sharp difference in the K/Na ratio was also found between cells of the endodermis and those of the adjacent cortex, and generally higher ratios of K/Na occurred in the stele than in the cortex. Estimation of the concentrations in the cytoplasm was at the limit of resolution of this technique, but it can be shown that the K/Na ratio in the cytoplasm was higher than that in the vacuole. In low salt roots, the K concentration in the cytoplasm was higher than that in the vacuoles. The results with the x-ray microprobe confirm other measurements based on flux analysis or analysis of small samples of the root.     

cMyBPC phosphorylation modulates the effect of omecamtiv mecarbil on myocardial force generation

Omecamtiv mecarbil (OM), a direct myosin motor activator, is currently being tested as a therapeutic replacement for conventional inotropes in heart failure (HF) patients. It is known that HF patients exhibit dysregulated β-adrenergic signaling and decreased cardiac myosin-binding protein C (cMyBPC) phosphorylation, a critical modulator of myocardial force generation. However, the functional effects of OM in conditions of altered cMyBPC phosphorylation have not been established. Here, we tested the effects of OM on force generation and cross-bridge (XB) kinetics using murine myocardial preparations isolated from wild-type (WT) hearts and from hearts expressing S273A, S282A, and S302A substitutions (SA) in the M domain, between the C1 and C2 domains of cMyBPC, which cannot be phosphorylated. At submaximal Ca²⁺ activations, OM-mediated force enhancements were less pronounced in SA than in WT myocardial preparations. Additionally, SA myocardial preparations lacked the dose-dependent increases in force that were observed in WT myocardial preparations. Following OM incubation, the basal differences in the rate of XB detachment (k_rel) between WT and SA myocardial preparations were abolished, suggesting that OM differentially affects the XB behavior when cMyBPC phosphorylation is reduced. Similarly, in myocardial preparations pretreated with protein kinase A to phosphorylate cMyBPC, incubation with OM significantly slowed k_rel in both the WT and SA myocardial preparations. Collectively, our data suggest there is a strong interplay between the effects of OM and XB behavior, such that it effectively uncouples the sarcomere from cMyBPC phosphorylation levels. Our findings imply that OM may significantly alter the in vivo cardiac response to β-adrenergic stimulation.     

Role of N-Cadherin cis and trans Interfaces in?the Dynamics of Adherens Junctions in Living Cells

Cadherins, Ca²⁺-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca²⁺-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca²⁺-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca²⁺-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins.     

Predictive Models for Escherichia coli Concentrations at Inland Lake Beaches and Relationship of Model Variables to Pathogen Detection

Predictive models, based on environmental and water quality variables, have been used to improve the timeliness and accuracy of recreational water quality assessments, but their effectiveness has not been studied in inland waters. Sampling at eight inland recreational lakes in Ohio was done in order to investigate using predictive models for Escherichia coli and to understand the links between E. coli concentrations, predictive variables, and pathogens. Based upon results from 21 beach sites, models were developed for 13 sites, and the most predictive variables were rainfall, wind direction and speed, turbidity, and water temperature. Models were not developed at sites where the E. coli standard was seldom exceeded. Models were validated at nine sites during an independent year. At three sites, the model resulted in increased correct responses, sensitivities, and specificities compared to use of the previous day''s E. coli concentration (the current method). Drought conditions during the validation year precluded being able to adequately assess model performance at most of the other sites. Cryptosporidium, adenovirus, eaeA (E. coli), ipaH (Shigella), and spvC (Salmonella) were found in at least 20% of samples collected for pathogens at five sites. The presence or absence of the three bacterial genes was related to some of the model variables but was not consistently related to E. coli concentrations. Predictive models were not effective at all inland lake sites; however, their use at two lakes with high swimmer densities will provide better estimates of public health risk than current methods and will be a valuable resource for beach managers and the public.