Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of P 0 mRNA in myelin-forming Schwann cells

Summary A biotinylated P ₀ glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelinforming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain P ₀ mRNA. Interestingly, P ₀ mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

Risk of AIDS related complex and AIDS in homosexual men with persistent HIV antigenaemia.

One hundred and ninety eight men seropositive for human immunodeficiency virus (HIV) antibody and 58 HIV antibody seroconverters were studied for an average of 19.3 (SEM 0.5) months to assess the relation between HIV antigenaemia and the risk of developing the acquired immune deficiency syndrome (AIDS) and AIDS related complex. Forty (20.2%) of the 198 HIV antibody seropositive men were antigen positive at entry and remained so during follow up. Eight (13.8%) of the 58 HIV antibody seroconverters and 20 (12.7%) of the remaining 158 HIV antibody seropositive men became antigen positive during follow up, resulting in an end point attack rate for HIV antigenaemia of 14.3%. AIDS related complex was diagnosed in 25 (15.8%) of the HIV antigen negative men and in 14 (20.7%) of the HIV antigen positive men. AIDS was diagnosed in 15 men, resulting in an end point attack rate for AIDS of 23.9% in the HIV antigen positive group and 1.3% in the antigen negative group. HIV antibody seropositive men without symptoms but with persistent HIV antigenaemia are at increased risk of developing AIDS and AIDS related complex.     

Pyridoxal 5'phosphate binding site of Escherichia coli beta cystathionase and cystathionine gamma synthase comparison of their sequences

The phosphopyridoxyl peptides of beta cystathionase and cystathionine gamma synthase of Escherichia Coli were identified after reduction, carboxymethylation and proteolysis of the holoenzymes. Their comparison with those obtained from rat liver gamma cystathionase (Fearon, C.W., Rodkey, J.A. and Abeles R.H. 1982. Biochemistry 21 3790-3794.) showed a high degree of homology between the three PLP binding sites with the presence of the tripeptide sequence: Thr-Lys(Pxy)-Tyr in their structure. This homology suggests that these enzymes of methionine metabolism have probably the same origin.     

A relationship between the inhibition of heparan sulfate and chondroitin sulfate synthesis and the inhibition of molting by selenate in the hemipteran Rhodnius prolixus

The insect Rhodnius prolixus synthesizes heparan sulfate and chondroitin sulfate after a blood meal containing [35S]-inorganic sulfate. A 40 to 80% inhibition of heparan sulfate synthesis was obtained when the meal was supplemented with 10(-5) and 10(-4) M sodium selenate respectively. Likewise an inhibition of the molting in the order of 30 to 60% was observed when the insects were fed with blood containing 10(-5) and 10(-4) M selenate respectively. The insects after a subsequent meal without selenate molted normally. Except for the inhibition of the ecdysis no gross physiological or morphological changes could be observed in the insects. Based on these and other findings the possible role of sulfated glycosaminoglycans in the control of cell growth is discussed.     

On the shoulders of giants: the discovery of atrial natriuretic factor

Investigations culminating at the beginning of this century clearly established that the cardiac muscle cell (cardiocytes) is differentiated for excitation, conduction, and contraction. All of the physiology and pathophysiology of the heart was developed subsequently based on this concept. However, morphological investigations in the mid 1950s suggested a secretory function for mammalian atrial cardiocytes. These cells contain storage granules, the specific atrial granules, which resemble granules found in polypeptide hormone-producing cells. The development of techniques for the study of these granules using a combined biochemical-morphological approach during the 1970s defined their general chemical nature and their behaviour under different experimental conditions. Because the number of atrial granules change dramatically following upsets of water and electrolyte balance, atrial muscle extracts were tested for effects on kidney function. In 1981, it was reported that atrial extracts contain a natriuretic factor (ANF) capable of inducing massive diuresis, increases in hematocrit, and lowering of blood pressure. It was demonstrated soon thereafter that ANF is stored within specific atrial granules. More recent work has defined ANF as a polypeptide hormone that appears to modulate or antagonize the renin-angiotensin-aldosterone system. Current work attempts to define the physiological and pathophysiological role for ANF as well as possible therapeutic uses.     

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G2 + M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram. The cohort can be described as the recruitment of cells from a pre-existing G0 compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G2 + M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.     

Feeding responses to solanaceous allelochemicals by larvae of the tobacco hornworm, Manduca sexta

Feeding responses of the oligophagous tobacco hornworm to allelochemicals prevalent in their host plants were determined in food choice-tests using filter paper discs laced with a test solution or water (control). Six solanaceous alkaloids, tomatine, tomatidine, solanine, solanocapsine, atropine and nicotine, were tested and only tomatine and solanocapsine were found to influence preference behavior. Solanocapsine (5 mM) deters feeding whereas tomatine (1 mM) stimulates feeding slightly. No synergistic effect of either tomatine or tomatidine with sucrose was found.The responses to tomatine are affected by previous feeding experience. Tomatine slightly stimulates feeding in larvae reared on tomato (Lycopersicon esculentum), but slightly deters feeding in larvae reared on Jerusalem cherry (Solanum pseudocapsicum). Such induced preference is absent for the other alkaloids tested, which indicates that these alkaloids do not by themselves induce preferences for the plants containing them.The non-alkaloid allelochemicals, chlorogenic acid, rutin, and 2-tridecanone also influenced food choice behavior. Chlorogenic acid is slightly stimulatory at its natural concentration (1mM), but strongly deterrent at higher concentrations. Rutin stimulates feeding in a concentration-dependent manner. Its activity must be due to the glycosylated structure, because both the aglycone (quercetin) and the sugar moiety (rutinose) are neutral. Removal of the glucose part of rutin, as in quercitrin, results in feeding deterrent activity. 2-Tridecanone is neutral at its concentration in cultivated tomato (1 mM), but strongly deterrent and toxic at higher concentrations. Preference behavior is not affected by solanesol, GABA, and a mixture of host plant compounds stimulatory for anothe solanaceous-specific feeder, the Colorado potato beetle (Leptinotarsa decemlineata).We conclude that the prevalent solanaceous alkaloids and other allelochemicals tested do not play important roles in food selection of the tobacco hornworm, although some may make small contributions.

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Résumé Des experiences de choix de chenilles oligophages de M. sexta ont été réalisees avec des disques de papier filtre imbiles d'eau ou de solutions des substances allélochimiques dominantes dans les plantes consommées. Sur les six alcaloïdes de solanées examinés: tomatine, tomatidine, solanine, solanocapsine, atropine et nicotine, seuls la tomatine et la solanocapsine ont influé sur le choix; la solanocapsine (5 mM) empêche la prise de nourriture, tandis que la tomatine (1 mM) la stimule légèrement. Aucun effet synergique de la tomatine ou de la tomatidine n'a été observé en présence de sucrose.La réponse à la tomatine est modifiée par la prise de nourriture antérieure. Elle stimule légèrement l'alimentation de chenilles élevées sur tomates (Lycopersicon esculentum), mais dissuade légèrement les chenilles élevées sur Solanum pseudocapsicum. II n'y a pas d'action induite semblable avec les autres alcalïdes examinés, ce qui indique que ces alcaloïdes ne peuvent pas induire par eux-mêmes de préférences pour les plantes qui les contiennent.Des substances allélochimiques non-alcaloïdes: acide chlorogénique, rutine, et 2-tridécanone, influent aussi sur le comportement de choix alimentaire. L'acide chlorogénique est légèrement stimulant à sa concentration naturelle (1 mM), mais fortement dissuasif aux concentrations supérieures. La rutine stimule la prise de nourriture en fonction de sa concentration. Son activité doit être due à sa structure glucosylate, puisqu'aussi bien l'aglycone (quercitine) que la moiteé sucrée (rutinose) sont neutres. La suppression de la partie glucose de la rutine, comme dans le cas de la quercitine, a un effet dissuasif. A sa concentration dans la tomate cultivée (1 mM), le 2-tridécanone est neutre, mais il est fortement dissuasif et toxique à des concentrations supérieures.Le comportement de choix n'est pas modifié par le solanésol, le GABA, et par un mélange de composés végétaux stimulant un consommateur spécifique de solanées, comme le doryphore (Leptinotarsa decemlineata).Nous pouvons conclure que les principaux alcaloïdes et autres substances allélochimiques des solanées que nous avons examinés n'interviennent pas d'une façon importante, mais peuvent avoir une influence secondaire, dans les choix alimentaires de Manduca sexta.

     

Effects of adriamycin on respiratory chain activities in mitochondria from rat liver, rat heart and bovine heart. Evidence for a preferential inhibition of complex III and IV

The inhibition of respiratory chain activities in rat liver, rat heart and bovine heart mitochondria by the anthracycline antibiotic adriamycin was measured in order to determine the adriamycin-sensitive sites. It appeared that complex III and IV are efficiently affected such that their activities were reduced to 50% of control values at 175 +/- 25 microM adriamycin. Complex I displayed a minor sensitivity to the drug. Of the complex-I-related activities tested, only duroquinone oxidation appeared sensitive (50% inhibition at approx. 450 microM adriamycin). Electron-transfer activities catalyzed by complex II remained essentially unaltered up to high drug concentrations. Of the activities measured for this complex, only duroquinone oxidation was significantly affected. However, the adriamycin concentration required to reduce this activity to 50% exceeded 1 mM. Mitochondria isolated from rat liver, rat heart and bovine heart behaved essentially identical in their response to adriamycin. These data support the conclusion that, in these three mitochondrial systems, the major drug-sensitive sites lie in complex III and IV. Cytochrome c oxidase and succinate oxidase activity in whole mitochondria exhibited a similar sensitivity towards adriamycin, as inner membrane ghosts, suggesting that the drug has direct access to its inner membrane target sites irrespective of the presence of the outer membrane. By measuring NADH and succinate oxidase activities in the presence of exogenously added cytochrome c, it appeared that adriamycin was less inhibitory under these conditions. This suggests that adriamycin competes with cytochrome c for binding to the same site on the inner membrane, presumably cardiolipin.     

An interstitial duplication of the X chromosome in a male allows physical fine mapping of probes from the Xq13-q22 region

Summary An insertional translocation into the proximal long arm of the X chromosome in a boy showing muscular hypotony, growth retardation, psychomotor retardation, cryptorchidism, and Pelizaeus-Merzbacher disease (PMD) was identified as a duplication of the Xq21–q22 segment by employing DNA probes. With densitometric scanning for quantitation of hybridization signals, 15 Xq probes were assigned to the duplicated region. Analysis of the duplication allowed us to dissect the X-Y homologous region physically at Xq21 and to refine the assignments of the loci for DXYS5, DXYS12, DXYS13, DXS94, DXS95, DXS96, DXS111, and DXS211. Furthermore, we demonstrated the presence of two different DXYS13, and DXS17 alleles in genomic DNA of our patient, suggesting that the duplication resulted from a meiotic recombination event involving the two maternal X chromosomes.     

Molecular and classical genetic analyses of his-3 mutants of Neurospora crassa. I. Tests for allelic complementation and specific revertibility

A collection of 81 his-3 mutants of Neurospora crassa was analyzed in assays for allelic complementation and specific revertibility. In these studies, the linearity of the complementation map of the his-3 cistron (Webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterns, or non-complementing. In the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment with the chemical mutagens N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino) acridine dihydrochloride, nitrous acid or hydroxylamine. The frequency of revertible mutants among the non-polarized complementing mutants was 96% (45/47), and 79% (15/19) for the polarized complementing and 79% (11/14) for the non-complementing mutants. The results of these classical genetic assays for allelic complementation and specific revertibility suggest a correlation between complementation pattern and presumptive genetic alterations at the molecular level among his-3 mutants similar to that found with ad-3B mutants induced by nitrous acid (Malling and de Serres, 1967), ethyl methanesulfonate (Malling and de Serres, 1968), or ultraviolet (Kilbey et al., 1971).