c-Src regulation of fibroblast growth factor-induced proliferation in murine embryonic fibroblasts

Activated fibroblast growth factor receptor 1 (FGFR1) propagates FGF signals through multiple intracellular pathways via intermediates FRS2, PLCgamma, and Ras. Conflicting reports exist concerning the interaction between FGFR1 and Src family kinases. To address the role of c-Src in FGFR1 signaling, we compared proliferative responses of murine embryonic fibroblasts (MEF) deficient in c-Src, Yes, and Fyn to MEF expressing either endogenous levels or overexpressing c-Src. MEF with endogenous c-Src had significantly greater FGF-induced DNA synthesis and proliferation than cells lacking or overexpressing c-Src. This was related directly to c-Src expression by analysis of c-Src-deficient cells transfected with and sorted for varying levels of a c-Src expression vector. This suggests an "optimal" quantity of c-Src expression for FGF-induced proliferation. To determine if this was a general phenomenon for growth factor signaling pathways utilizing c-Src, responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and lysophosphatidic acid (LPA) were examined. As for FGF, responses to EGF were clearly inhibited when c-Src was absent or overexpressed. In contrast, varying levels of c-Src had little effect on responses to PDGF or LPA. The data show that mitogenic pathways activated by FGF-1 and EGF are regulated by c-Src protein levels and appear to differ significantly from those activated by PDGF and LPA.     

Structural constraints on protein self-processing in L-aspartate-alpha-decarboxylase

Aspartate decarboxylase, which is translated as a pro-protein, undergoes intramolecular self-cleavage at Gly24-Ser25. We have determined the crystal structures of an unprocessed native precursor, in addition to Ala24 insertion, Ala26 insertion and Gly24-->Ser, His11-->Ala, Ser25-->Ala, Ser25-->Cys and Ser25-->Thr mutants. Comparative analyses of the cleavage site reveal specific conformational constraints that govern self-processing and demonstrate that considerable rearrangement must occur. We suggest that Thr57 Ogamma and a water molecule form an 'oxyanion hole' that likely stabilizes the proposed oxyoxazolidine intermediate. Thr57 and this water molecule are probable catalytic residues able to support acid-base catalysis. The conformational freedom in the loop preceding the cleavage site appears to play a determining role in the reaction. The molecular mechanism of self-processing, presented here, emphasizes the importance of stabilization of the oxyoxazolidine intermediate. Comparison of the structural features shows significant similarity to those in other self-processing systems, and suggests that models of the cleavage site of such enzymes based on Ser-->Ala or Ser-->Thr mutants alone may lead to erroneous interpretations of the mechanism.     

Cerebellar neurones: Differentiation and modulation of sensitivity to excitotoxic treatment

The neurite outgrowth and adhesion complex (NOAC), isolated from rabbit sera has been dissociated in its major components by reverse-phase chromatography in HPLC by using a C₁₈ column. SDS-PAGE analisys of the active fractions revealed the presence of three major bands of approximately 100, 70 and 50 kDa. Studies on the biological activity of NOAC were carried out on rat cerebellar granule cells. NOAC-cultured cells exhibit a marked resistance to excitotoxic stimuli carried by glutamate.     

Distribution of the molossinus allele of Sry, the testis-determining gene, in wild mice

When the Y chromosome of the laboratory inbred mouse strain C57BL/6 (B6) is replaced by the Y of certain strains of Mus musculus domesticus, testis determination fails and all XY fetuses develop either as hermaphrodites or XY females (XY sex reversal). This suggests the presence of at least two alleles of Sry, the male-determining gene on the Y:M. m. domesticus and B6. The B6 Y chromosome is derived from the Japanese house mouse, M. m. molossinus and therefore carries a molossinus Sry allele. As a first step to determine how the molossinus Sry allele evolved, its distribution pattern was determined in wild mice. The cumulative data of 96 M. musculus samples obtained from 58 geographical locations in Europe, North Africa, and Asia show the molossinus Sry allele is restricted to Japan and the neighboring Asian mainland and confirm that Japanese M. m. molossinus mice were derived in part from a race of M. m. musculus from Korea or Manchuria. Sry polymorphisms, as illustrated by the molossinus Sry allele, can serve as molecular markers for studies on the evolution of wild M. musculus populations and can help determine the role sex determination plays in speciation.      

Interactions of Photobleaching and Inorganic Nutrients in Determining Bacterial Growth on Colored Dissolved Organic Carbon

Abstract Bacteria are key organisms in the processing of dissolved organic carbon (DOC) in aquatic ecosystems. Their growth depends on both organic substrates and inorganic nutrients. The importance of allochthonous DOC, usually highly colored, as bacterial substrate can be modified by photobleaching. In this study, we examined how colored DOC (CDOC) photobleaching, and phosphorus (P) and nitrogen (N) availability, affect bacterial growth. Five experiments were conducted, manipulating nutrients (P and N) and sunlight exposure. In almost every case, nutrient additions had a significant, positive effect on bacterial abundance, production, and growth efficiency. Sunlight exposure (CDOC photobleaching) had a significant, positive effect on bacterial abundance and growth efficiency. We also found a significant, positive interaction between these two factors. Thus, bacterial use of CDOC was accelerated under sunlight exposure and enhanced P and N concentrations. In addition, the accumulation of cells in sunlight treatments was dependent on nutrient availability. More photobleached substrate was converted into bacterial cells in P- and N-enriched treatments. These results suggest nutrient availability may affect the biologically-mediated fate (new biomass vs respiration) of CDOC.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Patch-clamp recording of charge movement, Ca(2+) current, and Ca(2+) transients in adult skeletal muscle fibers

Intramembrane charge movement (Q), Ca(2+) conductance (G(m)) through the dihydropyridine-sensitive L-type Ca(2+) channel (DHPR) and intracellular Ca(2+) fluorescence (F) have been recorded simultaneously in flexor digitorum brevis muscle fibers of adult mice, using the whole-cell configuration of the patch-clamp technique. The voltage distribution of Q was fitted to a Boltzmann equation; the Q(max), V(1/2Q), and effective valence (z(Q)) values were 41 +/- 3.1 nC/&mgr;F, -17.6 +/- 0.7 mV, and 2.0 +/- 0.12, respectively. V(1/2G) and z(G) values were -0.3 +/- 0.06 mV and 5.6 +/- 0.34, respectively. Peak Ca(2+) transients did not change significantly after 30 min of recording. F was fit to a Boltzmann equation, and the values for V(F1/2) and z(F) were 6.2 +/- 0.04 mV and 2.4, respectively. F was adequately fit to the fourth power of Q. These results demonstrate that the patch-clamp technique is appropriate for recording Q, G(m), and intracellular [Ca(2+)] simultaneously in mature skeletal muscle fibers and that the voltage distribution of the changes in intracellular Ca(2+) can be predicted by a Hodgkin-Huxley model.     

Structural basis for the interaction of SARS‐CoV‐2 virulence factor nsp1 with DNA polymerase α–primase

The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)—the causative agent of coronavirus disease 2019 (COVID‐19)—are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS‐CoV‐2. The DNA polymerase α (Pol α)–primase complex or primosome—responsible for initiating DNA synthesis during genomic duplication—was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS‐CoV‐2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo‐electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS‐CoV‐2 interferes with Pol α''s putative role in the immune response during the viral infection.