The Kinetics of Vitamin D3 in the Osteoblastic Cell

Experimental evidence is presented on the translocation of vitamin D metabolite, 1,25-(OH)₂D₃, from the membrane to the nucleus in osteoblast progenitor cells. A mathematical model permitting traversal of the cytoplasm at either a fixed velocity or by diffusion is formulated in order to determine whether transport along the cytoskeletal tracks is more consistent with the observed spatial-temporal distribution than diffusion, and it is so found. The model includes reactions in the nucleus involving D₃ to form other compounds, such as protegerin, and thus also makes predictions of the concentrations of these compounds in various regions of the cell.     

The RecB Nuclease Domain Binds to RecA-DNA Filaments: Implications for Filament Loading

The E. coli RecBCD enzyme facilitates the loading of RecA onto single-stranded DNA produced by the combined helicase/nuclease activity of RecBCD. The nuclease domain of RecB protein, RecB^nuc, has been previously shown to bind RecA. Surprisingly, RecB^nuc also binds to phage and eukaryotic homologs of RecA, leading to the suggestion that RecB^nuc interacts with the polymerization motif that is present in all three proteins. This mode of interaction could only be with monomeric RecA, as this motif would be buried in filaments. We show that RecB^nuc binds extensively to the outside of RecA-DNA filaments. Three-dimensional reconstructions suggest that RecB^nuc binds to the ATP-binding core of RecA, with a displacement of the C-terminal domain of RecA. Solution experiments confirm that the interaction of RecB^nuc is only with the RecA core. Since the RecA C-terminal domain has been shown to be regulatory, the interaction observed may be part of the loading mechanism where RecB displaces the RecA C-terminal domain and activates a RecA monomer for polymerization.     

Reversible inhibitors of beta-glucosidase

A variety of reversible inhibitors of sweet almond beta-glucosidase were examined. These included simple sugars and sugar derivatives, amines and phenols. With respect to the sugar inhibitors and, indeed, the various glycoside substrates, the enzyme has what can be considered a "relaxed specificity". No single substituent on glucose, for example, is essential for binding. Replacement of a hydroxyl group with an anionic substituent reduces the affinity while substitution with a cationic (amine) substituent enhances the affinity. Amines, in general, are good inhibitors, binding more tightly than the corresponding alcohols: pKiRNH3+ = 0.645pKiROH + 1.77 (n = 9, r = 0.97). The affinity of a series of 10 primary amines was found to be strongly influenced by substituent hydrophobicity: pKi = 0.52 pi + 1.32 (r = 0.95). The major binding determinant of the glycoside substrates is the aglycon moiety. Thus, the Ki values of phenols are similar in magnitude to the Ks values of the corresponding aryl beta-glucoside. The pH dependence for the inhibition by various phenols indicates that it is the un-ionized phenol which binds to the enzyme when an enzymic group of pKa = 6.8 (+/- 0.1) is protonated. The affinity of the phenol inhibitor is dependent on its basicity with a Br?nsted coefficient for binding of beta = -0.26 (n = 14, r = 0.98). The pH dependence of the binding of two particularly potent beta-glucosidase inhibitors was also examined. 1-Deoxynojirimycin (1,5-dideoxy-1,5-imino-D-glucitol) has a pH-corrected Ki = 6.5 microM, and D-glucono-1,5-lactam has a pH-corrected Ki = 29 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Chlamydomonas cell wall: characterization of the wall framework

The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.     

Cloning and characterization of the gene for the '19 kDa' antigen of Mycobacterium bovis

Monoclonal antibody CMA134.1 reacted with a protein antigen of apparent molecular mass 22 kDa from Mycobacterium bovis and Mycobacterium tuberculosis, and with an apparently 24 kDa antigen of Mycobacterium kansasii, but not with other mycobacteria or related species. This antibody was used to screen a gene library of M. bovis in lambda gt11 and identified a recombinant clone that expressed a protein with an apparent molecular mass of 19-20 kDa. Gene expression occurred from the lac promoter in lambda gt11, but used an unidentified vector promoter, possibly that of the replication primer RNA, in the final plasmid construct. The sequence of an 840 bp fragment was determined and shown to code for a product of 15 kDa. This sequence is identical to that, independently determined, of a gene from M. tuberculosis, usually referred to as the 19 kDa antigen. The reasons for the apparent size discrepancies are discussed.     

Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens.

Products of the virB operon are proposed components of a membrane-associated T-DNA transport apparatus in Agrobacterium tumefaciens. Here we identified the virB10 gene product and raised specific antiserum to the protein. While the virB10 reading frame contains two potential ATG translation start sites located 32 codons apart, we found that only the downstream ATG was required for efficient VirB10 synthesis. Cellular localization studies and analysis of translational fusions with the Escherichia coli alkaline phosphatase gene (phoA) indicated that VirB10 was anchored in the inner membrane and contained a periplasmic domain. This work also demonstrated the utility of alkaline phosphatase as a reporter for secreted proteins in A. tumefaciens. Several high-molecular-weight forms of VirB10 were observed after treatment of A. tumefaciens whole cells or inner membranes with protein cross-linking agents, suggesting that VirB10 exists as a native oligomer or forms an aggregate with other membrane proteins. These results provide the first biochemical evidence that a VirB protein complex is membrane associated in A. tumefaciens.     

Programmed factor binding to simian virus 40 GC-box replication and transcription control sequences

Nuclear footprinting revealed a temporal program involving factor binding to the repetitive GC-box DNA elements present in the simian virus 40 regulatory region. This program specified ordered and directional binding to these tandem regulatory sequences in vivo during the late phase of infection. The program was interrupted by the DNA replication inhibitor aphidicolin or by inactivation of the viral replication factor simian virus 40 T antigen, suggesting a link between viral DNA replication and new factor binding. Measurements of DNA accumulation in viruses lacking either the distal or proximal halves of the GC-box region suggested that the region has a dual role in replication control. Overall, the data point to important relationships between DNA replication and factor binding to the GC-box DNA, a multifunctional regulatory region.     

Prostaglandins E1 and E2 enhance the stimulation of superoxide release by 1-oleoyl-2-acetylglycerol from human neutrophils

Superoxide release from human neutrophils was stimulated either by receptor activation (using fMet-Leu-Phe) or by activating, independently, each of the two pathways considered to be involved in signal transduction--calcium mobilization (using the ionophore, A23187) and protein kinase C activation (using phorbol myristate acetate or 1-oleoyl-2-acetylglycerol). Prostaglandin E1 (3 X 10(-5) M) decreased fMet-Leu-Phe-stimulated superoxide release, had no effect on superoxide release stimulated by A23187, or by phorbol myristate acetate, and markedly enhanced the superoxide release stimulated by 1-oleoyl-2-acetylglycerol. Similar enhancement was obtained with prostaglandin E2.     

Enhanced yeast immobilization by nutrient starvation

Saccharomyces uvarum NRRL Y1347 cells were immobilized in a porous support. Cell loadings of up to 600 mg dry cell/g support or 70 mg dry cell/cm³ support were obtained. Starvation in a marine environment increased the adhesion strength of immobilized cells.