Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

In vivo development and pathogenicity of Ascosphaera proliperda (Ascosphaeraceae) to the alfalfa leafcutting bee, Megachile rotundata

First instar larvae of the leafcutting bee, Megachile rotundata, were fed on either artificial or natural provisions containing spores of Ascosphaera proliperda. Two isolates were used as a source of inocula: one originated from in vitro isolates obtained while culturing what was thought to be pure spores of A. aggregata, the second originated from in vitro cultures from Denmark. Histological and scanning electron microscopy studies revealed that the spores germinated in the gut lumen and the developing hyphae invaded all tissues, after which they penetrated through larval integument and began the sexual phase of the life cycle aerially. Virtually all fungus-exposed larvae developed symptoms of disease regardless of source of inoculum, type of provision, and spore dose (1.5 × 10³ to 3 × 10⁶) per insect. It was concluded that the fungus was pathogenic to the alfalfa leafcutting bee under laboratory conditions and future studies should be conducted to determine its etiology, cross infectivity, and natural distribution in other bee taxa.     

Pathology of larval Eustrongylides in the rabbit

Three fishermen from Maryland who swallowed live bait-minnows developed severe abdominal pain within 24 hr; 2 required abdominal surgery. Larvae of the nematode Eustrongylides sp. were found in the peritoneal cavity of both (Guerin et al., 1982). In the current study, the lesions produced by Eustrongylides larvae were investigated in New Zealand white rabbits. None of these exhibited any signs of clinical illness; however, postmortem examination within 24 hr of inoculation revealed that larvae had migrated through the walls of the esophagus and stomach and viable larvae were recovered from the pleural and peritoneal cavities as well as from gastric contents. Necropsies performed at different intervals of time postinoculation showed that the migrating larvae had produced multi-focal peritonitis and multiple granulomata in the liver.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk^– cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.     

Effect of pulp pH on solid phase fermentation of fodder beets for fuel ethanol production

Summary The pH of fodder beet pulp was varied to see how this affected solid phase fermentation by yeast. The process is for fuel ethanol production. When pulp was adjusted to a pre-inoculation pH of 3.0–3.5, ethanol yields (78–85% of theoretical, averaging 8.9% v/v) and fermentation efficiencies (97–99%) were greatest, the fermentation time was the shortest (30–39 h) and no bacterial contamination occurred.     

Neonatal,maternal, and intrapartum factors and their relationship to cord-and maternal-plasma trace-element concentration

The study was carried out to examine the relationship of neonatal sex, birthweight, maternal parity, hemoglobin status, gestational age at term, and duration of labor to cord- and maternal-plasma zinc, copper, and iron levels, measured by atomic absorption spectrophotometry. Significant differences were observed between maternal-and neonatal-cord plasma concentrations of the different trace elements at term. Maternal parity had no significant influence on the distribution of plasma trace elements, except in the case of maternal plasma iron levels. However, maternal hemoglobin status was observed as an important covariate of maternal zinc and iron levels at term. Neonatal birthweight was observed to be an index of maternal plasma zinc status at term. On the other hand, although gestational age at term had no significant influence on maternal-plasma trace-element concentrations, it was observed to influence neonatal-cord plasma levels. Long durations of labor (≥18 h) were associated with relatively lower, but not significant, maternal-plasma iron levels, whereas neonatal-cord-plasma iron levels seemed to show sex differences.     

Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90°C

A novel type of bacterium has been isolated from various geothermally heated locales on the sea floor. The organisms are strictly anaerobic, rod-shaped, fermentative, extremely thermophilic and grow between 55 and 90°C with an optimum of around 80°C. Cells show a unique sheath-like structure and monotrichous flagellation. By 16S rRNA sequencing they clearly belong to the eubacteria, although no close relationship to any known group could be detected. The majority of their lipids appear to be unique in structure among the eubacteria. Isolate MSB8 is described as Thermotoga maritima, representing the new genus Thermotoga.Dedicated to Otto Kandler on the occasion of his 65th birthday Present addresses: University of South Dakota, Vermillion, USA; University of Illinois, Urbana, USA; Universität für Bodenkultur, Wien, Austria     

A Rapid Chromosome-Mapping Method for Cloned Fragments of Yeast DNA

A rapid and generally applicable method is described for mapping a cloned yeast DNA segment to the chromosome(s) from which it originated. The method is based upon the recent finding that the integration into a yeast chromosome of a segment of the 2 mu plasmid DNA results, in heterozygous diploids, in the specific loss of genetic information from the chromosome into which the 2 mu DNA was integrated (Falco et al. 1982). After verification of the accuracy of the method using several genes whose position was known in advance, the method was used to locate the yeast actin gene, which lies on the left arm of chromosome VI, about 50 cM distal to CDC4.