Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells.

GRP78 is a resident protein of the endoplasmic reticulum (ER) and a member of the glucose regulated protein (GRP) family. Many secretion incompetent proteins are found in stable association with GRP78 and are retained in the ER. Some proteins which are destined for secretion transiently associate with GRP78. To further increase our understanding of the role of GRP78 in secretion, we have stably overexpressed GRP78 in Chinese hamster ovary (CHO) cells and examined the effect on protein secretion and the stress response. GRP78 overexpressing cells treated with tunicamycin or A23187 exhibited a reduced induction of endogenous GRP78 and GRP94 mRNAs compared to wild-type CHO cells. This suggests that GRP78 overexpression either alleviates the stress or is directly involved in signaling stress-induced expression of GRPs. Transient expression of secreted proteins was used to measure secretion efficiency in the GRP78 overexpressing cells. Secretion of von Willebrand factor and a mutant form of factor VIII, two proteins which transiently associate with GRP78, was reduced by GRP78 overexpression. In contrast, secretion of M-CSF, which was not detected in association with GRP78, was unaffected. This indicates that elevated levels of GRP78 may increase stable association and decrease the secretion efficiency of proteins which normally transiently associate with GRP78. These results indicate that one function of GRP78 is selective protein retention in the ER.     

Molecular cloning and characterization of a complete Chinese hamster provirus related to intracisternal A particle genomes.

We report here the nucleotide sequence of a full-length Chinese hamster genomic proviral element, CHIAP34. CHIAP34 is 6,403 bp long with long terminal repeats of 311 bp at each end. The genetic organization of CHIAP34 was determined by comparison with intracisternal A particle (IAP) genetic elements from the mouse and Syrian hamster. Extensive homology at the nucleotide and deduced amino acid sequence levels was observed between CHIAP34 and the mouse and Syrian hamster IAP elements. CHIAP34 may represent a defective Chinese hamster IAP genetic element. The gag gene consists of 837 codons, of which 558 codons are in a single long open reading frame followed by several frameshifts. The pol gene begins with a -1 frameshift and consists of a long open reading frame of 753 codons followed by a short open reading frame of 103 codons. The putative env region contains multiple termination codons in all reading frames. CHIAP34 is representative of the predominant retroviral elements in the Chinese hamster ovary cell genome present at around 80 copies per haploid genome.     

Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

Canopy photosynthesis and its relationship to plant productivity in near-isogenic cotton lines differing in leaf morphology

A 2-year study was conducted to determine the relationships between plant canopy photosynthesis, canopy light interception, and plant productivity of cotton (Gossypium hirsutum L.) exhibiting differing leaf morphologies. The near-isogenic lines were from a single background (MD 65-11) and represented the leaf shapes Normal (small leaf lobing), Sub-Okra (intermediate leaf lobing), Okra (large leaf lobing), and Super Okra (severe leaf lobing). The F₁ of a cross Normal × Okra (intermediate leaf lobing) and the F₂ (segregating 1:2:1 for Normal Sub-Okra, and Okra, respectively) were also grown. Reduced plant canopies were produced by Okra and Super Okra lines, which translated into increased light penetration to the ground, and hence, in reduced canopy photosynthesis. Integrated canopy photosynthesis (ICAP) was significantly associated with light interception by the plant canopy. Part of the remaining variability in ICAP was associated with confounding factors associated with plant maturity and other unmeasured genotypic factors. Intermediate (F₁ and Sub-Okra) and normal leaf types displayed the largest ICAP values in both years. Lint production was positively related to ICAP (R² = 0.53). The combination of high ICAP values and competitive lint yields indicate that intermediate lobed leaf morphologies offer promise as productive sources of physiological variation for cotton germplasm development.     

Physical and surface properties of insect apolipophorin III

Apolipophorin III (apoLp-III) from Manduca sexta has a molecular weight of 18,100. Based on its hydrodynamic properties (sedimentation and diffusion coefficients, frictional ratio, intrinsic viscosity) and its behavior during gel permeation chromatography, we concluded that apoLp-III is a prolate ellipsoid with an axial ratio of about 3. The circular dichroic spectrum of apoLp-III suggests that the protein contains approximately 50% alpha-helix. At the air-water interface, apoLp-III forms a monolayer which is gaseous at surface pressures less than or equal to 1 dyne/cm. The isotherm of this phase yields an excluded molecular area of 3800 A2/molecule (23 A2/amino acid). At a surface pressure of 22.1 dynes/cm, the monolayer undergoes a phase transition reminiscent of a first-order phase transition of pure lipids. The monolayer can be compressed in this surface pressure range to an area per molecule of 480 A2 (2.9 A2/amino acid). Since a globular protein of molecular weight 18,100 could occupy an area of only about 2000 A2 when bound to a surface, it is suggested that in the expanded state, apoLp-III must unfold on the surface, whereas in the compressed state, the molecule is oriented with its minor axis parallel to the water surface. ApoLp-III binds with high affinity (Kd = 1.9 X 10(-7)M) to both phosphatidylcholine- and diacylglycerol-coated polystyrene beads. All of these results are consistent with the proposal that apoLp-III plays a key role in increasing the capacity of the insect lipoprotein, lipophorin, to transport diacylglycerol by stabilizing the increment of lipid-water interface that results from diacylglycerol uptake.     

Metabolic changes following eccentric exercise in trained and untrained men

The effects of one 45-min bout of high-intensity eccentric exercise (250 W) were studied in four male runners and five untrained men. Plasma creatine kinase (CK) activity in these runners was higher (P less than 0.001) than in the untrained men before exercise and peaked at 207 IU/ml 1 day after exercise, whereas in untrained men the maximum was 2,143 IU/ml 5 days after exercise. Plasma interleukin-1 (IL-1) in the trained men was also higher (P less than 0.001) than in the untrained men before exercise but did not significantly increase after exercise. In the untrained men, IL-1 was significantly elevated 3 h after exercise (P less than 0.001). In the untrained group only, 24-h urines were collected before and after exercise while the men consumed a meat-free diet. Urinary 3-methylhistidine/creatinine in the untrained group rose significantly from 127 mumol/g before exercise to 180 mumol/g 10 days after exercise. The results suggest that in untrained men eccentric exercise leads to a metabolic response indicative of delayed muscle damage. Regularly performed long distance running was associated with chronically elevated plasma IL-1 levels and serum CK activities without acute increases after an eccentric exercise bout.     

Molecular basis of host range variation in avian retroviruses.

Previous genetic analysis has localized the region of the Rous sarcoma virus (RSV) env gene responsible for host range specificity to that encoding the middle one-third of gp85. To better understand the host range determinants, the relevant regions of the genomes of infectious molecular clones of the transformation-defective Prague strain of RSV, subgroup B (Pr-RSV-B) and Rous-associated virus 0 (RAV-0) (subgroup E) were sequenced and compared with the sequence of Pr-RSV-C. This comparative analysis identified two variable regions of low amino acid sequence homology flanked by highly conserved amino acid sequences. The first variable region (hr1) begins at base 5654 in the Pr-RSV-C sequence and encodes 32 amino acids. The second variable region (hr2) begins at base 5846 and encodes 27 amino acids. To test the role of the variable regions in host range specificity, we determined the sequence of this region of the env gene of NTRE-4, a recombinant virus between Pr-RSV-B and RAV-0 which exhibits an extended host range. This analysis revealed that the recombinant subgroup-encoding region of NTRE-4 is composed of 200 bases of RAV-0 sequence, including hr2, flanked by sequences which are otherwise of Pr-RSV-B origin. This study indicates that hr1 and hr2 are the domains of gp85 responsible for host range determination in avian retroviruses.     

Large scale selection of aluminum-resistant mutants from plant cell culture: expression and inheritance in seedlings

Summary A large number of aluminum-resistant variants, selected from non-mutagenized homozygous diploid cell cultures of Nicotiana plumbaginifolia Viv., are characterized. Of 115 variants cloned and reselected from single cells, 67 retained Al resistance in callus cultures after 6–9 months of growth in the absence of Al. There was no association between Al resistance and callus growth in the absence of Al, suggesting that the Al-resistant phenotype is not detrimental in the absence of Al challenge and that Al resistance is not the result of increased vigor. Plants regenerated from initially resistant callus lines that subsequently lost their resistance failed, with one exception, to transmit resistance to their seedling progeny. Fertile plants were regenerated from 40 of the 67 variants that retained stable Al resistance in callus culture. All 40 transmitted Al resistance to their seedling progeny (selfed and backcrossed) in segregation ratios expected for a single dominant mutation. The selfed progeny of many variants also segregated for recessive lethal mutations which were attributed to independent mutations that occurred during cell culture.     

In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate.

The translation of poliovirus RNA in rabbit reticulocyte lysate was examined. Translation of poliovirus RNA in this cell-free system resulted in an electrophoretic profile of poliovirus-specific proteins distinct from that observed in vivo or after translation in poliovirus-infected HeLa cell extract. A group of proteins derived from the P3 region of the polyprotein was identified by immunoprecipitation, time course, and N-formyl-[35S]methionine labeling studies to be the product of the initiation of protein synthesis at an internal site(s) located within the 3'-proximal RNA sequences. Utilization of this internal initiation site(s) on poliovirus RNA was abolished when reticulocyte lysate was supplemented with poliovirus-infected HeLa cell extract. Authentic P1-1a was also synthesized in reticulocyte lysate, indicating that correct 5'-proximal initiation of translation occurs in that system. We conclude that the deficiency of a component(s) of the reticulocyte lysate necessary for 5'-proximal initiation of poliovirus protein synthesis resulted in the ability of ribosomes to initiate translation on internal sequences. This aberrant initiation could be corrected by factors present in the HeLa cell extract. Apparently, under certain conditions, ribosomes are capable of recognizing internal sequences as authentic initiation sites.     

Determinants for receptor interaction and cell killing on the avian retrovirus glycoprotein gp85

The virion envelope glycoprotein gp85 confers a high degree of subgroup specificity for interaction with distinct cell receptors. Specific subgroups of gp85 have been associated with a cytopathic virus-cell interaction, most likely resulting from reduced resistance to superinfection, which allows the buildup of excessive amounts of viral DNA. Previous nucleotide sequence analysis of the gp85 coding region identified small regions of variable amino acid sequence within a conserved framework. To define the role of these variable regions we constructed a series of molecular clones carrying novel combinations of variable regions from viruses. Analysis of rescued virus shows that receptor binding is determined by the interaction of two major regions and one minor region in the middle of gp85. Cytopathogenicity is not associated with any specific variable region but rather with the ability to recognize the subgroup B receptor on chicken cells.