The demonstration of B cells in pancreatic islets with Orcein

Summary Orcein stains granules in pancreatic islet cells selectively. The localization of Orcein-positive cells within islets differs from that of Grimelius-stained cells, but corresponds to the B cell type differentiated by Aldehyde Fuchsin.Usually there appear to be fewer Orcein-positive cells than Aldehyde Fuchsin-positive ones. This indicates either that Aldehyde Fuchsin is a more sensitive stain for B cells or that Orcein is a more selective stain for a B cell type subpopulation. The rationale of the Orcein reaction in B cells seems to depend on the oxidation of disulphide bonds present in insulin and its precursors rich in cystine.     

Simultaneous expression of a bacteriophage Mu transposase and repressor: a way of preventing killing due to mini-Mu replication

In vitro studies of bacteriophage Mu transposition have shown that the phage-encoded transposase and repressor bind the same sequences on the phage genome. We attempted to test that prediction in vivo and found that Mu repressor directly inhibits transposition. We also found that, in the absence of repressor, constitutive expression of Mu transposition functions pA and pB is lethal in Escherichia coli strains lysogenic for a mini-Mu and that this is the result of intensive replication of the mini-Mu. These findings have important consequences where such mini-Mus are used as genetic tools. We also tested whether in Erwinia chrysanthemi the effect of transposition functions on a resident mini-Mu was the same as in E. coli. We observed that expression of pA alone was lethal in E. chrysanthemi and that a large fraction of the survivors underwent precise excision of the mini-Mu.     

A synthetic strand of cardiac muscle: its passive electrical properties

The passive electrical properties of synthetic strands of cardiac muscle, grown in tissue culture, were studied using two intracellular microelectrodes: one to inject a rectangular pulse of current and the other to record the resultant displacement of membrane potential at various distances from the current source. In all preparations, the potential displacement, instead of approaching a steady value as would be expected for a cell with constant electrical properties, increased slowly with time throughout the current step. In such circumstances, the specific electrical constants for the membrane and cytoplasm must not be obtained by applying the usual methods, which are based on the analytical solution of the partial differential equation describing a one-dimensional cell with constant electrical properties. A satisfactory fit of the potential waveforms was, however, obtained with numerical solutions of a modified form of this equation in which the membrane resistance increased linearly with time. Best fits of the waveforms from 12 preparations gave the following values for the membrane resistance times unit length, membrane capacitance per unit length, and for the myoplasmic resistance: 1.22 plus or minus 0.13 x 10-5 omegacm, 0.224 plus or minus 0.023 uF with cm-minus 1, and 1.37 plus or minus 0.13 x 10-7 omegacm-minus 1, respectively. The value of membrane capacitance per unit length was close to that obtained from the time constant of the foot of the action potential and was in keeping with the generally satisfactory fit of the recorded waveforms with solutions of the cable equation in which the membrane impedance is that of a single capacitor and resistor in parallel. The area of membrane per unit length and the cross-sectional area of myoplasm at any given length of the preparation were determined from light and composite electron micrographs, and these were used to calculate the following values for the specific electrical membrane resistance, membrane capacitance, and the resistivity of the cytoplasm: 20.5 plus or minus 3.0 x 10-3 omegacm-2, l.54 plus or minus 0.24 uFWITHcm-minus 2, and 180 plus or minus 34 omegacm, respectively.     

Electron microscopic and cytochemical study on the role of Golgi elements and plasma membrane of enterocytes in the intestinal lipid transport

Summary Intestinal lipid absorption and transport were investigated in albino rats. The observations point towards the existence of a continuity between plasma membrane invaginations and elements of the Golgi complex on its mature face. They also suggest a segregation of lipid droplets by paired Golgi membranes and plasma membrane invaginations. The following way for lipid transport is deduced: lipid droplets moving inside the smooth endoplasmic reticulum accumulate progressively and are condensed in Golgi cisternae of the forming face. Their limiting membrane ruptures and liberated lipid droplets are segregated by paired Golgi membranes of the mature face or by plasma membrane invaginations. Subsequently the inner of the two segregating membranes disappears while the lipid droplet is moved towards the intercellular space inside a canal communicating with this space. The suggestion is made that the Golgi apparatus is of double origin: one component representing a terminal plication of the endoplasmic reticulum; the second one—a terminal plication of the plasma membrane invagination. This concept explains the ultrastructural and histochemical differences between Golgi membranes of the forming and mature faces of the complex.     

An elaboration on the syn-anti proton donor concept of glycoside hydrolases: electrostatic stabilisation of the transition state as a general strategy

An in silico survey of all known 3D-structures of glycoside hydrolases that contain a ligand in the -1 subsite is presented. A recurrent crucial positioning of active site residues indicates a common general strategy for electrostatic stabilisation directed to the carbohydrate's ring-oxygen at the transition state. This is substantially different depending on whether the enzyme's proton donor is syn or anti positioned versus the substrate. A comprehensive list of enzymes belonging to 42 different families is given and selected examples are described. An implication for an early evolution scenario of glycoside hydrolases is discussed.     

Functional Gene Hybridization Patterns of Terrestrial Ammonia-Oxidizing Bacteria

Abstract The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autotrophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct phylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosospira groups. Hybridization patterns were used to assess the potential of functional probes in non-PCR-based molecular analysis of natural AAO populations and their activity. The objective of this study was to obtain an overview of functional gene homologies by hybridizing probes derived from N. europaea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with 32P to Southern blots containing genomic DNA from four Nitrosospira representatives. Probes were specific for genes encoding ammonia monooxygenase (amoA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy). These probes produced hybridization signals, at low stringency (30 degreesC), with DNA from each of the four representatives; signals at higher stringency (42 degreesC) were greatly reduced or absent. The hybridization signals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional genes in the ammonia oxidation pathway have diverged between the Nitrosomonas and Nitrosospira groups. The hao probe produced the most consistent hybridization intensities among the Nitrosospira representatives, suggesting that hao sequences would provide the best probes for non-PCR-based molecular analysis of terrestrial AAOs. Since N. europaea can also denitrify, an additional objective was to hybridize genomic DNA from AAOs with probes for Pseudomonas genes involved in denitrification. These probes were specific for genes encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and Nitrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in their mechanisms and capacities for denitrification.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Cytidine monophosphate as substrate for the electron microscopic visualization of alkaline phosphatase activity

Summary Since in histochemical reactions the liver alkaline phosphatase discloses only low activity with respect to beta-glycerophosphate, the authors have replaced the latter by cytidine monophosphate. With this substrate they obtained much better results: greater sensibility permitting to reduce the incubation time, and much more precise localization of the enzyme activity, at the ultrastructural level. Details of the method are given with a discussion of its specifity. The method was applied on liver tissue of normal albino rats and animals of the same strain with ligated common bile duct.This study was supported by a grant from the Fonds voor Wetenschappelijk Geneeskundig Onderzoek.     

Tooth Retrospective Dosimetry Using Electron Paramagnetic Resonance: Influence of Irradiated Dental Composites

In the aftermath of a major radiological accident, the medical management of overexposed individuals will rely on the determination of the dose of ionizing radiations absorbed by the victims. Because people in the general population do not possess conventional dosimeters, after the fact dose reconstruction methods are needed. Free radicals are induced by radiations in the tooth enamel of victims, in direct proportion to dose, and can be quantified using Electron Paramagnetic Resonance (EPR) spectrometry, a technique that was demonstrated to be very appropriate for mass triage. The presence of dimethacrylate based restorations on teeth can interfere with the dosimetric signal from the enamel, as free radicals could also be induced in the various composites used. The aim of the present study was to screen irradiated composites for a possible radiation-induced EPR signal, to characterize it, and evaluate a possible interference with the dosimetric signal of the enamel. We investigated the most common commercial composites, and experimental compositions, for a possible class effect. The effect of the dose was studied between 10 Gy and 100 Gy using high sensitivity X-band spectrometer. The influence of this radiation-induced signal from the composite on the dosimetric signal of the enamel was also investigated using a clinical L-Band EPR spectrometer, specifically developed in the EPR center at Dartmouth College. In X-band, a radiation-induced signal was observed for high doses (25-100 Gy); it was rapidly decaying, and not detected after only 24h post irradiation. At 10 Gy, the signal was in most cases not measurable in the commercial composites tested, with the exception of 3 composites showing a significant intensity. In L-band study, only one irradiated commercial composite influenced significantly the dosimetric signal of the tooth, with an overestimation about 30%. In conclusion, the presence of the radiation-induced signal from dental composites should not significantly influence the dosimetry for early dose assessment.