Species-dependent features of Dogiel type II neurones in the mammalian enteric nervous system.

Although autonomic gastrointestinal reflex movements, which occur in all mammalian species, have been described almost a century ago, little was known on the mechanisms underlying this behaviour. Recently, however, intrinsic primary afferent neurones, functioning as the first relay in the reflex arches embedded in the intestinal wall, have been identified in the guinea pig ileum. In guinea pig, such neurones display a Dogiel type II morphology and behave electrophysiologically as slow AHP neurones. In other gastrointestinal regions, in both guinea pig and rat, Dogiel type II cells are also encountered, but the strong correlation with slow AHP neuronal features seems less strict. In large mammals, a correlation of the cellular morphology with intracellular el ectrophysiological recordings has only been obtained in the pig small intestine. Surprisingly, in these experiments aberrant electrophysiological behaviour of Dogiel type II neurones is even more striking since the majority of these cells display electrophysiological features considered typical of S neurones. Furthermore, in those rare cases in which a slow afterhyperpolarization (AHP) could be recorded in porcine Dogiel type II cells, its amplitudes were negligible. This has led us to the conclusion that the differences in electrophysiological behaviour of neurones with comparable morphology in different species are most probably due to the modulating influence of the neurotransmitter substances present. This seems to be the most likely hypothesis in view of the considerable differences in neurotransmitter content of neurones with comparable functions throughout the species.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Association of mRNA and eIF-2 alpha with the cytoskeleton in cells lacking vimentin

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2 alpha to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2 alpha shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.     

An improved method for light- and electron microscopial studies of nematode/fungal interactions

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys ologospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.     

A Kdp-like,high-affinity,K+-translocating ATPase is expressed during growth of Rhodobacter sphaeroides in low potassium media

Cells of the purple non-sulphur bacterium Rhodobacter sphaeroides express a high-affinity K⁺ uptake system when grown in media with low K⁺ concentrations. Antibodies againts the catalytic KdpB protein or the whole KdpABC complex of Escherichia coli crossreact with a 70.0 kDa R. sphaeroides protein that was expressed only in cells grown in media with low K⁺ concentrations. In membranes derived from R. sphaeroides cells grown with low K⁺ concentrations (induced cells), a high ATPase activity could be detected when assayed in Tris-HCl pH 8.0 containing 1 mM MgSO₄. This ATPase activity increased upon addition of 1 mM KCl from 166 to 289 mol ATP hydrolysed x min^-1 x g protein^-1 (1.7-fold stimulation). The K⁺-stimulated ATPase activity was inhibited approximately 93% by 0.5 mM vanadate but hardly by N,N-dicyclohexylcarbo-diimide (DCCD). These results indicate that the inducible K⁺-ATPase in R. sphaeroides resembles the Kdp K⁺-translocating ATPase of Escherichia coli. This Kdp-like transport system is also expressed in R. capsulatus and Rhodospirillum rubrum during growth in media with low K⁺ concentrations suggesting a wide distribution of this transport system among phototrophic bacteria.Abbreviations electrical potential difference across the cytoplasmic membrane - pH pH difference across the cytoplasmic membrane - BSA bovine serum albumine - PAGE polyacrylamide gel electrophoresis - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - PMSF phenyl-methyl-sulfonyl fluoride - DCCD N,N-dicyclohexylcarbodiimide - AIB 2--aminoisobutyric acid - TMG methyl--d-thiogalactopyranoside     

Trans replication and high copy numbers of wheat dwarf virus vectors in maize cells.

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Three-dimensional organization and topographical features of the myenteric plexus (Auerbach) in the porcine small intestine: scanning electron microscopy after enzymatic digestion and HCl-hydrolysis

The present scanning electron microscopical (SEM) study was initiated to visualize the surface topography of Auerbach's plexus in the ileum of the pig. After enzymatic digestion of the connective-tissue components of the tunica muscularis and the tunica serosa followed by glutaraldehyde fixation, HCl-hydrolysis and stripping off either the longitudinal or circular smooth muscle layer, the three-dimensional architecture and topographical features of the myenteric plexus can clearly be observed. In this way, ganglia, primary, secondary and tertiary strands, and single nerve fibres can be demonstrated. Individual nerve cells, which are incompletely covered by glial cells and by remnants of the basal lamina, can be recognized in the centre and periphery of the ganglia and adjacent to primary and secondary nerve strands.     

The synthesis of some polyether bridged diphosphines and their reaction with Rh(COD)acac

The polyether bridged diphosphines,

(n = 1,2) have been prepared in 60–70% yield by reduction of the corresponding diphosphinedioxides with Si₂Cl₆ or (i-Bu)₂AlH. These diphosphinedioxides have been prepared in 75–90% yield by reaction of two equivalents of the appropriate

with one equivalent of di- and triethylene glycol ditosylate.In general, reaction between the diphosphines, Rh(COD)acac and HClO₄ gives a mixture of species, cis-[Rh(COD)($\text{PP}$)] [ClO₄] being the main complex. This complex reacts with CO to η³-trans- [$\text{Rh(CO)(}\text{PO}$$\text{P}$)] [ClO₄].