In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.     

In situ measurement of methane oxidation in groundwater by using natural-gradient tracer tests.

Methane oxidation was measured in an unconfined sand and gravel aquifer (Cape Cod, Mass.) by using in situ natural-gradient tracer tests at both a pristine, oxygenated site and an anoxic, sewage-contaminated site. The tracer sites were equipped with multilevel sampling devices to create target grids of sampling points; the injectate was prepared with groundwater from the tracer site to maintain the same geochemical conditions. Methane oxidation was calculated from breakthrough curves of methane relative to halide and inert gas (hexafluroethane) tracers and was confirmed by the appearance of 13C-enriched carbon dioxide in experiments in which 13C-enriched methane was used as the tracer. A Vmax for methane oxidation could be calculated when the methane concentration was sufficiently high to result in zero-order kinetics throughout the entire transport interval. Methane breakthrough curves could be simulated by modifying a one-dimensional adevection-dispersion transport model to include a Michaelis-Menten-based consumption term for methane oxidation. The Km values for methane oxidation that gave the best match for the breakthrough curve peaks were 6.0 and 9.0 microM for the uncontaminated and contaminated sites, respectively. Natural-gradient tracer tests are a promising approach for assessing microbial processes and for testing in situ bioremediation potential in groundwater systems.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Role of cysteines 640, 656, and 661 in steroid binding to rat glucocorticoid receptors.

The involvement of a vicinally spaced dithiol group in steroid binding to the glucocorticoid receptor has been deduced from experiments with the thiol-specific reagent methyl methanethiolsulfonate and the vicinal dithiol-specific reagent sodium arsenite. The vicinally spaced dithiol appears to reside in the 16-kDa trypsin fragment of the receptor, which is thought to contain 3 cysteines (Cys-640, -656, and -661 of the rat receptor) and binds hormone with an approximately 23-fold lower affinity than does the intact 98-kDa receptor. We now report that the steroid binding specificity of preparations of this 16-kDa fragment and the intact receptor are virtually identical. This finding supports our designation of the 16-kDa fragment as a steroid-binding core domain and validates our continued use of this tryptic fragment in studies of steroid binding. To identify the cysteines which comprise the vicinally spaced dithiol group, and to examine further the role of cysteines in steroid binding, a total of five point mutant receptors were prepared: cysteine-to-serine for each suspected cysteine, cysteine-to-glycine for Cys-656, and the C656,661S double mutant. Unexpectedly, each receptor with a single point mutation still bound steroid. Even the double mutant (C656,661S) bound steroid with wild type affinity. These results suggest that none of these cysteines are directly required either for steroid binding to the glucocorticoid receptor or for heat shock protein 90 association with the receptor. However, the presence of Cys-656 was obligatory for covalent labeling of the receptor by [3H]dexamethasone 21-mesylate. Studies with preparations of the 98 and 16 kDa forms of these mutant receptors revealed both that Cys-656 and -661 comprise the vicinally spaced dithiols reacting with arsenite and that any two of the three thiols could form an intramolecular disulfide after treatment with low concentrations of methyl methanethiolsulfonate. These data, in conjunction with those from experiments on the effects of steric bulk on various receptor functions, support a model for the ligand binding cavity of the receptor that involves all three thiols in a flexible cleft but where thiol-steroid interactions are not essential for binding.     

Relationship of 25-hydroxyvitamin D3 side chain structure to biological activity.

27-nor-25-Hydroxyvitamin D3, 26,27-bisnor-25-hydroxyvitamin D3, and 22-27-hexanor-20-hydroxyvitamin D3 and the corresponding 5,6-trans isomers have been synthesized. All compounds were tested for their ability to induce intestinal calcium transport and bone calcium mobilization in normal and anephric rats. The 27-nor- and 26,27-bisnor-25-hydroxyvitamin D3 analog are capable of stimulating intestinal calcium transport and bone calcium mobilization in normal rats but are 10 to 100 times less active than 25-hydroxyvitamin D3. Although these analogs are inactive in anephric rats, their corresponding 5,6-trans isomer are capable of stimulating both intestine and bone activity in these animals. The 22-27-hexanor-20-hydroxyvitamin D3 and its corresponding 5,6-trans isomer are incapable of stimulating either intestinal calcium transport or bone calcium mobilization. These results suggest that minor alterations in the side chain significantly decrease the biopotency of 25-hydroxyvitamin D3. Since these analogs are biologically active in normal but not in anephric animals, it appears that the kidney 1alpha-hydroxylation is necessary for activity. Since 22-27-hexanor=20-hydroxyvitamin D3 and its corresponding 5,6-trans analog are biologically inactive, it is likely that at least part of the side chain is necessary for 25-hydroxyvitamin D3 to stimulate intestinal calcium transport and bone calcium mobilization.     

Substructure of the glomerular slit diaphragm in freeze-fractured normal rat kidney

In the renal glomerulus, the narrow slits between adjacent epithelial podocytes are bridged by a diaphragm (2, 8, 11). In rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde (TAG), it has recently been discovered that this diaphragm has a highly ordered, isoporous substructure (9). It consists of a regular array of alternating cross bridges extending from the podocyte plasma membranes to a centrally running filament. This zipperlike pattern results in two rows of rectangular pores, approximately 40 X 140 A in cross section, dimensions consistent with the proposed role of the diaphragm as an important filtration barrier to plasma proteins (6). In the present study, we found in freeze-cleaved and in freeze-etched normal rat glomeruli that the surface of the slit diaphragm has an appearance conforming to the pattern found in sectioned material.     

Strain differences in tolerance induction to human gamma-globulin subclasses: dependence on macrophages.

BALB/c and DBA/2 mice differ with respect to ease of tolerance induction with HGG, BALB/c mice being the resistant strain. When tested for susceptibility to tolerance induction with individual IgG subclasses, both strains were easily rendered unresponsive with IgG₁ and IgG₂ and less so with IgG₄. A strain difference appeared with IgG₃, where only BALB/c mice showed complete resistance to tolerance induction. Mixtures of the IgG subclasses Showed that IgG₁ and IgG₂ accounted for most of the tolerance to whole HGG seen in both strains, while addition of IgG₃ to the mixture made DBA/2 completely tolerant but reversed the trend toward tolerance in the BALB/c mice. By rosette assay it was found that BALB/c macrophages had receptors for IgG₃ (and to a lesser extent for IgG₄). These results are consistent with the hypothesis that resistance to tolerance induction with HGG is dependent on the presence of a receptor on the macrophage for a minor IgG subclass.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.