Peptidergic neurons of the crab,Cardisoma carnifex,in defined culture maintain characteristic morphologies under a variety of conditions

Summary Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, superlarge, found occasionally, has a soma diameter greater than 40 m and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with l-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26° C instead of 22° C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K⁺]_o medium ([K⁺]_o=15–110 mM; standard=11 mM) has no long-term effect, but growth is arrested by [K⁺]_o greater than 30 mM. Cultures were also grown in media in which [Ca²⁺]_o ranged from 0.1 M to 26 mM (standard=13 mM). Outgrowth occured from all neuronal types in all [Ca²⁺]_o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.     

Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn)

Engagement of the clonotypic antigen receptor (TCR) on T lymphocytes provokes an activation response leading to cell proliferation and lymphokine secretion. To examine the molecular basis of T cell signaling, we generated transgenic animals in which a lymphocyte-specific nonreceptor protein-tyrosine kinase p59fyn(T) is 20-fold overexpressed in developing T lineage cells. Thymocytes from these mice, analyzed using both cellular and biochemical assays, were remarkably hyperstimulable. Moreover, the responsiveness of normal thymocytes to TCR-derived signals correlated well with the extent to which p59fyn was expressed in these cells. Overexpression of a catalytically inactive form of p59fyn substantially inhibited TCR-mediated activation in otherwise normal thymocytes. These effects are unique to p59fyn; overexpression of a closely related T cell-specific tyrosine kinase, p56lck, elicits dramatically different phenotypes. Our results suggest that p59fyn is a critically important component of the TCR signal transduction apparatus.     

Use of Oospore Formulations of Pythium oligandrum for Biological Control of Pythium Damping-off in Cress

Oospore preparations of Pythium oligandrum produced by liquid and solid-substrate fermentations were evaluated for biocontrol activity against Pythium damping-off in cress in artificially infested sand and naturally infested soil. Oospore biomass preparations from liquid fermentation of six isolates of P. oligandrum were equally effective in reducing damping-off in sand when tested as seed-coatings, whereas this type of preparation of a single isolate formulated as a kaolin dust, on Perlite and as alginate pellets incorporated into sand gave little or no control. None of the formulations containing oospores produced by solid-substrate fermentation incorporated into sand had any effect. In soil, a formulation containing oospores produced in a barley-Perlite solid-substrate fermentation and all oospore-biomass formulations which were prepared increased seedling survival, but none of these were as effective as a propamocarb HCl drench.     

Cellular heterogeneity and insulin-like growth factor I immunoreactivity among epiphysial growth plate chondrocytes in the pig

The localization of insulin-like growth factor I (IGF-I, also called somatomedin C) production in porcine epiphysial growth plates of the distal humerus was studied by immunohistochemistry. Counterstaining with Alcian blue-van Gieson demonstrated two cell types (blue and red cells) in the germinal (reserve), proliferating and hypertrophic zones; only those chondrocytes of the proliferative and hypertrophic zones that stained red were also immunoreactive to the antibody to IGF-I. The results indicate that there exists a functional heterogeneity among the chondrocytes of both the proliferative and hypertrophic zones of growth cartilage and that IGF-I is locally produced in only the red cells of these zones. Because the red cells of the germinal zone were not immunoreactive, the results suggest that the red cells of the germinal zone and the red cells of the proliferative and hypertrophic zones are also functionally distinct.     

Phospholipases modulate the rat testicular androgen biosynthetic pathway in vitro

The role of membrane phospholipids in testicular androgen biosynthesis was investigated by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Androgen biosynthesis in untreated rat testicular microsomes was examined by monitoring the temporal appearance of pregnenolone metabolites and was found to proceed through the 4-ene route. When phospholipase A2 was included, the 5-ene steroids 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) were formed in greater quantities, and the production of 4-ene steroids was reduced indicating that the conversion of 5-ene steroids to the 4-ene configuration was inhibited by phospholipase A2 treatment. Phospholipase C, in addition to inhibiting this step, also inhibited the conversion of C21 steroids to C19 steroids. When the enzymatic steps were measured individually, phospholipase A2 inhibited 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) with an ED50 of 73 mU/ml but had no effect on the activities of 17-hydroxylase, C-17, 20 lyase, or 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). However, though phospholipase C treatment inhibited 3 beta-HSD-Isomerase, it caused less inhibition (the ED50 value was 149 mU/ml). Furthermore, 17-hydroxylase and C-17, 20 lyase activities were also inhibited by phospholipase C treatment (ED50 values were 410 and 343 mU/ml, respectively), but no effect on 17 beta-HSD was observed. The differences in the apparent phospholipid requirements of the steroidogenic enzymes provides the possibility that the metabolic fate of pregnenolone may be regulated by changes in the phospholipid composition of the microenvironment.     

The inhibition of muscle contraction by adenosine 5' (beta, gamma-imido) triphosphate and by pyrophosphate.

We have studied the inhibition of the contraction of glycerinated rabbit psoas muscle caused by ligands that bind to the ATPase site of myosin. Two ligands, adenosine 5' (beta, gamma-imido) triphosphate (AMPPNP) and pyrophosphate (PPi), decreased the force and stiffness developed in isometric contractions and the velocity of shortening of isotonic contractions. The force exerted by isometric fibers was measured as a function of MgATP in the presence and absence of a constant concentration of the ligands. As the MgATP concentration decreased, the inhibition of tension caused by the ligand increased, reaching approximately 50% at 25 microM MgATP and either 2 mM MgPPi or 2 mM MgAMPPNP. The maximum velocity of shortening was also measured as a function of MgATP concentration in the presence of 1 and 2 mM MgPPi and 2.5 and 5 mM MgAMPPNP. Both ligands acted as pure competitive inhibitors with Ki = 3.0 mM for PPi and 5.1 mM for MgAMPPNP. These data show that both ligands are weak inhibitors of the contraction of fibers. The results provided information on the energetics of actin-myosin-ligand states that occur in the portion of the cross-bridge cycle where MgATP binds to myosin. A simple analysis of the inhibition of velocity suggests that MgAMPPNP binds to the actomyosin complex at this step of the cycle with an effective affinity constant of approximately 2 X 10(2) M-1.     

Suppression of prostaglandin F-2 alpha release and delay of luteolysis after active immunization against oxytocin in the goat

Active immunization against oxytocin significantly prolonged the oestrous cycle in 3 out of 4 goats; the mean (+/- s.e.m.) cycle length was 29.1 +/- 1.7 days (n = 12) compared to 19.4 +/- 0.6 days (n = 9) in control animals. During Days 10-21 of the cycle in the 3 responsive goats, peripheral plasma concentrations of progesterone and oxytocin were steady and those of 13,14-dihydro-15-keto-prostaglandin F-2 alpha were very low (50-100 pg X ml-1) with no marked pulsatile activity. The major effect of immunization would appear to be suppression of the synthesis of the uterine luteolysin PGF-2 alpha, thus confirming that endogenous oxytocin has a facilitatory role in luteolysis via prostaglandin production.     

Effects of calmodulin and lipoxygenase inhibitors on LH (lutropin)- and LHRH (luliberin)-agonist-stimulated steroidogenesis in rat Leydig cells.

The results of this study, carried out with purified rat Leydig cells, indicate that there are no major differences in the stimulating effects of lutropin (LH) and luliberin (LHRH) agonists on steroidogenesis via mechanisms that are dependent on Ca2+. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. All three calmodulin inhibitors used (calmidazolium, trifluoperazine and chlorpromazine) were shown to block LH- and LHRH-agonist-stimulated steroidogenesis. This probably occurred at the step of cholesterol transport to the mitochondria. Similarly, three lipoxygenase inhibitors (nordihydroguaiaretic acid, BW755c and benoxaprofen), inhibited both LH- and LHRH-agonist-stimulated steroidogenesis. The amounts of the inhibitors required were similar for LH- and LHRH-agonist-stimulated steroidogenesis. Steroidogenesis stimulated by the Ca2+ ionophore A23187 was also inhibited, but higher concentrations of the inhibitors were required. Indomethacin (a cyclo-oxygenase inhibitor) increased LHRH-agonist-stimulated steroidogenesis;this is consistent with the role of the products of arachidonic acid metabolism via the alternative, lipoxygenase, pathway. The potentiation of LH-stimulated testosterone production by LHRH agonist was unaffected by indomethacin or by lipoxygenase inhibitors at concentrations that inhibited LH-stimulated testosterone production by 75-100%. It was not possible to eliminate a role of calmodulin in modulating the potentiation, although higher concentrations of the inhibitors were generally required to negate the potentiation than to inhibit LH- or LHRH-agonist-stimulated testosterone production.     

Women doctors in urban general practice: the patients.

A large study from a representative sample of general practitioners in Manchester showed that women doctors saw more women patients than men doctors, especially in the childbearing age group. They saw a similar range of diagnoses as men doctors, though they saw more women patients for cervical smears, contraception, and breast disorders. Preventive health care may not be adequately provided for these in practices without a woman partner.     

An estimate of unique DNA sequence heterozygosity in the human genome

Summary Patients with recessive X-linked ichthyosis Patients with recessive X-linked ichthyosis (RXLI), one hereditary form of scaly skin, lack activity of the enzyme steroid sulfatase in all tissues studied. To investigate the molecular defect underlying the lack of enzyme activity, we prepared antisera against normal enzyme by injecting normal placental microsomal suspensions or partially purified steroid sulfatase into rabbits. Antibody activity was assessed by immunoprecipitation of detergent solubilized steroid sulfatase. In addition, we prepared rabbit antisera against RXLI placental microsomal suspensions. To detect immunologically cross-reactive material in patients' placentas, extracts were studied by immunoblot techniques and by competition with normal enzyme for antibody binding. Patients' extracts did not contain immunoreactive material co-migrating on electrophoresis with purified enzyme nor did they inhibit immunoprecipitation of normal enzyme. Sera from rabbits immunized with RXLI placental microsomes contain no antibodies to normal steroid sulfatase, as judged by their failure to immunoprecipitate normal enzyme or to react with normal steroid sulfatase on immunoblot. Thus the mutation in RXLI appears to reduce steroid sulfatase enzyme protein as well as enzyme activity. Portions of this material have appeared in abstract form in Clinical Research 31:564A, 1983 and 32:138A, 1984