Mobilization of a Sym plasmid from a fast-growing cowpea Rhizobium strain.

A large Sym plasmid from a fast-growing cowpea Rhizobium species was made mobilizable by cointegration with plasmid pSUP1011, which carries the oriT region of RP4. This mobilizable Sym plasmid was transferred to a number of Rhizobium strains, in which nodulation and nitrogen fixation functions for symbiosis with plants of the cowpea group were expressed.     

Distribution of calcium during interphase and mitosis as observed by ion microscopy

The ion microscope, based on secondary ion mass spectrometry, has been used to demonstrate the distribution of calcium in the root tip cells of two plant species, Allium cepa and Vicia faba. Interphase nuclei showed higher intensities of calcium than cytoplasm, while nucleoli exhibited higher calcium intensities than the rest of the nucleoplasm. The chromosomes showed high intensities of calcium at all stages of mitosis. Calcium was also detected in the cell plate and phragmoplast region of dividing cells. It appears that during prophase calcium concentrates in the condensing chromosomes, and during telophase it is transferred to nucleoli. These observations suggest that chromosomes may serve as a reservoir of calcium during mitosis.     

Absence of female-specific protein in the hemolymph of stable fly Stomoxys calcitrans (L.) (Diptera: Muscidae)

Changes, during the reproductive cycle, in fat body, hemolymph, and ovarian proteins of the stable fly Stomoxys calcitrans were characterized quantitatively and qualitatively using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein content of all three tissues increased after blood feeding. Fat body protein increased first, followed by hemolymph and ovarian proteins. SDS-PAGE failed to identify vitellogenin in both female hemolymph and fat body samples. No single protein or group of proteins predominated at any stage of the reproductive cycle. Comparisons between male and female stable fly hemolymph and fat body proteins failed to detect female-specific proteins. Female-specific proteins, however, were detected in the hemolymph of four other species of Diptera.     

The interaction of human erythrocyte band 3 with cytoskeletal components

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of ³⁵SO₄. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.     

Isolation and characterization of three new classes of transformation-deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination

A bifunctional enzyme, L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and EC 1.1.1. . . , respectively), was discovered in cells of Rhodopseudomonas sphaeroides Y, which accounts for the ability of this organism to grow on L-(+)-malate. The enzyme was purified 110-fold to homogeneity with a yield of 51%. During the course of purification, including ion-exchange chromatography and preparative gel electrophoresis, both enzyme activities appeared to be in association. The ratio of their activities remained almost constant [1:10, L-(+)-tartrate dehydrogenase/D-(+)-malate dehydrogenase (decarboxylating)] throughout all steps of purification. Analysis by polyacrylamide gel electrophoresis revealed the presence of a single protein band, the position of which was coincident with both L-(+)-tartrate dehydrogenase and D-(+)-malate dehydrogenase (decarboxylating) activities. The apparent molecular weight of the enzyme was determined to be 158,000 by gel filtration and 162,000 by ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 38,500, indicating that the enzyme consisted of four subunits of identical size. The isoelectric point of the enzyme was between pH 5.0 and 5.2. The enzyme catalyzed the NAD-linked oxidation of L-(+)-tartrate as well as the oxidative decarboxylation of D-(+)-malate. For both reactions, the optimal pH was in a range from 8.4 to 9.0. The activation energy of the reaction (delta Ho) was 71.8 kJ/mol for L-(+)-tartrate and 54.6 kJ/mol for D-(+)-malate. NAD was required as a cosubstrate, and optimal activity depended on the presence of both Mn2+ and NH4+ ions. The reactions followed Michaelis-Menten kinetics, and the apparent Km values of the individual reactants were determined to be: L-(+)-tartrate, 2.3 X 10(-3) M; NAD, 2.8 X 10(-4) M; and Mn2+, 1.6 X 10(-5) M with respect to L-(+)-tartrate; and D-(+)-malate, 1.7 X 10(-4) M; NAD, 1.3 X 10(-4); and Mn2+, 1.6 X 10(-5) M with respect to D-(+)-malate. Of a variety of compounds tested, only meso-tartrate, oxaloacetate, and dihydroxyfumarate were effective inhibitors. meso-Tartrate and oxaloacetate caused competitive inhibition, whereas dihydroxyfumarate caused noncompetitive inhibition. The Ki values determined for the inhibitors were, in the above sequence, 1.0, 0.014, and 0.06 mM with respect to L-(+)-tartrate and 0.28, 0.012, and 0.027 mM with respect to D-(+)-malate.     

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.     

Ultraviolet-Stimulated KHCO(3) Efflux from Rose Cells: Regulation of Cytoplasmic pH

Suspension-cultured cells of Rosa damascena that have been irradiated with ultraviolet light (254 nanometers, 2.1 × 10⁴ joules per square meter) rapidly lose K⁺ and HCO₃⁻ ions to the medium. If the HCO₃⁻ is derived from respiratory CO₂ inside the cell, then loss of HCO₃⁻ should be accompanied by an acidification of the cytoplasm. Estimates of the pH of control and ultraviolet-irradiated cells by ³¹P-nuclear magnetic resonance spectroscopy indicated that, following irradiation, the pH of both cytoplasm and vacuole dropped by 0.2 to 0.3 units. This change was not as great as was predicted from the observed HCO₃⁻ loss. Analysis of nitrogenous compounds in the cell suggested that reduction of nitrate and synthesis of γ-aminobutyric acid absorbed some of the protons formed by the synthesis and dissociation of bicarbonate.     

Comparison of vascular hemodynamics in experimental models of microvascular anastomoses

Blood flow was measured in the canine saphenous artery using electromagnetic flowmetry. Significant increase in blood flow was noted after occlusion of the distal femoral artery. However, after raising a saphenous island flap there was no significant change in the blood flow before and after distal femoral artery occlusion. The flap peripheral resistance and blood flow were compared after end-to-end and end-to-side anastomosis and no statistical difference was noted.     

Status of reduced glutathione in primary cultures of rat hepatocytes and the effect on conjugation of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide

The intracellular level of reduced glutathione (GSH) and GSH conjugation have been investigated in primary cell cultures of hepatocytes isolated from control rats, phenobarbitone (PB) and 3-methylcholanthrene (MC) treated rats. The data demonstrate that in all cell cultures the GSH concentrations show a triphasic pattern: (i) within 1 h of culture an initial marked decrease to 50% of the levels found in fresh hepatocytes; (ii) recovery of GSH concentrations to above the levels observed in fresh cells. This occurs after 6 h in culture with control cells and after 10-24 h with cells from either PB or MC treated rats and was most prominent in cells from PB-treated rats. (iii) A slow decline to between 30 and 40 nmol GSH/mg protein from 24 to 96 h in culture. Synthesis of GSH was slower in cultured cells from PB treated rats and this was confirmed by the resynthesis rates when diethylmaleate (DEM) was used to deplete GSH. The formation of GSH conjugates with racemic 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was measured in control cells in suspension and after 3 and 24 h in culture. Despite the decrease in GSH concentrations observed between 1 and 4 h after culture, the conjugation rates were not decreased.