Galactose Transport in Saccharomyces cerevisiae III. Characteristics of Galactose Uptake in Transferaseless Cells: Evidence Against Transport-Associated Phosphorylation

The characteristics of the inducible galactose transport system in bakers' yeast were studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaseless cells. Transferaseless cells transport galactose at the same initial rate as wild-type cells and accumulate a mixture of free galactose and galactose-1-phosphate. The addition of ¹⁴C-labeled galactose to cells preloaded with unlabeled galactose and galactose-1-phosphate results in a higher rate of labeling of the free-sugar pool than of the galactose-1-phosphate pool. These results support other evidence that galactose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and that phosphorylation is an intracellular event after uptake of the free sugar.     

Specificity of gentamicin accumulation by rat renal cortex.

The accumulation of gentamicin by rat renal cortex $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ was not inhibited by probenecid, tetraethylammonium, cephalosporins nor α-aminoisobutyric acid, but was significantly blocked by other aminoglycosides (neomycin, tobramycin and kanamycin). The data suggest that specific binding sites for aminoglycosides are present on the surface or in cells of the renal proximal tubule.     

Determination of free and esterified cholesterol in human serum by high-performance liquid chromatography and optical activity detection

A separation and detection scheme is presented for the determination of free, esterified and total cholesterol in human serum. Separation is accomplished by reversed-phase high-performance liquid chromatography and the eluate is monitored by the laser-based optical activity detector. The method is simple, accurate and has the advantage of specificity and selectivity when compared with the many methods commonly used.     

Intermolecular triplex formation distorts the DNA duplex in the regulatory region of human papillomavirus type-11.

A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts.     

Physiological characteristics of NG2-expressing glial cells

Antibodies against the chondroitin sulfate proteoglycan NG2 label a subpopulation of glial cells within the CNS, which have a small cell body and thin radiating processes. Physiological recordings from these small cells in acute brain slices have revealed that they possess unique properties, suggesting that they may comprise a class of glial cells distinct from astrocytes, oligodendrocytes, or microglia. NG2-expressing glial cells (abbreviated as “NG2 cells” here) have a moderate input resistance and are not dye- or tracer-coupled to adjacent cells. They express voltage-gated Na⁺, K⁺and Ca²⁺conductances, though they do not exhibit regenerative Na⁺or Ca²⁺action potentials due to the much larger K⁺conductances present. In addition to voltage-gated conductances, they express receptors for various neurotransmitters. In the hippocampus, AMPA and GABA_Areceptors on these cells are activated by release of transmitter from neurons at defined synaptic junctions that are formed with CA3 pyramidal neurons and GABAergic interneurons. These rapid forms of neuron-glial communication may regulate the proliferation rate of NG2 cells or their development into mature oligodendrocytes. These depolarizing inputs may also trigger the release of neuroactive substances from NG2 cells, providing feedback regulation of signaling at neuronal synapses. Although the presence of Ca²⁺permeable AMPA receptors provides a pathway to link neuronal activity to Ca²⁺dependent processes within the NG2 cells, these receptors also put these cells at risk for glutamate-associated excitotoxicity. This vulnerability to the sustained elevation of glutamate may underlie ischemic induced damage to white matter tracts and contribute to cerebral palsy in premature infants.     

Cyclic GMP-dependent and cyclic AMP-dependent protein kinases,protein kinase modulators and phosphodieterases in arteries and veins of dogs Distribution and effects of arteriovenous fistula and arterial occlusion

Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.     

Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies

The ornithine transcarbamoylase catalyzed reaction and its inhibition by L-norvaline have been investigated between pH 5.5 and 10.5. The steady-state turnover rate (kcat) of the enzyme from Escherichia coli increases with pH and plateaus above pH 9. Its change with pH conforms to a single protonation process with an apparent pKa of 7.3. The effect of pH on the apparent Michaelis constant (KMapp) of L-ornithine suggests that this diamino acid in its cationic form is not the substrate. Treating only the zwitterions of ornithine as substrate, the pH profile of the pseudo-first-order rate constant (kcat/KMz) of the reaction is a bell-shaped curve characterized by pKa's of 6.2 and 9.1 and asymptotic slopes of +/- 1. Similar pKa's (6.3 and 9.3) are obtained for the pKi profile of zwitterionic L-norvaline, a competitive inhibitor. The pKi profile further indicates that the alpha-amino group of the inhibitor must be charged for binding. Together, these pH profiles provide sufficient information to suggest that only the minor zwitterionic species of ornithine, H2N(CH2)3CH(NH3+)COO-, binds the enzyme productively. The selection of this substrate form by the enzyme leads to a Michaelis complex in which ornithine is poised for nucleophilic attack. Following such binding, the need for deprotonation of the delta-NH3+ group is avoided, and transcarbamoylation becomes energetically more feasible. Reaction schemes accounting for the effects of pH are proposed for the enzymic reaction.     

Effects of oxygen and chloramphenicol on Frankia nitrogenase activity

The kinetics of asymbiotic nitrogenase activity in three strains of the actinomycete Frankia were studied. Decay rates for enzyme activity were determined by adding chloramphenicol to active acetylene-reducing cells and measuring the time required for all activity to cease. Synthesis rates were measured by bubbling oxygen through actively-reducing cells (which totally destroyed all activity) and then measuring the time required for activity to return to normal. Decay rates (t _1/2) for these three strains were approximately 30 to 40 min. Synthesis rates were slower and initial nitrogenase activities were recorded about 110 min (DDB 011610) or 210 min (DDB 020210 and WgCc1.17) after return to air-equilibrated cultures. Frankia strain WgCc1.17 showed a greater sensitivity to oxygen and nitrogenase activity was totally lost when cells were bubbled only with atmospheric concentrations of oxygen. The results presented here indicate that nitrogenase activity turnover time is relatively rapid, on the order of minutes rather than hours or days. However, regulation of nitrogenase activity will differ from one strain to another and asmmbiotic characterization will be useful for understanding nitrogenase regulation in the bacterial-plant symbiosis.Contribution no. 879 from the Battelle-Kettering Laboratory     

The Identity of Hexokinase Activities from Mitochondrial and Cytoplasmic Fractions of Rat Brain Homogenates

Cytoplasmic hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from the soluble fraction of a rat brain homogenate by a procedure that included a unique affinity elution of the enzyme from Blue Dextran-Sepharose. The purified enzyme was examined with respect to properties in which the impure cytoplasmic enzyme has been reported to differ from the solubilized mitochondrial enzyme. These included the ability to bind to mitochondria, inhibition by quercetin, effect of pH on activity, and kinetics. In all regards the purified mitochondrial and cytoplasmic enzymes appeared identical. In addition, comparative peptide maps after partial proteolysis showed no detectable differences. These results do not support the view that there exist distinct mitochondrial and cytoplasmic forms of hexokinase, the latter being permanently relegated to a cytoplasmic location and unable to participate in a dynamic equilibrium with the mitochondrially-bound enzyme. Alternatives are proposed to explain previous results that had been interpreted as indirect evidence for the existence of a distinct cytoplasmic hexokinase.     

THE NACREOUS WALLS OF SIEVE ELEMENTS IN SEAGRASSES

The nacreous walls of sieve elements occur in seagrasses in all three genera of the family Zosteraceae and the genus Halodule of the family Cymodoceaceae but are absent from another eight seagrass genera belonging to the families Hydrocharitaceae, Cymodoceaceae, and Posidoniaceae. They occur in leaf blades, leaf sheaths, rhizomes, and erect stems but are not present in root tissues. The nacreous wall is uneven along the inner limits reflecting irregular thickness. The wall consists of hemicellulose or pectin and cellulose, but no protein, lignin, or lipid. Ultrastructurally, the wall contains parallel microfibrils or loose fibrils embedded in an amorphous matrix. Open pores occur in sieve plates and branching plasmodesmata are present in enlarged sieve areas. Mitochondria, endoplasmic reticulum, and plastids are also present in these sieve elements.