Protein tyrosine phosphorylation in cardiovascular system

Treatment of rat parotid acinar cells with sodium orthovanadate (an inhibitor of protein tyrosine phosphatase) caused a dose-dependent inhibition of phosphatase activity as measured by the hydrolysis of para nitrophenylphosphate (pNPP). Inclusion of 50 M sodium orthovanadate inin vitro gland cultures prevented the amylase secretion from both untreated control and isoproterenol-stimulated parotid acinar cells. Four different tyrosine-phosphorylated proteins with M_r 40, 45, 70 and 95 kDa, respectively, were identified in secretory granule preparations from rat parotid glands by immunoblot using a monospecific antibody for phosphotyrosine. An increase in the phosphorylation levels of these phosphoproteins was noted in the presence of 50 M sodium orthovanadate, suggesting that a protein tyrosine phosphatase (PTPase) is involved in parotid gland protein dephosphorylation reactions. Using antibody to Syp (a PTPase belonging to class 1D), a major fraction of subcellular activity was found to be associated with secretory granule membranes. These results suggest the possible involvement of a PTPase (Syp) in parotid gland secretory mechanisms.     

Reactions of the vaginal plate and the vaginal epithelium to oestradiol monopropionate given once to 5 or 26-day old rats

Summary Five-day old rats were given a single injection of varying amounts of oestradiol monopropionate (OeM) in propylene glycol (0.5, 5, 30 and 150 g) and 26-day old rats were given a single injection of 5 or 150 g OeM. The rats were sacrificed at varying times after the injection and the changes in the vaginal plate and the vaginal epithelium proper were studied. As indicated by the cornification of the epithelium the reaction to OeM is stronger in the vaginal plate than in the vaginal epithelium proper. The response of the vaginal epithelium to 5 or 150 g OeM is most extensive in the rats injected at 26 days of age. With 150 g OeM a cornification of the vaginal epithelium was obtained in rats injected when 26 days old but not in those injected when 5 days old. The reactions in the vaginal plate were compared with those found previously after injections of testosterone propionate. Acknowledgement. This investigation was supported by grants from the Swedish Medical Research Council (12 X-579-03, K67-14X-571-03) and from the Swedish Cancer Society (67:46).     

The structure and differentiation of granulated metrial gland cells of the pregnant mouse uterus

Summary A study was made with the light and electron microscopes of the granulated metrial gland cells of the decidua basalis of the pregnant mouse uterus, up to day 11 of pregnancy. The granulated metrial gland cells are large, up to 50 in diameter, mono- or binucleate and the glycogen rich cytoplasm typically contains many large glycoprotein granules which may be up to 5 in diameter. Morphological evidence is described in support of a lymphocyte-like cell being the precursor to the granulated metrial gland cell. This differentiation sequence is similar to that already proposed in the rat but differences between the ultrastructure of the mature metrial gland cells of rats and mice were noted.     

Failure of intracerebral BCG cell wall preparation to prevent glioma induction by avian sarcoma virus in rats

Summary Five-day-old Fischer 344 rats were inoculated intracerebrally with BCG cell walls attached to oil (BCG-CW) or with oil emulsion alone. Doses of BCG-CW of 40 g in 0.01 ml, 200 g in 0.05 ml, and 400 g in 0.1 ml were used, with similar volumes of oil emulsion for comparison. Twentyfive days later hydrocephalus, intracerebral granulomas, and arachnoid granulomas were found at autopsy. The frequency and severity of these abnormalities were related to the presence of BCG-CW in the inoculum and the dose administered. There were minimal abnormalities with 40 g BCG-CW.Two microliters of Avian sarcoma virus (ASV) suspension was inoculated intracerebrally into 5-day-old rats to induce intracerebral gliomas. One hour later 40 g BCG-CW in 0.01 ml, 80 g BCG-CW in 0.02 ml, or similar volumes of oil emulsion alone were inoculated intracerebrally in the region of the virus injection site. BCG-CW or oil emulsion alone failed to prevent induction and growth of the ASV-induced brain tumors. Survival curves of the BCG-CW inoculated animals were similar to that obtained for control animals injected with ASV and otherwise untreated. The oil emulsion decreased survival times compared with control animals.     

Mechanisms of Long-Lasting Depression of Synaptic Transmission in the CA1 Region of the Rat Hippocampus

Low-frequency tetanic stimulation (2 sec^-1, 5 min) of Schaffer collaterals (SchC) in superfused slices of the dorsal hippocampus of 12- to 15-day-old rats was demonstrated to evoke homosynaptic long-lasting depression (LLD) of synaptic transmission. The same procedure applied to hippocampal slices of mature (8-week-old or older) rats failed to elicit LLD. Low-frequency tetanic stimulation of the alveus in hippocampal slices, applied under conditions of intensified NMDA glutamate receptor functioning, led to the development of heterosynaptic LLD of synaptic transmission in the SchC–dendrites of the CA1 pyramidal neurons system. Both LLD cases were either absent or weakened when hippocampal slices were treated with a competitive blocker of the NMDA glutamate receptors, D-2-amino-5-phosphonovalerate (50 M). Morphine hydrochloride (10 M), as well as inhibitors of calmodulin and calcineurin (trifluoroperasine and cyclosporin A in concentrations of 1 and 50 M, respectively), interfered with induction of LLD or decreased its intensity. A blocker of the L-type voltage-dependent Ca²⁺ channels, nifedipine (10 M), did not influence homosynaptic LLD, but decreased heterosynaptic depression. Both types of depression of synaptic transmission were facilitated upon application of substances possessing a nootropic activity, 1 mM pyracetam or 5 M carbacetam. A blocker of NO synthase, N-nitro-L-arginine (10 M) did not alter either type of LLD. When hippocampal slices were influenced with a blocker of the A1 adenosine receptors, 1,3-dipropyl-8-phenylxanthine (1 M, 15 min), both LLD forms were intensified, and the development of homosynaptic LLD of synaptic transmission became possible in hippocampal slices of mature rats. When hippocampal slices were treated with an inhibitor of protein kinase C, polymyxin B (50 M, 15 min), intensification of LLD and, in particular, the development of homosynaptic LLD of synaptic transmission were observed. When an inhibitor of phospholipase A2, mepacrine (25 M, 15 min), was applied to hippocampal slices, both forms of LLD of synaptic transmission were significantly suppressed.     

Callus induction and shoot regeneration in Sempervivum tectorum

Leaf and shoot explants of Sempervivum tectorum L., taken from 14- and 30-day-old plants germinated in vitro, have been studied by using Murashige-Skoog and White basal media with cytokinins (benzyladenine, kinetin) and auxins (indoleacetic acid, naphthaleneacetic acid, indolebutyric acid) in various concentrations. Explants taken from 14-day-old plants died but 30-day-old leaves and shoots produced yellow and soft, as well as green and hard calluses on Murashige-Skoog medium with 4.4–8.8 M benzyladenine and 0.57 M indoleacetic acid. Shoot organogenesis was induced from green, hard callus in a medium with 2.2 M benzyladenine plus either 1.1 M indoleacetic acid or 2.5 M indolebutyric acid. Whole plants were grown on Murashige-Skoog medium without plant growth regulators. On the other hand, White medium was not suitable for raising Sempervivum tectorum in vitro.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - MS Murashige-Skoog - NAA napthaleneacetic acid - W White     

Quantitative observations of the effect of sex-steroids on the postnatal development of LH-cells

Summary The effects of sex-steroids on the LH-cell development in neonatal rats were studied. The cells were stained immunohistochemically by applying anti-HCG serum. On the second day after birth some of the animals of both sexes were gonadectomized and simultaneously injected with testosterone or estradiol (50 or 200 g). The remaining animals were either gonadectomized or injected with either one of the sex-steroids. The LH-cell numbers in each group were determined on the 12th day of age from serially cut histological sections of the pituitary. In castrated males the number of LH-cells was about twice that of the intact animals. In the so-called sex-zone, LH-cells tended to be hypertrophied in castrates. These alterations in the appearance of LH-cells did not occur after ovariectomy. In gonadectomized animals injected with sexsteroids (200 g), the cells were markedly reduced in number and size, both in males and females. Testosterone injection (50 g) into intact newborn animals also suppressed the numerical development of LH-cells, especially in females. These alterations were particularly evident in the sex-zone in both sexes. Thus the present findings show that sex-steroids may be involved in sexual differences in morphological development of LH-cells in newborn rats.     

Inhibition of the Mitochondrial Respiratory Chain by Phenylalanine in Rat Cerebral Cortex

Phenylketonuria (PKU) is biochemically characterized by the accumulation of phenylalanine (Phe) and its metabolites in tissues of affected children. Neurological damage is the clinical hallmark of PKU, and Phe is considered the main neurotoxic metabolite in this disorder. However, the mechanisms of neurotoxicity are poorly known. The main objective of the present work was to measure the activities of the mitochondrial respiratory chain complexes (RCC) and succinate dehydrogenase (SDH) in brain cortex of Wistar rats subjected to chemically induced hyperphenylalaninemia (HPA). We also investigated the in vitro effect of Phe on SDH and RCC activities in the cerebral cortex of 22-day-old rats. HPA was induced by subcutaneous administration of 2.4 mol/g body weight -methylphenylalanine, a phenylalanine hydroxylase inhibitor, once a day, plus 5.2 M/g body weight phenylalanine, twice a day, from the 6th-21st postnatal day. The results showed a reduction of SDH and complex I + III activity in brain cortex of rats subjected to HPA. We also verified that Phe inhibited the in vitro activity of complexes I + III, possibly by competition with NADH. Considering the importance of SDH and RCC for the maintenance of energy supply to brain, our results suggest that energy deficit may contribute to the Phe neurotoxicity in PKU.     

Changes in the Activity of Cathepsin B in the Rat Brain Under Conditions of Hemic Hypoxia

We studied the effects of experimental hemic hypoxia of medium severity on the activity of lysosomal cathepsin B in different brain structures of the mother rats and their offspring. Hypoxia was evoked by daily i.p. injections of 60 g/kg sodium nitrite into pregnant rat females (from the 5th to the 19th day of pregnancy). It resulted in noticeable changes in the activity of cathepsin B in the neocortex, cerebellum, midbrain, and pons Varolii of the hypoxic mother rats and their offspring. These changes differed in both the trend and the degree of expression depending on the duration of the post-hypoxic period.     

Role of histamine and cyclic nucleotides in implantation in the rabbit

Summary The effect of histamine on cAMP and cGMP levels in day 6 (144 h post coitum) rabbit blastocysts was determined. Histamine at 200 M and 1000 M concentrations stimulated the increased formation of cAMP in vitro, whereas stimulation of cGMP occurred only in the presence of 1000 M histamine. Furthermore, intrauterine injection of RMI-12330A (50 g or 500 g/uterine horn), an inhibitor of adenylyl cyclase, on day 5 of pregnancy interrupted embryro development and implantation of the embryo. The drug was also effective in reducing the cAMP level in the endometrial cells in vitro. A relationship between histamine and cyclic nucleotide changes in embryo development and implantation is suggested.     

Polyamines and proline are affected by cadmium stress in nodules and roots of soybean plants

The effect of moderate (50 M) and high (200 M) doses of Cd were studied in relation to polyamine (Pas) metabolism, proline level and the glutamine synthetase/glutamate synthase system (GS/GOGAT) activity in nodules and roots of soybean plants during 6 days of treatment. The lower Cd concentration increased putrescine (Put) in both nodules and roots, while 200 M Cd increased Spm only in nodules and Put in roots. Spermidine (Spd) decreased in roots under both Cd concentrations. Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were both involved in Put biosynthesis in roots. In nodules, Put formation could mainly be attributed to ODC activity. Diamine oxidase (DAO) activity was severely reduced by 50 and 200 M Cd either in nodules or roots. The GS/GOGAT system activity was depressed either with 50 or 200 M Cd, but most significantly with the highest metal concentration. Under 200 M Cd, GS activity decayed to 25% or 60% of the control in nodules and roots, respectively, while GOGAT decreased 85% in nodules and 79% in roots by day 4 of treatment. Ammonium increased greatly in nodules (200% over the controls) and roots (100%) under 200 M Cd. Proline concentration increased significantly in nodules and roots under both Cd treatments, more markedly under 200 M Cd. The relationship between Pas and proline accumulation and nitrogen assimilation is discussed.     

Location of ethylene production in cotton flowers and dehiscing fruits

Summary Over half of the ethylene produced by 1-day-old cotton flowers came from the combined stigma, style, and stamens. These tissues produced 0.0050 l/flower·h compared to 0.0024 and 0.0010 l/flower·h produced by the petals and ovary, respectively. Walls of dehiscing cotton fruits produced 0.052 l ethylene/fruit·h. This was approximately 50% more than seeds plus fiber which produced 0.033 l/fruit·h.     

Karyometry of nuclei in liver cells

Summary In untreated adult male albino rats nuclear volume and the percentage of binucleate cells were determined in the first layer of hepatocytes adjacent to hepatic venous branches of varying diameters (<40 m, 40 m–80 m, 80 m–120m, 120 m–160 m, >160 m), and in the third and fourth layer of hepatocytes in the remainder of the perivenous parenchyma. In the first layer of hepatocytes adjacent to the vascular structures means of nuclear volume are significantly lower and percentage of binucleate cells significantly higher than in the cells of the remainder of the perivenous parenchyma. Within each area measured distribution curves of nuclear volume classes were homogeneous but showed heterogeneity in comparison with each other. The morphometric data presented in this study strongly support the opinion of the heterogeneity of liver cells in the perivenous zone, as previously postulated on the basis of histochemical investigations.Supported by the Deutsche Forschungsgemeinschaft (Hi 318/2-1)     

Myelogenesis and estimation of the number of axons in the anterior commissure of the chick (Gallus gallus)

Summary By use of light- and electron microscopy the anterior commissure of the chick was studied at different times during development. Between the 19th day of incubation and the 35th day after hatching the cross-sectional area of the anterior commissure, as determined from mid-sagittal sections, undergoes a 6-fold increase in size. Thereafter the area remains fairly constant. The total number of fibres in the anterior commissure was estimated to be 89000. The full complement of fibres is already present by the 19th day of incubation. Myelogenesis occurs mainly between the 19th day of incubation and the 35th day after hatching, concomitant with the increase in cross-sectional area. From the 35th day after hatching, myelinated fibres comprise approximately 40% of the total number of fibres. The median diameter of unmyelinated fibres is about 0.35–0.40 m. The median diameters of myelinated axons and fibres are 0.8–1.0 m and 1.1–1.3 m, respectively.     

The distribution of short chain carboxylic acids in eutrophic drainage channels

The concentration of the C₂–C₅ carboxylic acids in the water column and sediment of shallow, eutrophic, drainage channels (Lewes Brooks, UK) were measured by gas chromatography. The concentrations of these acids were negatively correlated with dissolved oxygen. The highest concentrations of acetate (up to 200 M), propanoate (up to 38 M) and butanoate (up to 1.2 M) were measured during the summer in the water above the sediment, which became hypoxic during this period. Both acetate and propanoate concentrations declined at night. High concentrations of the acids were also recorded in reedbeds, which were also hypoxic. Only acetate was detected in the sediment pore water (up to 168 M). Its concentration declined during the autumn and winter and with increasing depth within the sediment.Author for Correspondence     

Effects of Lead on Adenylate Cyclase Activity in Rat Cerebral Cortex

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC₅₀ = 2.5 ± 0.1 M). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 M Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 M) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 M), lead (3 M) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 M). In membranes preincubated with 10 M lead, the addition of 10 M Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 M lead + 10 M Gpp(NH)p, Gpp(NH)p (10 M) or forskolin (10 M) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.     

Growth and morphogenesis of immature embryos of sweet potato (Ipomoea batatas L.) in vitro

Sweet potato (Ipomoea batatas L.) embryos excised from the fertilized ovules of 6- to 12-day-old capsules were cultured on MS medium supplemented with NAA, BA, GA separately and in combinations. GA was found essential for initial morphogenesis of globular and heart stages. Seedlings were recovered from late globular stage onwards but recovery was best from advanced embryo stages. Differentiated embryos produced multiple shoots on MS medium +1M NAA÷2M BA +0.5M GA.     

Callus induction and plant regeneration in Vetiveria zizanioides

Callus induction was obtained from basal parts of Vetiveria zizanioides Stapf. leaves cultured on Murashige and Skoog (MS) medium supplemented with 9.0 M 2,4-dichlorophenoxyacetic acid (2,4-d), 5.7 M indoleacetic acid (IAA) and 4.6 M kinetin. Calli were maintained on MS medium with the addition of 0.9 M 2,4-d and 2.3 M kinetin. Shoot formation was obtained from fast growing 14-day-old callus on the same basal medium supplemented with 0.9 M 2,4-d and 9.3 M kinetin. Embryo-like structures were observed. When transferred to basal medium, shoots readily developed roots. Fully developed regenerated plants were then successfully established in soil.     

Regional and subcellular distribution of taurine-synthesizing enzymes in the rat central nervous system

The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94–99 mol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44–51 mol/h/g; cerebellum, 71 mol/h/g; pons-medula and spinal cord, 94 and 60 mol/h/g, respectively. The distribution of CSD activity expressed as mol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14–21 mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8–13 mol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, colliculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.     

Pre-epithelial mucus layer in the colon of conventional and germ-free rats

Summary The pre-epithelial mucus layer (PML) and epithelial mucins were studied by mucin histochemistry in 10m-thick celloidinstabilized cryostat sections in the proximal and distal colon of conventional and germ-free rats aged 120 and 350 days. No continuous PML was found in the proximal colon. A continuous mucus blanket, of fairly homogenous thickness, was observed in the distal colon, where the PML-thickness was 40±24 m at 120 days of age and 44±22 at 350 days of age in conventional rats, and 25±17m (120 days) and 22±10 (350 days) in germ-free rats. The stainability of the PML by periodic acid-Schiff and Alcian Blue at pH 2.5 and 1.0 was stronger in conventional rats than in germ-free rats, indicating higher concentrations of mucosubstances and of acid and sulphated mucins, respectively. The PML of the conventional rat distal colon showed a stratified structure of up to eight sublayers. In the distal colon of germ-free rats, the whole gut wall thickness was reduced 47% compared to the conventional rat (germ-free: 185±73m, conventional: 350±115m). No stratification of the PML was observed. The presence of intestinal microflora obviously had a strong influence on the thickness, compactness, mucin content, mucin composition and structure of the pre-epithelial mucus layer.