Antigenic similarity between cytoplasmic tetrodotoxin-sensitive protein and neuronal membrane proteins

Membrane proteins with a molecular weight of 290, 180, and 55 kDa were isolated using immunosorbent attached to sepharose and rabbit antibodies to cytoplasmic tetrodotoxin-sensitive protein from beef brain gray matter. A technique used for research into voltage-dependent sodium channels was applied to reconstruction of these proteins and investigation of toxin-dependent sodium flows through the lipoprotein membrane. Findings are interpreted as evidence of the similarity between cytoplasmic tetrodotoxin-sensitive protein and that of sodium channels at the cell membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev; A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 485–489, July–August, 1989.     

Interactions of three sequentially expressed genes control temporal and spatial specificity in Aspergillus development

Aspergillus nidulans brlA, abaA, and wetA form a dependent pathway that regulates asexual reproductive development. The order in which these genes are expressed determines the outcome of development. Expression of brlA in vegetative cells leads to activation of abaA and wetA, cessation of vegetative growth, cellular vacuolization, and spore formation. By contrast, expression of abaA in vegetative cells does not result in conidial differentiation but does lead to activation of brlA and wetA, cessation of vegetative growth, and accentuated cellular vacuolization. brlA, abaA, and wetA act individually and together to regulate their own expression and that of numerous other sporulation-specific genes. We propose that the central pathway controlling development is largely autoregulatory. The timing and extent of expression of the regulatory genes and their targets are determined as development proceeds by intrinsically controlled changes in the relative concentrations of regulatory gene products in the various conidiophore cell types.     

A restricted cytoplasmic region of IL-2 receptor beta chain is essential for growth signal transduction but not for ligand binding and internalization

The functional, high affinity form of interleukin-2 receptor (IL-2R) is composed of two receptor components, the IL-2R alpha (p55) and IL-2R beta (p70-75) chains. Unlike the IL-2R alpha chain, the IL-2R beta chain contains a large cytoplasmic domain that shows no obvious tyrosine kinase motif. In the present study, we report the establishment of a system in which the cDNA-directed human IL-2R beta allows growth signal transduction in a mouse pro-B cell line. This system enabled us to identify a unique region within the cytoplasmic domain of the human IL-2R beta chain essential for ligand-mediated signal transduction. We also demonstrate that certain cytoplasmic deletion mutants in the IL-2R beta chain, although deficient in signal transduction, can still form high affinity IL-2R in conjunction with endogenous mouse IL-2R alpha chain; the mutants are still able to internalize the ligand as well.     

Comparisons of eight commercial on-farm milk progesterone tests

To evaluate eight commercial on-farm milk progesterone kits, milk samples (50 ml each of foremilk and postmilk strippings) were collected during the estrous cycle from 10 cycling Holstein cows for 24 consecutive days. Relative concentrations of progesterone were classified as low or high by comparison with standard progesterone samples supplied with each kit. The concentration of progesterone in each milk sample was determined by radioimmunoassay (RIA). Accuracy of classification into low or high levels by commercial tests was determined by the percentage of similarity with RIA values using discriminant analysis. Accuracy of the eight tests ranged from 89.0 to 98.9% for low progesterone, 74.8 to 85.6% for high progesterone, and 80.3 to 87.3% for all samples (n = 238). The percentage of fat in milk or an interaction of the percentage of milkfat by day of estrous cycle influenced commercial test results for all tests except Accufirm and Calfcheck. Progesterone levels, estimated by the test-kits, were low from 1.5 +/- 0.5 to 2.8 +/- 0.9 days before estrus (X +/- SEM) and until 4.0 +/- 0.6 to 5.9 +/- 1.3 days after estrus. These data support the principle that a single low progesterone sample cannot be used to determine proper timing of insemination. All eight commercial kits can be used to determine accurately the relative concentrations of progesterone in milk samples.     

The diagnosis of short penis as a cause of impotentia coeundi in bulls

Short penis condition was diagnosed as the cause of impotentia coeundi in 10 bulls, aged 2.5 to 5 yr. The diagnosis was based on observation of service attempts, measurement of the extended penis, and elimination of other causes of impotence. Measurements of the penis were made under general inhalation anesthesia or pudendal nerve block. These measurements were then compared with those of 10 control bulls, matched for age and breed and having no history of impotence; the latter measurements were likewise obtained under general anesthesia, pudendal nerve block or sedation. with propionyl promazine. Similar measurements were obtained from 10 yearling bulls under propionyl promazine sedation. Measurements obtained under general anesthesia or pudendal nerve block in the same bull were usually similar and repeatable, while phenothiazine tranquillization produced incomplete and variable relaxation of the retractor penis muscles. The dimension best correlated with impotence due to short penis was the distance from the tip of the extended penis to the preputial orifice in its resting position. In 10 bulls in which short penis was diagnosed, this distance was 10 to 22 cm, while in 10 control bulls with no history of impotence it was 25 to 42 cm. The distances from the tip of the extended penis to the preputial reflection (fornix) and to the neck of the scrotum were also shorter in affected than in control bulls. Although observation of service ability remains the cornerstone of diagnosis of short penis, a presumptive diagnosis can be made if penile protrusion of less than 25 cm can be obtained in an adult bull under general anesthesia or pudendal nerve block. Phenothiazine tranquillization is suitable for screening examinations but not for definitive diagnosis.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.     

Use of benzodiazepines to manipulate the circadian clock regulating behavioral and endocrine rhythms

Extensive studies have now been carried out demonstrating that the systemic administration of the short-acting benzodiazepine, triazolam, can have pronounced effects on both behavioral and endocrine circadian rhythms. For example, three daily injections of triazolam can phase-advance the circadian rhythm of pituitary luteinizing hormone release and locomotor activity by about 2-3 h in female hamsters maintained in constant light. Triazolam has also been found to facilitate the rate of reentrainment of the activity rhythm following an 8-hour advance or delay in the light-dark cycle. Limited studies with other short-acting benzodiazepines indicate that the effects of triazolam on the circadian system of hamsters can be generalized to this class of drugs. Recent studies in humans indicate that treatment with triazolam can alter the time it takes for human endocrine rhythms to become reentrained following an 8-hour delay in the sleep-wake and light-dark cycle. Such findings raise the possibility that short-acting benzodiazepines may prove useful in reducing the symptoms associated with 'jet-lag' and rotating shift-work schedules as well as in the treatment of various physical and mental illnesses that have been associated with a disorder of biological timekeeping.     

Production of trichothecene and non-trichothecene mycotoxins by Fusarium species isolated from maize in Minnesota

Eighty-two cultures of Fusarium species isolated in 1986 from moldy maize in Minnesota were each cultured on rice for 4 weeks and found to produce the following mycotoxins: F. graminearum isolates, deoxynivalenol (DON, 4–225 g/g), 3-acetyldeoxynivalenol (3-ADON, 2–4g/g), 15-acetyldeoxynivalenol (15-ADON, 1–35 g/g) and zearalenone (ZEA, 5–4350 g/g); F. moniliforme, fusarin C (detectable amounts to 1000 g/g); F. mòniliforme, F. oxysporum, F. proliferatum and F. subglutinans isolates, moniliformin (15–6775 g/g); F. moniliforme, F. proliferatum, and F. subglutinans isolates, fusaric acid (detectable amounts). Other mycotoxins screened for in each rice sample and not detected were T-2 toxin, HT-2 toxin, neosolaniol, T-2 tetraol, nivalenol, fusarenon-X, scirpenols, alpha and beta trans-zearalenols, wortmannin, and fusarochromanone. The rat feeding bioassay indicated that other, unidentified toxins may be present.     

Two distinct O-methyltransferases in aflatoxin biosynthesis

The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G1 were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (O-methyltransferase I) and then the C-7-OH groups are methylated to produce O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited O-methyltransferase I activity but not that of O-methyltransferase II. Furthermore, these O-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium.