Studies on H-Y antigen in different cell fractions of the testis during pubescence

Summary Various cell types of the rat testis during pubescence, including germ, Sertoli, and Leydig cells, were partially enriched. The fractions were tested for the presence, binding, and secretion of H-Y antigen. The main results are: Immature germ cells are H-Y antigen-negative until the late diploid stages, and late primary spermatocytes or spermatids become positive; the somatic cells of the gonad are positive at all ages examined (18 days old to adulthood). Secretion of H-Y antigen is restricted to the Sertoli cell fraction. Binding of externally supplied antigen takes place on Leydig cells; the Sertoli cell surface will be saturated because of active secretion; there is no binding to germ cells. Thus, immature germ cells seem to be the only H-Y antigen-negative cells of the male organism, and the Sertoli cells seem to be the only ones to secrete H-Y antigen.     

Yeast invertase: Subcellular distribution and possible relationship between the isoenzymes

The intracellular invertase ofSaccharomyces cerevisiae is mainly found in a soluble form (91–95%), while only minor amounts are found bound to the internal (4–8%) and plasma membranes (less than 1%). In the processes of derepression or repression, inhibition of RNA or protein synthesis, or in the presence of 2-deoxy-d-glucose, the levels of the membrane-bound and external activities are modified in a way in which their relation is clear, while the soluble enzyme does not change at all. These results, together with the fact that the membrane-bound and the external enzymes are glycoproteins, suggest a precursor-product relationship between the enzymic forms.     

THE EFFECT OF ISCHAEMIA AND RECIRCULATION ON PROTEIN SYNTHESIS IN THE RAT BRAIN

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L-[1–¹⁴C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post-ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L-[1–¹⁴C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.
Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post-ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.     

The cell cycle in the small intestine transplanted beneath the kidney capsule in syngenic mice

Summary Transplantation of a small fragment of the ileum beneath the kidney capsule in syngenic mice results in the formation of a cyst lined with proliferating intestinal epithelium. The duration of the cell cycle in this epithelium was determined (using tritiated thymidine and the FLM method) as 14.5 h, as compared with 11.5 h in the intestinal epithelium in situ. We conclude that the intestinal content has little effect on the cell cycle of epithelial cells of the small intestine.     

Chlamydia trachomatis in cell culture

Summary Trachoma organisms of serotype B were grown serially in irradiated cells (McCoy, BHK-21, Microbiological Associates, and BHK-21, Lister) and tested for infectivity in monolayers of five mammalian cell lines (BHK-21, CHO, HeLa S₃, McCoy and OWMK) and two diploid strains (ST/BTL and WI-38). All cell types had low susceptibility to chlamydial infection but the number of inclusions increased when the inoculum was centrifuged onto the monolayers, or when the cells were irradiated. Infection was higher in non-irradiated CHO than in irradiated CHO in three out of a total of six experiments. Inclusion number was increased 300 times in HeLa S₃ and up to three times in the other cell types after treatment with diethylaminoethyl-dextran (DEAE-D). Serial passage of Chlamydia trachomatis serotype B (strain Har-36) in CO₆₀ McCoy and CO₆₀ BHK-21 Lister resulted in partial adaptation of the strain to the host cell. The phenomenon of adaptation of serotype B to McCoy compensated for the lower susceptibility of this cell revealed when McCoy cells were inoculated with trachoma elementary bodies grown in BHK-21 Lister or in chick embryo yolk sac. Trachoma organisms of immunotypes A, B and C prepared in yolk sac produced more inclusion-forming units per ml in CO₆₀ BHK-21 Lister than in CO₆₀ McCoy. This research was supported by a grant from the National Eye Institute (EI-00812-08), and by the Arabian American Oil Company. The paper is dedicated to the memory of Francis B. Gordon, who pioneered research methods for the cultivation of trachoma and inclusion conjunctivitis (TRIC) agents in cell culture. Dr. Gordon patiently studied tables and photographs which accompany this text when he visited our laboratory on the day prior to his sailing to England on the ill-fated voyage in which he and Mrs. Gordon perished (October 1973).     

Ouabain-insensitive Na+-stimulated ATPase activity of basolateral plasma membranes from guinea-pig kidney cortex cells: II. Effect of Ca2+

The ouabain-insensitive, Mg²⁺-dependent, Na⁺-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca²⁺ to the assay medium. The Ca²⁺ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily ‘deactivated’ by reducing the free Ca²⁺ concentration of the assay medium to values lower than 1 μM; and as a stable component, which can be ‘deactivated’ by preincubating the membranes for periods of 3–4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca²⁺. The $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca²⁺ on the Na⁺-stimulated ATPase is on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, and not on the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺. The activating effect of Ca²⁺ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca²⁺ concentration may modulate the ouabain-insensitive, Na⁺-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na⁺ accompanied by Cl^? and water that these cells show, and to which the Na⁺-ATPase has been associated as being responsible for the energy supply of this mode of Na⁺ extrusion.     

Activation of Protein Kinase C by Phorbol Esters and Arachidonic Acid Required for the Optimal Potentiation of Glutamate Exocytosis

Abstract: The effects of arachidonic acid and phorbol esters in the Ca²⁺-dependent release of glutamate evoked by 4-aminopyridine (4-AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations (500 n M ) of 4β-phorbol dibutyrate (4β-PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 4β-PDBu (1–50 n M ) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis.     

Epidermal growth factor receptor: Elements of intracellular communication

While EGF has an important function in cell growth regulation, the molecular mechanisms by which intracellular signal connect the EGF: receptor complex on the plasma membrane with the initiation of DNA synthesis and mitogenesis is not well understood. The discovery that rasGAP, PI-3 kinase and PLC-gamma 1 are substrates for the EGF receptor tyrosine kinase has provided a beginning in understanding the biochemistry underlying growth factor receptor transduction.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.