Immunological studies of the relationship between the antigenic change of Ascaris oocytes and the junctional fluid of the fertilization chamber

Antigenic change on the surface of immature oocytes of Ascaris occurred during passage through the oviduct. Using immunodiffusion and immunoelectrophoretic techniques we examined the possible relationship between the antigenic change of immature oocytes and the junctional fluid (JF) which is present in the fertilization chamber. From the immunodiffusion it was found that the anti-immature oocyte serum (A-I serum) had more immunoprecipitation arcs than did the anti-mature oocyte serum (A-M serum) when reacted against the junctional fluid. This indicated that A-I serum contained more immunoglobulins that reacted with junctional fluid than did the A-M serum. This result was substantiated by using immunoelectrophoretic analysis and two dimension crossed immunoelectrophoresis. Our results suggest that during the migration toward the oviduct-uterine junction the immature oocytes might shed surface proteins into the luminal fluid. Alternatively, the membrane surface of mature oocytes may be altered by the interaction with the substances present in the junctional fluid.     

New mutation affecting the synthesis of some membrane proteins and sporulation in Bacillus subtilis.

A new mutation, mpo, which affects the synthesis of some membrane proteins and sporulation in Bacillus subtilis was identified. The mpo mutation was tightly linked to the overproduction of membrane proteins MP32 and MP18 (molecular weights of 32,000 and 18,000, respectively) and the temperature-sensitive sporulation phenotype. Genetic analysis showed that the mpo mutation maps between the spoIIIB and lys loci.     

Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.     

A recessive mutation leading to vertebral ankylosis in zebrafish is associated with amino acid alterations in the homologue of the human membrane-associated guanylate kinase DLG3.

We describe the characterization of the zebrafish homologue of the human gene DLG3. The zebrafish dlg3 gene encodes a membrane-associated guanylate kinase containing a single PDZ domain. This gene was cloned using a gene-trap construct inserted in the gene's first intron. The insertion co-segregates with a viable mutation called humpback (hmp), which leads to formation of ankylotic vertebrae in adult fishes. Insertion and mutation have both been mapped to chromosome 12, in a segment which is syntenic with region p12 to q12 of human chromosome 17. The hmp mutant phenotype, however, appears to be due to two point mutations in the guanylate kinase domain rather than to the transgene insertion itself. The results of this study are discussed in the light of the possible function of the guanylate kinase domain.     

Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function; tests for receptor-stimulated phosphatidylinositol turnover and for modulation of agonist binding by guanylylimidodiphosphate showed functional coupling to be present several days prior to the onset of synapse formation. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes.

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.     

Stem cell function during plant vascular development

While many regulatory mechanisms controlling the development and function of root and shoot apical meristems have been revealed, our knowledge of similar processes in lateral meristems, including the vascular cambium, is still limited. Our understanding of even the anatomy and development of lateral meristems (procambium or vascular cambium) is still relatively incomplete, let alone their genetic regulation. Research into this particular tissue type has been mostly hindered by a lack of suitable molecular markers, as well as the fact that thus far very few mutants affecting plant secondary development have been described. The development of suitable molecular markers is a high priority in order to help define the anatomy, especially the location and identity of cambial stem cells and the developmental phases and molecular regulatory mechanisms of the cambial zone. To date, most of the advances have been obtained by studying the role of the major plant hormones in vascular development. Thus far auxin, cytokinin, gibberellin and ethylene have been implicated in regulating the maintenance and activity of cambial stem cells; the most logical question in research would be how these hormones interact during the various phases of cambial development.     

Nucleoside exchange catalysed by the cytoplasmic 5''-nucleotidase.

The inhibition of the cytoplasmic 5'-nucleotidase (EC 3.1.3.5) by its product, inosine, was studied with a partially purified preparation of the enzyme from rat liver. Inhibition of Pi production was found to be due to exchange of the inosine moiety between inosine and IMP. Exchange was not catalysed by reversal of the hydrolytic reaction, suggesting, instead, the mediation of an enzyme-phosphate intermediate. Two models for the catalytic mechanism are proposed and rate equations for the dependence of Pi production on inosine concentration are derived. The experimentally determined dependence was consistent with a mechanism in which hydrolysis of the enzyme-phosphate intermediate occurred only when it was unoccupied by inosine. This conclusion suggests that inosine analogues that cannot participate in exchange should inhibit the enzyme. Such inhibitors might be useful in defining the enzyme's physiological role or as pharmacological agents to decrease breakdown of purine nucleotides. The possibility that nucleoside exchange provides an alternative route for the phosphorylation of mutagenic or cytotoxic nucleoside analogues should also be considered.