Metabolic changes in coho and chinook salmon resulting from acute insufficiency in pancreatic hormones

Acute deficiency in pancreatic peptides (insulin, somatostatin-25, glucagon, and glucagon-like peptide) was invoked for 9-12 hr in coho, Oncorhynchus kisutch, and chinook, O. tshawytscha, salmon by administration of specific antisera raised against purified salmon hormones. Insulin-deficient fish were hyperglycemic, had diminished glycogen content in the liver (Plisetskaya et al., '88a, elevated liver triacylglycerol lipase activity, and higher concentration of plasma triiodothyronine (T3) compared to a control group of fish injected with nonspecific rabbit serum. After immunoneutralization of somatostatin-25, fish remained normoglycemic, with higher liver glycogen content, decreased lipase activity, and elevated plasma levels of insulin, while the levels of T3 declined. The induced deficiency in glucagon family peptides led to comparatively smaller changes: liver glycogen content was increased after anti-glucagon-like peptide (aGLP) injection and transient hyperglycemia was apparent following anti-glucagon (aGLU) administration. Circulating levels of insulin remained unaffected for at least 9 hr following aGLU and aGLP treatments. The velocity of pyruvate kinase at 2.5 mM phosphoenolpyruvate (V2.5) was depressed, especially after the combined administration of aGLU + aGLP. The effectiveness of immunoneutralization experiments was greatly dependent on the particular stage of the fish life cycle. Antisera against fish pancreatic peptides proved to be a suitable tool in the studies of hormonal regulation of fish metabolism.     

Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection.

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.     

Molecular Mapping of Uncharacteristically Small 5q Deletions in Two Patients with the 5q- Syndrome: Delineation of the Critical Region on 5q and Identification of a 5q- Breakpoint

Molecular mapping techniques have defined the region of gene loss in two patients with the 5q- syndrome and uncharacteristically small 5q deletions (5q31-q33). The allelic loss of 10 genes localized to 5q23-qter (centromere-CSF2-EGR1-FGFA-GRL-ADRB2-CSF1R-SPARC-GLUH1-NKSF1-FLT4-telomere) was investigated in peripheral blood cell fractions. Gene dosage experiments demonstrated that CSF2, EGR1, NKSF1, and FLT4 were retained on the 5q- chromosome in both patients and that FGFA was retained in one patient, thus placing these genes outside the critical region. GRL, ADRB2, CSF1R, SPARC, and GLUH1 were shown to be deleted in both patients. The proximal breakpoint is localized between EGR1 and FGFA in one patient and between FGFA and ADRB2 in the other, and the distal breakpoint is localized between GLUH1 and NKSF1 in both patients. Pulsed-field gel electrophoresis was used to map the 5q deletion breakpoints, and breakpoint-specific fragments were detected with FGFA in the granulocyte but not the lymphocyte fraction of one patient. This study has established the critical region of gene loss of the 5q- chromosome in the 5q- syndrome, giving the location for a putative tumor-suppressor gene in the 5.6-Mb region between FGFA and NKSF1.     

Growth hormone stimulates hepatic lipid mobilization in rainbow trout,Oncorhynchus mykiss

Rainbow trout were used to characterize the direct influence of growth hormone on hepatic lipid mobilization in vitro. Liver was removed from fish fasted 72 h, sliced into 1-mm³ pieces and incubated in Hanks' medium containing ovine or salmon growth hormone (0.28 ng·ml^-1–28 g·ml^-1). Lipid mobilization, resulting from triacylglycerol hydrolysis, was evaluated by fatty acid and glycerol release into culture medium. Control rates of fatty acid release and glycerol release were 0.95±0.16 (mean ± SE) and 0.88±0.28 mol·l^-1·mg fresh weight, respectively. Both ovine growth hormone (28 ng·ml^-1) and salmon growth hormone (28 ng·ml^-1) stimulated fatty acid release into culture medium, increasing rates by 112% and 97%, respectively, during the course of a 24-h incubation. Glycerol release was similarly augmented by growth hormone treatment. Growth hormone-stimulated metabolite release became evident within 12 h and persisted for up to 72 h. The presence of amino acids in the culture medium potentiated the lipolytic action of growth hormone. Ovine growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 108% increase in glycerol, release over rates observed in the absence of amino acids. Salmon growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 44% increase in glycerol, release over rates observed in the absence of amino acids. Ovine growth hormone (28 ng·ml^-1) also stimulated gluconeogenesis, as indicated by a 75% increase in phosphoenolpyruvate carboxykinase activity in liver pieces incubated in the presence of amino acids. These results indicate that growth hormone directly stimulates lipid breakdown in the liver of trout and that amino acids potentiate growth hormone-stimulated lipolysis.Abbreviations AA amino acid(s) - dGDP deoxy-guanosine diphosphate - ED ₅₀ 50% effective dose - FA fatty acid(s) - fw fesh weight - GH growth hormone - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MS-222 tricaine methanesulfonate - MEM minimum essential medium - oGH ovine growth hormone - PEPCK phosphoenolpyruvate carboxykinase - PK_c protein kinase C - rpm revolutions per minute - sGH salmon growth hormone - TG triacylglycerol - w/v weight per volume This paper was presented in abstract form at the Annual Meeting of the American Society of Zoologists, Dec. 26–30, 1991, Atlanta, GA     

Female Gametophyte Development in Maize: Microtubular Organization and Embryo Sac Polarity

The developmental stages of the maize embryo sac were correlated with the corresponding silk lengths of ear florets in the female inflorescence. The development of embryo sacs in the ovules of spikes occurs in a gradient pattern with the initiation of the embryo sac beginning at the base of the ear and progressing to the top. At the beginning of meiosis, the presence of conspicuous cortical microtubules coincides with the extensive elongation of the megasporocyte. The spindles at metaphase I and II align along the long axis of the megasporocyte leading to the linear alignment of the dyad and tetrad of megaspores. During megagametogenesis, micropylar and chalazal nuclei of the embryo sac undergo synchronized divisions and migration at the second and third mitosis. Radiate perinuclear microtubules are present during the interphase of the second and third mitosis, and inter-sister nuclear microtubules occur at the late four-nucleate embryo sac. The configuration and orientation of the spindles, phragmoplasts, and pairs of nuclei result in precise positioning of the nuclei. The fusion of the polar nuclei and the formation of a microtubule organizing center-like structure in the filiform apparatus occur right after the first division of the antipodal cells. The different patterns of organization of microtubules in the cells of the mature embryo sac reflect their structural adaptations for their future function.     

Mutational analysis of morphogenesis of the maize embryo

Morphogenesis of the maize embryo is controlled by many genes. A group of 51 embryo-specific (emb) mutations representing at least 45 independent mutation events and many different gene loci have been isolated from active Robertson's Mutator stocks. The authors have reported previously that the embryo phenotype of 27 of these mutations, characterized by examining mature embryos in fresh dissection. The maximal development capacity of the 24 emb mutations are reported here which have not been reported previously. All result in retarded embryos that are morphologically abnormal. Three of the mutants are blocked during the first phase of morphogenesis, the period in which the basal-apical asymmetry is established and the embryo is regionalized into suspensor and embryo proper. Nineteen mutants are blocked during the second phase, the period in which radial asymmetry appears, the embryonic axis is established at a different angle than the original basal-apical axis of the zygote and the vegetative organ primordia of the adult plant make their first appearance. Two mutants are blocked or altered during the third phase, the period in which vegetative structures are elaborated. Some of the mutants affected in the first phase of morphogenesis may have defective mechanisms for establishing basal-apical asymmetry, including possibly the asymmetric distribution of morphogenic determinants. Similarly, some of the mutants affected in the second phase may be altered in the mechanisms establishing radial asymmetry and the origin of the meristems. Mutations of the first type may act as early as the first cell division when the zygote undergoes a transverse division, while mutations of the second type are likely to act during the proembryo and transition stages. Both types include mutations affecting embryo pattern formation. Mutations affecting the third phase of morphogenesis may identify genes regulating reiterative morphogenic processes of vegetative plant development and events of embryo maturation. This group of 24 mutations is like that reported previously in representing genes that are crucial to embryo morphogenesis.     

New Insights into the Role of the Maize Ameiotic1 Locus

In maize the am1-1 mutant allele results in both the male and female meiocytes undergoing mitosis in place of the meiotic divisions. A second mutant allele am1-praI enables both the male and female meiocytes to proceed to the early zygotene stage of meiotic prophase I before being blocked. Here we report on three new alleles that allow all male meiocytes to undergo mitosis but in female meiocytes approximately one quarter (am1-2), one half (am1-485), or all (am1-489) of them are blocked at an abnormal interphase stage. Previous analysis has shown that am1-praI is dominant to am1-1 in male meiocytes. Cytological analysis of heteroallelic combinations in female meiocytes now indicates a dominance relationship of am1-praI > am1-1 > am1-2/am1-485 > am1-489. The evidence provided by the female phenotypes of the new mutant alleles suggest that, whereas the normal am1 allele is required for the meiocytes to proceed through meiosis, a partially functional allele may be required for their diversion into a mitotic division. The partial or complete blockage of mitosis in female meiocytes carrying the new am1 alleles rules out the possibility that the mitotic division of mutant meiocytes reflects a simple default pathway for cells that cannot initiate meiosis. This locus may have a dual function.     

A silver impregnation technique for the study of steroid receptors in central nervous system tissue prepared for autoradiography

The commonly used silver stains were found to be unsatisfactory for nervous tissue processed for autoradiography. A silver impregnation procedure for central nervous system tissues prepared for the autoradiographic study of steroid receptors is described. The procedure is a combination of several silver and reticular strains made up in solutions containing dimethylsulfoxide. The technique clearly distinguishes perikarya of neurons, brain nuclei and fiber tracts without substantial loss of silver grains, and thus greatly facilitates the identification of steroid receptor nuclei at all levels of the central nervous system.     

Inhibition of nitrogen fixation in alfalfa by arsenate, heavy metals, fluoride, and simulated Acid rain

The acute effects of aqueous solutions of As, Cd, Cu, Pb, F, and Zn ions at concentrations from 0.01 to 100 micrograms per milliliter and solutions adjusted to pH 2 to 6 with nitric or sulfuric acid were studied with respect to acetylene reduction, net photosynthesis, respiration rate, and chlorophyll content in Vernal alfalfa (Medicago sativa L. cv. Vernal). The effects of the various treatments on acetylene reduction varied from no demonstrable effect by any concentration of F⁻ and 42% inhibition by 100 micrograms Pb²⁺ per milliliter, to 100% inhibition by 10 micrograms Cd²⁺ per milliliter and 100 micrograms per milliliter As, Cu²⁺, and Zn²⁺ ions. Zn²⁺ showed statistically significant inhibition of activity at 0.1 micrograms per milliliter. Acid treatments were not inhibitory above pH 2, at which pH nitric acid inhibited acetylene reduction activity more than did sulfuric acid. The inhibition of acetylene reduction by these ions was Zn²⁺ > Cd²⁺ > Cu²⁺ > AsO₃⁻ > Pb²⁺ > F⁻. The sensitivity of acetylene reduction to the ions was roughly equal to the sensitivity of photosynthesis, respiration, and chlorophyll content when Pb²⁺ was applied, but was 1,000 times more sensitive to Zn²⁺. The relationship of the data to field conditions and industrial pollution is discussed.