Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Nonadditive interactive effects of polychlorinated biphenyl congeners in rats: role of the 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor

Administration of 3,3',4,4',5,5'-hexa-,3,3',4,4',5-penta-, and 2,3,3'4,4'5-hexa-chlorobiphenyl to immature male Wistar rats caused a thymic atrophy at high dose levels (1.25, 1.0, and 100 mumol/kg, respectively) and induced the hepatic cytochrome P-448 dependent monooxygenases (benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase) at both high and low (0.25, 0.01, and 5 mumol/kg, respectively) doses. In contrast, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) (300 mumol/kg) did not elicit any of these effects but elevated hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein levels (threefold) as previously reported. The effects of hepatic receptor modulation by 2,2',4,4',5,5'-HCBP (300 mumol/kg) on the enzyme induction activities of 3,3'4,4',5-penta-, 3,3'4,4',5,5'-hexa-, and 2,3,3',4,4',5-hexa-chlorobiphenyl were dose-dependent; no interactive effects were observed at high (toxic) doses of these compounds, whereas apparent synergistically increased hepatic microsomal monooxygenase induction activities were noted at the lower submaximal induction doses. It was concluded that the increased responsiveness of the rats was due to elevated hepatic 2,3,7,8-TCDD receptor levels.     

Incorporation of 35SO 4 2− into sediments of three New York lakes

Intact sediment cores were obtained from three New York lakes in May, July, and October 1981. Radioactive S (as ³⁵SO ₄ ²⁻ ) was added to the overlying water and cores were incubated without atmospheric exchange for one week near lake bottom temperatures. Headspace flux of 0₂ as an index of sediment respiration rates varied among lakes and seasonally within lakes. Acidic South Lake had the lowest respiration rate at all seasons and also the smallest net incorporation of the ³⁵SO ₄ ²⁻ . Summer net isotope transformation into ester sulfate and non-HI reducible S (pyrite and C-bonded S) constituents was 88.6%, 89.4%, and 59.7% of total sediment isotope for Oneida, Deer, and South, respectively. Seasonal variation of net isotope incorporation was observed in each lake as were differences in ³⁵SO ₄ ²⁻ partitioning into major S pools. Of the S constituents analyzed, HCl digestible S (volatile sulfides) was the smallest pool, while ester sulfate and non-HI reducible S together accounted for greater than 50% of S isotope transformation in all lakes. In addition, ester sulfate is the major product of dissolved SO ₄ ²⁻ transformation and its formation results in less alkalinity generation than the formation of non-HI reducible S constituents. Thus ester sulfate transformation processes must be considered in calculating alkalinity generation by lake sediments. Financial support provided by Office of Water Research Technology (Project No. 13-096-NY). Financial support provided by Office of Water Research Technology (Project No. 13-096-NY).     

Characterization, Localization, and Biosynthesis of an Interstitial Retinol-Binding Glycoprotein in the Human Eye

Abstract: Human eyes contain an M_r 135K retinol-binding protein that is analogous to interstitial retinol-binding protein (IRBP) in the subretinal space of bovine eyes. It is a glycoprotein, because it binds ¹²⁵I-concanavalin A, ¹²⁵I-wheat germ agglutinin and ¹²⁵I- Lens culinaris hemagglutinin. It does not bind Ricinus communis agglutinin I. After desialation, it binds Ricinus communis agglutinin I, but loses its capacity to bind wheat germ agglutinin. These observations, coupled with the known specificities of these lectins, suggest that at least one of the oligosaccharide chains is a sialated, biantennary complex type containing fucose. Both by direct analysis of dissected ocular tissues and by immunocytochemistry it was shown that human interstitial retinol binding protein is an extracellular protein that is confined predominantly to the subretinal space. Monkey retinas incubated in vitro in medium containing [³H]leucine were shown to synthesize and secrete this protein into the medium, a conclusion that was confirmed by immunoprecipitation with an immunoglobulin fraction prepared from rabbit antibovine IRBP serum. Virtually no other labeled proteins were detectable in the medium. It is concluded that interstitial retinol-binding protein meets many of the requirements for a putative transport protein implicated in the transfer of retinol between the pigment epithelium and retina during the visual cycle, and that the neural retina may play an important role in regulating its amount in the subretinal space.     

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.     

High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

Molecular Cloning of a Lobster Gαq Protein Expressed in Neurons of Olfactory Organ and Brain

Abstract: We have isolated from an American lobster ( Homarus americanus ) olfactory organ cDNA library a clone, hGα_q, with >80% identity to mammalian and arthropod Gα_q sequences. In brain and olfactory organ, hGα_q mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. Gα_q protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hGα_q plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hGα_q mRNA species (6 kb) in the olfactory organ, and the localization of hGα_q mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hGα_q is to mediate olfactory transduction.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Distribution of interstitial retinol-binding protein (IRBP) in the vertebrates

Immunoblots of interphotoreceptor matrix preparations from 20 species belonging to six vertebrate classes were probed with antibodies against bovine interstitial retinol-binding protein (b-IRBP). Each preparation displayed an immunoreactive protein band. In the Osteichthyes, the apparent Mr of this band was 67,600 +/- 2,700 (mean +/- SD, n = 8). In two of the Osteichthyes, the band was resolved into a closely spaced doublet. Including previously published data for five mammals and one amphibian, species from the other classes (Chondrichthyes, one species; Amphibia, four species; Reptilia, one species; Aves, one species; Mammalia, nine species) had IRBPs with Mr that averaged 2.0 times that of the Osteichthyes, namely 134,200 +/- 8,600 (mean +/- SD, n = 17). Frog IRBP was very similar to mammalian IRBP in terms of its immunohistochemical distribution (determined with rabbit anti-frog IRBP antibodies), its molecular weight (sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography), retinol- and concanavalin A-binding ability, and because it was synthesized and secreted in vitro by the isolated retina but not by the pigmented layers of eye. Goldfish IRBP apparently binds exogenous (3H)-retinol but does not bind concanavalin A and has about half the Mr of frog IRBP. The occurrence of IRBP-like proteins cross-reacting with anti b-IRBP antibodies in the interphotoreceptor matrix of all six major vertebrate classes is consistent with the hypothesis that IRBP is an important element in the vertebrate visual cycle.