Photoreceptor membrane breakdown in the spider Dinopis: GERL differentiation in the receptors

Summary In the spider Dinopis, retinae of the posterior median eyes synthesise new photoreceptor membrane in bulk at dusk and destroy it at dawn (Blest, 1978). During the dawn period, there is a rapid, anticipatory differentiation of unusual organelles from the rough endoplasmic reticulum (RER) in the intermediate segments of the receptors. This system is classified as GERL. Its products appear to play a role in the autolysis of photoreceptor membrane. Consistent topographical relationship to Golgi bodies has not been determined. Circumscribed regions of RER whorls first reorganise to yield fenestrated profiles; these differentiate further to a number of structures by condensation and loss of ribosomes. Smooth tubular profiles are termed rigid tubules to indicate their probable homology with the rigid lamellae of vertebrate secretory cells. More complex smooth multilocular bodies are also produced. Evidence is discussed which implies that both rigid tubules and multilocular bodies give rise to condensing vacuoles. These, in turn, pinch off coated vesicles assembled as Nebenkerne. Some rigid tubules are transported to the interrhabdomeral cytoplasm of the receptive segments. At late stages of differentiation, rigid tubules, multilocular bodies and Nebenkerne give strong, positive responses to zinc iodide-osmium tetroxide (ZIO) treatment; early stages and both cis and trans Golgi components do not. GERL differentiation is independent of immediate illumination of the retina at dawn. It is suggested to mediate the lysis of membrane degradation products by the production of hydrolases.We thank Professor D.T. Anderson F.R.S. for our use of field facilities at the Crommelin Biological Field Station of Sydney University at Warrah, Pearl Beach, N.S.W., and Andrew and Sally Austin and Sally Stowe for help in the field. We are indebted to Rod Whitty and the Electron Microscopy Unit for advice and support throughout these studies     

Modulation of drug permeability in Chinese hamster ovary cells. Possible role for phosphorylation of surface glycoproteins.

We have previously reported that the uptake of colchicine and other drugs in Chinese hamster ovary (CHO) cells can be greatly enhanced by the addition of metabolic inhibitors such as cyanide (See, Y.P., Carlsen, S.A., Till, J.E. and Ling, V. (1974) Biochim. Biophys. Acta 373, 242-252). This has led us to postulate the presence of an active drug permeability barrier in these cells. In this paper we provide evidence for the dependence of this permeability barrier on intracellular ATP levels. Colchicine-resistant mutants of CHO cells exhibiting a reduced drug permeability, however, can maintain this drug permeability barrier at much lower ATP levels, suggesting that they possess an altered active drug permeability barrier. We have also observed a membrane-associated protein kinase-phosphoprotein phosphatase system in the isolated membranes of mutant and wild-type cells. Differences in the intrinsic protein phosphorylation patterns between the membranes of these cells have led us to conclude that the control of the drug permeability barrier may be mediated via the phosphorylation of at least two high molecular weight surface glycoproteins.     

Endorphins are located in the intermediate and anterior lobes of the pituitary gland, not in the neurohypophysis.

Immunocytofluorescence techniques with well characterized anti-sera to α-endorphin and β-endorphin show presence of these two peptides in all cellular elements of the pars intermedia of the rat hypophysis, and in discrete cells of the pars distalis (adenohypophysis) at the complete exclusion of the neurohypophysis (pars nervosa, posterior lobe).     

Differentiation of aflatoxins from territrems.

Three methods were adopted for differentiation of aflatoxins B1 and B2 from territrems A and B. They were as follows. (i) Then-layer chromatography coupled with chemical confirmation. A significant decrease in the Rf value of trifluoroacetic acid-treated aflatoxin B1 developed in chloroform-acetone (85:15, vol/vol) was satisfactory in differentiating this toxin from the other three. (ii) High-pressure liquid chromatography monitored synchronously at two wavelengths, 365 and 335 nm. The ratio derived from this double-wavelengh detection could serve as an indicator of the presence of each toxin. (iii) Velasco's flurotoxin meter method, which is used for the determination of aflatoxins within the range of 0 to 50 ng/ml, was not significantly affected by territrems even when they were present in quantities at the microgram-per-milliliter level.     

Dominance of colchicine resistance in hybrid CHO cells

Intraspecific hybrids of colchicine-sensitive with colchicine-resistant (CHR) Chinese hamster ovary cells were constructed, using six different colchicine-resistant clones from two independent series. In each instance, colchicine resistance was expressed in an incompletely dominant manner. Some hybrid clones were examined further for the expression of the pleiotropic CHR phenotype and for the cell surface P glycoprotein. These features of the colchicine-resistant phenotype were also expressed coordinately.     

Somatic embryogenesis and plant regeneration in an interspecific hybrid of Oryza

An embryogenic callus was obtained from immature panicle of an interspecific hybrid (Oryza sativa x O. latifolia) F₁. The medium consisted of HE salts supplemented with 2,4-D, NAA (each 2 mg/l), kinetin (3 mg/l), yeast extract (1360 mg/l) and casein hydrolyzate (300 mg/l). The callus was milk-white in colour compact and granulate in texture. Various developmental stage of embryoid, such as globular, heart-shape, scutellum-shape and mature embryoid were observed in an embryogenic callus. Plantlets were successfully regenerated from 1-month-old callus with more than 80% regenerational frequency in each subculture for 12 passages.     

Studies on the physical state of water in living cells and model systems. I. The quantitative relationship between the concentration of gelatin and certain oxygen-containing polymers and their influence upon the solubility of water for NA+ salts

The quantitative relationships between the concentrations of solutions of gelatin, polyvinylpyrrolidone, poly(ethylene oxide), polyvinylmethylether, and poly(ethylene glycol), and their ability to reduce the solubility of water for Na citrate are presented. The data in general are in harmony with the polarized multilayer theory of protein (and polymer) dominated water in vitro and in living cells.     

Isolation and characterization of a succinimide variant of methionyl human growth hormone.

Deamidation of asparagine and glutamine residues, isomerization of aspartic acid side chains, and racemization of the L- to the D-form of the amino acids are common spontaneous chemical reactions known to occur in proteins. Previous studies have implicated succinimides as intermediates in these reactions; however, the evidence has been indirect. Our results demonstrate, for the first time, the presence of a succinimide intermediate in an intact protein. The succinimide (cyclic imide) variant was isolated from thermally stressed recombinant methionyl human growth hormone (hGH) by high performance anion-exchange chromatography, further purified by reversed-phase high performance liquid chromatography, and analyzed by tryptic mapping. A later eluting tryptic peptide, compared with the native T12 peptide (residues 128-134, Leu-Glu-Asp-Gly-Ser-Pro-Arg), was analyzed by mass spectrometry (MS). This variant had a protonated molecular mass of 755.3 atomic mass units (u), as compared with 773.3 u for the native T12 peptide. A difference of 18 u, a loss of water, is consistent with the formation of a succinimide intermediate at Asp-130 of methionyl hGH. MS/MS analysis of the cyclic imide-containing peptide verified that the modification occurred at Asp-130. A difference of 18 u was also observed for the intact cyclic imide methionyl hGH variant (22,238 u), as measured by electrospray mass spectrometry, compared with native methionyl hGH (22,256 u).     

Adrenalectomy Potentiates Immediate Early Gene Expression in Rat Brain

Administration of kainate or pentylenetetrazole increased c-fos, c-jun, junB, and junD mRNA levels in rat brain in a dose-dependent manner. Kainate increased these mRNA levels predominantly in the hippocampus, and pentylenetetrazole was more effective in the cortex. Adrenalectomy (3 days) was used to eliminate endogenous glucocorticoid hormones. Adrenalectomy significantly potentiated kainate-induced increases, compared with increases caused by kainate (4 mg/kg) alone, in the hippocampal mRNA levels of c-fos and junB by 6.5-fold and of junD by twofold and tended to augment c-jun mRNA. Corticosterone administration blocked the potentiated stimulation of these mRNA levels caused by adrenalectomy. Adrenalectomy also significantly increased pentylenetetrazole-induced levels of c-fos mRNA in the cortex. These results demonstrate that glucocorticoids modulate immediate early gene expression in the brain, raising the possibility that this interaction contributes to interneuronal and interindividual differences in responses to stimuli and to the effects of stress- or disease-induced changes in glucocorticoid concentrations.     

Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.

Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.