Effect of acetazolamide on cerebral blood flow and capillary patency.

This study investigated the effects 2 h after administration of acetazolamide on cerebral blood flow and the pattern of cerebral capillary perfusion. Arterial blood pressure, heart rate, arterial blood gases, and pH were recorded in two groups of rats along with either regional cerebral blood flow or the percentage of capillary volume per cubic millimeter and number per square millimeter perfused as determined in cortical, thalamic, pontine, and medullary regions of the brain. Blood pressure, heart rate, and arterial PCO2 were not significantly different between the rats receiving acetazolamide (100 mg/kg) and the controls. Arterial blood pH was significantly lower in the acetazolamide rats. Blood flow increased significantly in the cortical (+ 102%), thalamic (+ 89%), and pontine (+ 88%) regions receiving acetazolamide. In control rats, approximately 60% of the capillaries were perfused in all of the examined regions. The percentage of capillaries per square millimeter perfused was significantly greater in the cortical (+ 52%), thalamic (+ 49%), and pontine (+ 47%) regions of acetazolamide rats compared with controls. In the medulla the increases in blood flow and percentage of capillaries perfused were not significant. Thus in the regions that acetazolamide increased cerebral blood flow, it also increased the percentage of capillaries perfused.     

Structural studies of the lysozyme coded by the pneumococcal phage Cp-1. Conformational changes induced by choline

The CPL-1 lysozyme coded by the pneumococcal phage Cp-1 has been overproduced in Escherichia coli under the control of a modified lipoprotein lactose promoter. This result has provided the conditions to analyse the CPL-1 secondary structure by circular dichroism (CD). The CD spectra recorded in the far-ultraviolet region showed, at neutral pH, two minima at 210 nm and 230 nm and a shoulder at 217 nm, whereas two bands at 260 nm and 295 nm were observed in the near-ultraviolet region. It has been estimated, by using the CDPROT program, that the protein is composed of 19% alpha-helix, 32% beta-sheet, 28% beta-turn and 21% random coil. Minor changes in the CD spectra were detected either when the pH was varied over 6-10 or when the ionic strength was increased to 1 M NaCl. Choline, a well known modulator of the enzyme activity that is present in the pneumococcal cell wall, induced remarkable changes in the intensities of the bands at 210, 230 and 295 nm, with the appearance of an unusual positive band at 225 nm. The conformational change was reversible and correlated with the competitive inhibitory effect of choline on the lysozyme activity, supporting, by a new and direct experimental approach, the basic role of choline in the recognition of the cell wall substrate. The analyses of the secondary structure prediction and the CD data reported here are compatible with the two-domain structure of CPL-1 reinforce our hypothesis that the C-terminal region is directly involved in the binding of the enzyme to the pneumococcal teichoic and lipoteichoic acids.     

MULTIVARIATE PHENOTYPIC DIFFERENTIATION AMONG BOTTLENECK LINES OF THE HOUSEFLY

Multivariate phenotypic differentiation in eight morphometric traits was examined in bottleneck lines of the housefly initiated with one, four, or 16 pairs of flies from a natural outbred population. Differentiation was assessed using a Mahalanobis' distance metric in units of additive genetic variance and covariance estimated from the ancestral population (i.e., generalized genetic distance). This distance metric was partitioned into contributions of size and shape to total distance. Bottleneck lines of all sizes diverged significantly from the ancestral line, but the direction of these shifts differed among the lines of different initial founding size. Those populations founded with single pairs diverged from the ancestral line mostly in shape; the 16-pair lines differentiated almost entirely in size, and the four-pair lines were intermediate in the relative contribution of shape to differentiation from the control. Bottlenecks serve to alter the genetic relationships among traits within the derived populations and in doing so could promote speciation by permitting differentiation of the populations along evolutionary trajectories less accessible to the base population.     

Sequence of the Streptococcus pneumoniae bacteriophage HB-3 amidase reveals high homology with the major host autolysin.

We have sequenced a DNA fragment containing the pneumococcal bacteriophage HB-3 hbl gene, which codes for the phage lytic amidase. A remarkable nucleotide similarity (87.1%) between the lytA gene, coding for the pneumococcal amidase, the major autolysin of Streptococcus pneumoniae, and the hbl gene was found. This similarity completely disappeared outside the open reading frames coding for both amidases. The hbl gene transformed amidase-deficient strains of S. pneumoniae to the wild-type phenotype, and Southern blotting experiments provided evidence for recombination between donor and recipient genes. A comprehensive evaluation of these and previous results on the peptidoglycan hydrolases of S. pneumoniae and its bacteriophages suggested that recombination mechanisms participate in the evolution of the genes coding for these enzymes.     

PHYLOGENETIC IMPLICATIONS OF DIFFERENCES IN NUMBER OF PLASTID PHOSPHOGLUCOSE ISOMERASE ISOZYMES IN NORTH AMERICAN COREOPSIS (ASTERACEAE: HELIANTHEAE: COREOPSIDINAE)

Enzyme electrophoresis was employed to ascertain the number of loci encoding plastid phosphoglucose isomerase (PGI) in species representing all sections of North American Coreopsis. Several species from each of the closely related genera Bidens, Coreocarpus, Cosmos, and Thelesperma were also examined. Species in nine of the 11 sections of North American Coreopsis have two isozymes for plastid PGI, and nearly all species examined in the four other genera also have two (one species has three) isozymes. Since most diploid vascular plants have one plastid PGI isozyme, a gene duplication probably occurred in an ancestor that is common to Coreopsis and the other four genera. That is, two isozymes represent the ancestral number for Coreopsis. The two sections (Electra and Anathysana) apparently lacking the duplication are closely related woody plants restricted largely to Mexico. One gene encoding plastid PGI ostensibly was silenced in a common ancestor of these two sections. This is concordant with other data suggesting a close relationship between the two sections, i.e., they appear to represent a monophyletic group. The electrophoretic data also indicate that 1) the enigmatic monotypic section Silphidium is more closely related to eastern North American sections and not derived from section Electra; and 2) section Anathysana is not ancestral to the three California sections Leptosyne, Pugiopappus, and Tuckermannia; rather, it represents a terminal element closely related to and possibly derived from section Electra.     

Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135

A number of Pseudomonas species have been identified which accumulate a polyhydroxyalkanoate containing mainly 3-hydroxydecanoate monomers from sodium gluconate as the sole carbon source. One of these, Pseudomonas sp. strain NCIMB 40135, was further investigated and shown to accumulate such a polyhydroxyalkanoate from a wide range of carbon sources (C₂ to C₆); however, when supplied with octanoic acid it produced a polyhydroxyalkanoate containing mainly 3-hydroxyoctanoate monomers. Polymer synthesis occurred in batch culture after cessation of growth due to exhaustion of nitrogen. In continuous culture under nitrogen limitation up to 16.9% (wt/wt) polyhydroxyalkanoate was synthesized from glucose as the carbon source. The monomer units are mainly of the R-(−) configuration. Nuclear magnetic resonance studies confirmed the composition of the polymer. Differential scanning calorimetry suggested that the solvent-extracted polymer contained a significant proportion of crystalline material. The weight-average molecular weight of the polymer from glucose-grown cells was 143,000.     

The association of FAD with the cytochrome b−245 of human neutrophils

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b₋₂₄₅ at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b₋₂₄₅ the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b₋₂₄₅ and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b₋₂₄₅ of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b₋₂₄₅ and support a scheme for electron transport thus: [Formula: see text]     

Effects of temperature on the size and shape of Escherichia coli cells

Two substrains of Escherichia coli B/r were grown to steady-state in batch cultures at temperatures between 22 and 42° C in different growth media. The size and shape of the cells were measured from light and electron micrographs and with the Coulter channelizer. The results indicate that cells are shorter and somewhat thicker at the lower temperatures, especially in rich growth media; cell volume is then slightly smaller. A lower temperature was further found to increase the relative duration of the constriction period. The shapes of the cell size distributions are indistinguishable, indicating that the pattern of growth of the cells is the same at all temperatures. The adaptation of the cells to a temperature shift lasted several generations, indicating that the morphological effects of temperature are mediated by the cell's physiology.     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Summary As a part of a microfluorometric investigation of the nucleoproteins of nuclei whose chromatin displays varying degrees of condensation, a comparison was made of mouse small thymocyte and hepatocyte nuclei stained with the acidic dye, brilliant sulfaflavine, at pH 2.8. These estimates of total protein content were compared with measurements obtained in similarly stained nuclei after extraction either with 0.4 N H₂SO₄ to remove all histones or with 0.35 M NaCl to remove nucleoplasmic proteins and some loosely bound non-histone chromosomal proteins. Treatment with 5% TCA at 60°C was used to remove nucleic acids and to reverse the effects of formaldehyde fixation. In all instances, the fluorescence of 2c hepatocyte nuclei greatly exceeded that of similarly treated thymocyte nuclei. While extraction with 0.4 N H₂SO₄ resulted in reductions of as much as 75% of the total fluorescence of small thymocyte nuclei, the losses of fluorescence in 2c hepatocyte nuclei amounted to only 20–30%. Nevertheless, the absolute values of fluorescence lost in both types of nuclei were very similar. After extraction in 0.35 M NaCl, thymocyte nuclei displayed slightly greater fluorescence than control thymocyte nuclei, while the total fluorescence of hepatocyte nuclei declined. In hepatocyte nuclei extracted with TCA, with and without treatment with 0.35 M NaCl, two populations of diploid nuclei were apparent: one corresponding to parenchymal cell nuclei and the other comprised of non-parenchymal cell nuclei. Only single diploid populations were visible in acid-extracted material. The ratios of 4c2c, 8c4c, and 8c2c hepatocyte nuclei in control, acid-extracted, and NaCl-extracted groups were generally lower than the expected 224 values. These results indicate that total nuclear histones may be estimated microfluorometrically by computing the difference between acid-extracted and unextracted preparations treated in otherwise equivalent ways. In addition, despite very similar absolute losses of fluorescence after removal of histones in thymocyte and 2c hepatocyte nuclei, the proportion of total protein ascribable to histones is much greater in thymocyte nuclei than in 2c hepatocyte nuclei — or, conversely, the percentage of total protein attributable to non-histone proteins is much greater in 2c hepatocyte nuclei than in thymocyte nuclei.     

Effect of angiotensin II and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E₂ (PGE₂) and 6 keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE₂ and 6-keto-PGF_1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE₂ and 6-keto-PGF_1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI₂ is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE₂ and 6-keto-PGF_1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.