The bacterial flora of the intestine of Ascaris suum and 5-hydroxytryptamine production

Representative facultative anaerobes of the bacterial flora from the intestine of female Ascaris suum were isolated and identified. The number of bacteria in the intestine was approximately 4 X 10(9) per g wet weight of intestine. Seventeen of 19 of the isolated colonies were found to secrete 5-hydroxytryptamine in culture. Holding A. suum in an antibiotic-containing medium did not affect the levels of 5-hydroxytryptamine in the worm, which were 231 +/- 14 ng/g in antibiotic-media as compared to 250 +/- 16 ng/g in control media. This implied that the bacteria may not be contributing to the level of 5-hydroxytryptamine in the tissues of A. suum.     

Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii

Candida yeasts rapidly form peroxisomes of simple function and composition when grown on methanol. Because the induction is both massive and rapid, this system may be useful for a detailed dissection of peroxisomal biogenesis. We report procedures to express peroxisomal proteins in cells and spheroplasts of Candida boidinii to stabilize peroxisomes in a lysate of spheroplasts and to obtain an enriched peroxisomal fraction. With these techniques we have been able to study the assembly of alcohol oxidase, a homo-octomeric flavoprotein, into the organelle in vivo. The primary translation product of alcohol oxidase comigrates on sodium dodecyl sulfate-polyacrylamide gels with the mature subunit. Pulse-chase experiments indicate that the newly synthesized monomer of alcohol oxidase has a half-life of about 20 min in intact cells and 13 min in spheroplasts before conversion to octomer. The monomer first appears in a high speed supernatant of a lysate of spheroplasts and then chases into a purified peroxisomal fraction before or during its octomerization. There is no detectable intermediary organelle involved in this process.     

Arrangement of MP26 in lens junctional membranes: Analysis with proteases and antibodies

Summary The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21–22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2–3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately fourfifths of the primary sequence “protected” by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.     

A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast

We have made deletions of the HIS4 5' noncoding region in vitro and inserted these deletions into the yeast genome by transformation. Deletions that extend from -588 to -235 have no detectable effects on either promoter or regulatory functions. Deletions that extend to -138 affect promoter function, but are still regulated by the general control of amino acid biosynthesis. A deletion that extends to -136 cannot derepress HIS4 mRNA in response to the general control. This deletion removes all copies of the sequence 5'-TGACTC-3', which appears at positions -194, -182 and -138 in strains without the deletion. The importance of at least one copy of this repeat for regulation of HIS4 is shown by the reappearance of this sequence in revertants of the -136 deletion that have regained the regulatory response. The fact that deletion of this sequence leads to the inability to derepress suggests that HIS4 is under positive control.     

Survey for mitochondrial-desmosome complexes in differentiating epithelia

Summary Mitochondria are frequently found to be closely associated with the plaques of desmosomes in a variety of columnar or cuboidal epithelia of fetal or early postnatal mammals (mouse, rat, human being). The organs in which mitochondrial-desmosome complexes were found include stomach, small intestine, pancreas, kidney, epididymis, seminal vesicle, coagulating gland, thyroid gland. The association has not been observed in simple squamous epithelium (vascular endothelium). Mitochondria lie quite close to desmosomes in the stratum spinosum of stratified squamous mucous epithelium of fetal animals and also to axo-dendritic synapses in still poorly differentiated central nervous system. Mitochondria have also been detected close to attachment sites in ectoderm of the early frog gastrulae. Here there is as yet no visible plaque material.We suggest that the mitochondria may provide energy or some chemical for the formation of the plaque. This hypothesis does not explain why the complexes are not found in poorly differentiated epithelia from older animals.Dedicated to Professor Berta V. Scharrer on her 60th birthday, with affection and admiration. — This study was supported by U.S.P.H.S. research grants NB-05219 and GM-10757 from the National Institutes of Health.     

Systematic placement of the basidiomycetous yeastCystofilobasidium lari-marini comb. nov. as predicted by rRNA nucleotide sequence analysis

Based on similarities in basidial morphology and nucleotide sequences of the V3 variable region in the large sub-unit ribosomal RNA, the yeastLeucosporidium lari-marini is considered phylogenetically related to the genusCystofilobasidium. Therefore the new combinationCystofilobasidium lari-marini is proposed.     

Plasticity in cultured carotid body chemoreceptors: Environmental modulation of GAP-43 and neurofilament

In this study we use dissociated cell cultures of the rat carotid body to investigate the adaptive capabilities of endogenous oxygen chemoreceptors, following chronic stimulation by various environmental factors. These oxygen chemoreceptors are catecholamine-containing glomus cells, which derive from the neural crest and resemble adrenal medullary chromaffin cells. Using double-label immunofluorescence, we found that chronic exposure of carotid body cultures to hypoxia (2% to 10% oxygen) caused a significant fraction of tyrosine hydroxylase-positive (TH+) glomus cells to acquire detectable immunoreactivity for growth-associated protein gap-43. The effect was dose-dependent and peaked around an oxygen tension of 6%, where approximately 30% of glomus cells were GAP-43 positive. Treatment with agents that elevate intracellular cyclic adenosine monophosphate (cAMP) (i.e., dibutyryl cAMP or forskolin) also markedly stimulated GAP-43 expression. Since hypoxia is known to increase cAMP levels in glomus cells, it is possible that the effect of hypoxia on GAP-43 expression was mediated, at least in part, by a cAMP-dependent pathway. Unlike hypoxia, however, cAMP analogs also stimulated neurofilament (NF 68 or NF 160 kD) expression and neurite outgrowth in glomus cells, and these properties were enhanced by retinoic acid. Nerve growth factor, which promotes neuronal differentiation in related crest-derived endocrine cells, and dibutyryl cGMP were ineffective. Thus, it appears that postnatal glomus cells are plastic and can express neuronal traits in vitro. However, since hypoxia stimulated GAP-43 expression, without promoting neurite outgrowth, it appears that the two processes can be uncoupled. We suggest that stimulation of GAP-43 by hypoxia may be important for other physiological processes, e.g., enhancing neurotransmitter release or sensitization of G-protein–coupled receptor transduction. © 1995 John Wiley & Sons, Inc.     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.