Patterns of early and late discharges in somatotopically identified precentral neurons in awake monkeys in response to somatic inputs.

Extracellular recordings were made of single neurons in precentral cortex of awake monkeys. These neurons were somatotopically identified with respect to their responses to inputs from single joints or their somatic surround. Many of these neurons exhibited early (less than 50 ms) and late (greater than 50 ms) discharges in response to flexion or extension torques delivered about the wrist. With the monkey in a mode requiring opposition to the injected torque, all responsive neurons showed a parallel excitatory or inhibitory modification in the early and late discharges. This was true both for cells identified as wrist (flexion-extension) neurons and those identified as nonwrist (flexion-extension) neurons. These findings indicate that the reflex and voluntary components of percentral discharge invariably show a congruent functional response to a torque disturbance, for this particular instruction set.     

Branched chain amino acid aminotransferase isoenzymes of Pseudomonas cepacia

Pseudomonas cepacia grew rapidly using a mixture of all three branched chain amino acids as carbon source, but failed to use individual branched chain amino acids as sole carbon source. Extracts of bacteria grown on branched chain amino acids had between 2- and 3-fold higher levels of -ketoglutarate-dependent branched chain amino acid aminotransferase activity than extracts of glucose-grown bacteria. The increase in enzyme activity was due to the presence of a second aminotransferase not detected in extracts of glucose-grown bacteria. The enzyme, which presumably plays a role in branched chain amino acid degradation, had an apparent molecular weight (mol. wt.) of 75,000. The other aminotransferase was formed constitutively and apparently functions in synthesis of branched chain amino acids. It was more stable than the 75,000 mol.wt. enzyme, and was purified to homogeneity and found to be a 180,000 mol.wt. oligomer containing 6 subunits of approximately 30,000 mol.wt. Antiserum prepared against the purified enzyme inhibited its activity but failed to influence the activity of the 75,000 mol.wt. aminotransferase, suggesting that the two isoenzymes are encoded by different genes.     

Uridine diphosphate galactose 4-epimerase: nucleotide and 8-anilino-1-naphthalenesulfonate binding properties of the substrate binding site.

Escherichia coli UDP-galactose 4-epimerase in its native form (epimerase.NAD) binds 8-anilino-1-naphthalenesulfonate (ANS) at one tight binding site per dimer with a dissociation constant of 25.9 +/- 2.1 micrometer at pH 8.5 and 27 degrees C. This appears to be the substrate binding site, as indicated by the fact that ANS is a kinetically competitive reversible inhibitor with a Ki of 27.5 micrometer and by the fact that ANS competes with UMP for binding to the enzyme. Upon binding at this site the fluorescence quantum yield of ANS is enhanced 185-fold, and its emission spectrum is blue shifted from a lambdamax of 515 to 470.nm, which suggests that the binding site is shielded from water and probably hydrophobic. Competitive binding experiments with nucleosides and nucleotides indicate that nucleotide binding at this site involves coupled hydrophobic and electrostatic interactions. The reduced form of the enzyme (epimerase.NADH) has no detectable binding affinity for ANS. The marked difference in the affinities of the native and reduced enzymes for ANS is interpreted to be a manifestation of a conformational difference between these enzyme forms.     

A simple dichromatic densitometer for repeated measurements of cardiac output in small animals.

The construction of a simple device for continuous monitoring and recording of plasma indocyanine green concentration in small animals is described. The apparatus is designed to hold a vascular bed such as the ear or the omentum and to transilluminate it with white light from a fiber optic bundle. The transmitted light is divided by a dichroic mirror. Appropriate filters select a narrow band of frequencies from each segment. There are two photovoltaic cells, one sensitive to the dye and the other insensitive to it. Both are approximately equally reactive to changes in transilluminence caused by changes in blood content or hematocrit and are relatively insensitive to changes in oxygen saturation of the transilluminated blood. Reproducible estimates of cardiac output have been obtained with the injection of 300 mug indocyanine green in a volume of 20 mul with the densitometer applied to the small intestine or to the ear of the animal. The device responds linearly to increasing plasma indocyanine green concentrations and can be calibrated with less than 0.1 ml of blood.     

Immobilization of lymphocytes at surfaces by lectins

Phytohemagglutinin (PHA) and Concanavalin A (ConA) cause normal chicken lymphocytes to adhere to glass and plastic surfaces. Pokeweed mitogen (PWM) does not cause adherence. The effect of ConA was studied in detail. The reaction begins within 15 min at 25 °C and proceeds to completion by 2 h. It is independent of pH and resembles in this respect the spontaneous adherence which can occur in protein-depleted suspensions of chicken lymphocytes. It is distinguished from spontaneous adherence by conducting the reaction in 1% or more serum protein; high concentrations exert a slight restraint, which can be overcome by increasing the concentration of ConA. The reaction is slightly greater at high cell concentrations, is inhibited by 3 mM sodium cyanide, and is effectively blocked by 3 mM iodoacetamide and the α-methyl- -glycosides of glucose and mannose. The reaction is not affected by 2-deoxy- -glucose or N-acetyl glucosamine. Adherent lymphocytes detach when the lectin solution is replaced with lectin-free saline; they readhere when reexposed to ConA or to alloantibody directed against lymphocyte surface antigen. At low concentrations of ConA the large lymphocytes of the bursa of Fabricius adhere more rapidly than the small lymphocytes of the blood and thymus. Mouse lymph node lymphocytes adhere in the same manner as small chicken lymphocytes.     

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

Specific conversion of s4 U to U in Escherichia coli tRNA by iodate oxidation.

The minor nucleoside 4-thiouridine in Escherichia coli tRNA is transformed selectively to uridine by iodate oxidation at acidic pH. The four major nucleotides were found to be inert under these conditions. The iodate oxidation appears to be more specific than the previous conversion methods reported, and has the advantage that it does not affect the chargeability of most tRNA.     

Simultaneous tissue profiling of eicosanoid and endocannabinoid lipid families in a rat model of osteoarthritis

We describe a novel LC method for the simultaneous and quantitative profiling of 43 oxylipins including eicosanoids, endocannabinoids, and structurally related bioactive lipids with modified acyl groups. The LC-MS/MS method uses switching at a defined time between negative and positive electrospray ionization modes to achieve optimal detection sensitivity for all the lipids. The validated method is linear over a range of 0.01–5 nmol/g (0.1–50 nmol/g for 2-arachidonoyl glycerol) with intra- and interday precision and accuracy between 1.38 and 26.76% and 85.22 and 114.3%, respectively. The method successfully quantified bioactive lipids in different tissue types in the rat, including spinal cord, dorsal root ganglia (DRGs), knee joint, brain, and plasma. Distinct regional differences in the pattern of lipid measured between tissue types were observed using principle component analysis. The method was applied to analyze tissue samples from an established preclinical rat model of osteoarthritis (OA) pain and showed that levels of 12-hydroxyeicosatetraenoic acid were significantly increased in the OA rat knee joint compared with controls, and that 15-hydroxyeicosatetraenoic acid was significantly increased in the DRGs in the model of OA compared with controls. The developed LC-MS/MS method has the potential to provide detailed pathway profiling in tissues and biofluids where the disruption of bioactive oxylipins may be involved in disease states.