A mutation in VP4 defines a new step in the late stages of cell entry by poliovirus.

During the entry of poliovirus into cells, a conformational transition occurs within the virion that is dependent upon its binding to the cell surface receptor. This conformational rearrangement generates an altered particle of 135S, results in the extrusion of capsid protein VP4 and the amino terminus of VP1 from the virion interior, and leads to the acquisition of membrane-binding properties by the 135S particle. Although the subsequent fate of VP4 is unknown, its apparent absence from purified 135S particles has long suggested that VP4 is not directly involved during virus entry. We report here the construction by site-specific mutagenesis of a nonviable VP4 mutant that upon transfection of the cDNA appears to form mature virus particles. These particles, upon interaction with the cellular receptor, undergo the 135S conformational transition but are defective at a subsequent stage in virus entry. The results demonstrate that the participation of VP4 is required during cell entry of poliovirus. In addition, these data indicate the existence of additional stages in the cell entry process beyond receptor binding and the transition to 135S particles. These post-135S stages must include the poorly understood processes by which nonenveloped viruses cross the cell membrane, uncoat, and deliver their genomes into the cytoplasm.     

A kinetic model for the deactivation of pyruvate decarboxylase (PDC) by benzaldehyde

To provide further understanding of the biotransformation of benzaldehyde to L-phenylacetyl carbinol (L-PAC), an intermediate in L-ephedrine production, a kinetic model has been developed for the deactivation of pyruvate decarboxylase (PDC) by benzaldehyde. The model confirms that deactivation is first order with respect to benzaldehyde concentration and exhibits a square root dependency on time. The model covers the range of benzaldehyde concentrations 100–300 mM, as it has been shown previously that 200 mM benzaldehyde can produce L-PAC concentrations up to 190 mM (28.6 g/L) using partially purified PDC from Candida utilis.     

Pseudomonas aeruginosa B-band O-antigen chain length is modulated by Wzz (Ro1).

The wbp gene cluster, encoding the B-band lipopolysaccharide O antigen of Pseudomonas aeruginosa serotype O5 strain PAO1, was previously shown to contain a wzy (rfc) gene encoding the O-antigen polymerase. This study describes the molecular characterization of the corresponding wzz (rol) gene, responsible for modulating O-antigen chain length. P. aeruginosa O5 Wzz has 19 to 20% amino acid identity with Wzz of Escherichia coli, Salmonella enterica, and Shigella flexneri. Knockout mutations of the wzz gene in serotypes O5 and O16 (which has an O antigen structurally related to that of O5) yielded mutants expressing O antigens with a distribution of chain lengths differing markedly from that of the parent strains. Unlike enteric wzz mutants, the P. aeruginosa wzz mutants continued to display some chain length modulation. The P. aeruginosa O5 wzz gene complemented both O5 and O16 wzz mutants as well as an E. coli wzz mutant. Coexpression of E. coli and P. aeruginosa wzz genes in a rough strain of E. coli carrying the P. aeruginosa wbp cluster resulted in the expression of two populations of O-antigen chain lengths. Sequence analysis of the region upstream of wzz led to identification of the genes rpsA and himD, encoding 30S ribosomal subunit protein S1 and integration host factor, respectively. This finding places rpsA and himD adjacent to wzz and the wbp cluster at 37 min on the PAO1 chromosomal map and completes the delineation of the O5 serogroup-specific region of the wbp cluster.     

Membrane potentials, electrolyte contents, cell pH, and some enzyme activities of fibroblasts

The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was -10.2 +/- 0.20 mV (n = 390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3',5'-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10(-5) to 10(-4) M), hydrocortisone (10(-7) to 10(-6) M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl- concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [14C] dimethyloxazolidine-2, 4-dione, and 3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activities of Na+, K+-, HCO3(-)-, and Ca++, Mg++-ATPases in these cultured cells were 19.0 +/- 2.1, 13.6 +/- 2.1, and 6.6 +/- 1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, K+, Cl-, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl-, and H+ are actively transported out of the cells and K+ into the cells.     

Proteolytic modifications of the carboxyl-terminal region of H-2Kk

Conditions were established for the generation of limited proteolysis products from purified H-2Kk in high yield (greater than 70%). Chymotrypsin, trypsin, or papain treatment in buffer containing Nonidet P-40 resulted in removal of discrete segments from the H-2 heavy chain without detectable alteration of the beta 2-microglobulin. The Mr = 47,400 heavy chain was converted to products with Mr = 44,200, 42,800, or 40,600 by treatment with chymotrypsin, trypsin, or papain, respectively. Papain digestion removed both the hydrophilic carboxyl terminus and the hydrophobic regions. The size, detergent binding properties, and products resulting from subsequent papain treatment demonstrated that chymotrypsin or trypsin removed segments of the hydrophilic carboxyl-terminal region of the heavy chain while leaving the hydrophobic (membrane-spanning) and glycosylated NH2-terminal regions intact. Chymotrypsin and trypsin caused rapid and extensive degradation of the H-2Kk heavy chain when treatment was done in buffer containing deoxycholate, suggesting that the protein undergoes partial, but readily reversible, denaturation in this detergent. This may account for the elution of H-2K and D antigens from monoclonal antibody affinity columns by deoxycholate-containing buffers.     

The stacking of chloroplast thylakoids: Quantitative analysis of the balance of forces between thylakoid membranes of chloroplasts,and the role of divalent cations

An analysis is made of the van der Waals dispersion attractive forces and electrostatic repulsive forces between the grana thylakoid membranes of chloroplasts. These forces are determined for negatively charged surfaces with a pK_a value of 4.7 for a bulk pH of 7.0 with a range of mono- and divalent cation concentrations and intermembrane spacing in the range 10 to 80 Å. For equilibrium under dark conditions, it is concluded that either there is extensive electrostatic binding of divalent cations (Mg²⁺) to the negatively charged membrane groups (phospholipid, sulfolipid, and protein carboxyl), or a redistribution of these groups between stacked and unstacked regions must be invoked.