Expression, localization and effects on virulence of the cysteine-rich 8 kDa protein of Potato mop-top virus.

Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.

The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 A resolution.

We present the three-dimensional structure of Trichoderma reesei endoglucanase 3 (Cel12A), a small, 218 amino acid residue (24.5 kDa), neutral pI, glycoside hydrolase family 12 cellulase that lacks a cellulose-binding module. The structure has been determined using X-ray crystallography and refined to 1.9 A resolution. The asymmetric unit consists of six non-crystallographic symmetry-related molecules that were exploited to improve initial multiple isomorphous replacement phasing, and subsequent structure refinement. The enzyme contains one disulfide bridge and is glycosylated at Asp164 by a single N-acetyl glucosamine residue. The protein has the expected fold for a glycoside hydrolase clan-C family 12 enzyme. It contains two beta-sheets, of six and nine strands, packed on top of one another, and one alpha-helix. The concave surface of the nine-stranded beta-sheet forms a large substrate-binding groove in which the active-site residues are located. In the active site, we find a carboxylic acid trio, similar to that of glycoside hydrolase families 7 and 16. The strictly conserved Asp99 hydrogen bonds to the nucleophile, the invariant Glu116. The binding crevice is lined with both aromatic and polar amino acid side-chains which may play a role in substrate binding. The structure of the fungal family 12 enzyme presented here allows a complete structural characterization of the glycoside hydrolase-C clan.

The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV genome, undergoes structural changes in naturally infected and experimentally inoculated plants.

Molecular data on Potato mop-top virus (PMTV), genus Pomovirus, is currently mostly based on analysis of two Scottish isolates, PMTV-S and PMTV-T. Here we report the complete sequence of "the coat protein (CP) encoding RNA" of an isolate of PMTV obtained from the field in Sweden. Our data show that this RNA (3134 nt) is the second largest of the three RNA species in the tripartite PMTV genome, and it should, therefore, be referred to as RNA 2. This nomenclature is consistent with other pomoviruses. The sequence of the readthrough domain (RT) of RNA 2 was determined also in two additional field isolates of PMTV from Finland and Denmark. All three isolates contained a novel, 109 nucleotides long sequence at the 3'-end of the RT, which has not been found in PMTV-S and PMTV-T. Hence, our data suggest that the RNA 2 sequences previously described for the isolates PMTV-T and PMTV-S may represent deletion derivatives. The C-proximal half of RT contained many amino acid (aa) differences among the isolates, in contrast to only few aa differences in the N-proximal part of RT. Deletion variants of RNA 2 were generated from the Nordic isolates in potato tubers infected in the field, and in the mechanically inoculated test plants. All deletions started within a short region (18 nt) and removed 558-940 nt from the 3'-end of RT region. This study for the first time describes the full-length sequence of the "CP-encoding RNA" (RNA2) of PMTV, and reveals considerable aa variability and occurrence of deletion variants of RT in the field isolates of PMTV.

Mutations that affect ligand binding to the Escherichia coli aspartate receptor: implications for transmembrane signaling.

Three arginine residues of the binding site of the Escherichia coli aspartate receptor contribute to its high affinity for aspartate (K(d) approximately 3 microm). Site-directed mutations at residue 64 had the greatest effect on aspartate binding. No residue could substitute for the native arginine; all changes resulted in an apparent K(d) of approximately 35 mm. These mutations had little impact on maltose responses. At residue Arg-69, a lysine substitution was least disruptive, conferring an apparent K(d) of 0.3 mm for aspartate. Results obtained for an alanine mutant were similar to those with cysteine and histidine mutants (K(d) approximately 5 mm) indicating that side chain size was not an important factor here. Proline and aspartate caused more severe defects, presumably for reasons related to conformation and charge. The impact of residue 69 mutations on the maltose response was small. Mutations at Arg-73 had similar effects on aspartate binding (K(d) 0.3-7 mm) but more severe consequences for maltose responses. Larger side chains resulted in the best aspartate binding, implying steric considerations are important here. Signaling in the mutant proteins was surprisingly robust. Given aspartate binding, signaling occurred with essentially wild-type efficiency. These results were evaluated in the context of available structural data.

Chemotaxis receptors: a progress report on structure and function.

Recent biochemical and structural studies have provided many new insights into the structure and function of bacterial chemoreceptors. Aspects of their ligand binding, conformational changes, and interactions with other members of the signaling pathway are being defined at the structural level. It is anticipated that the combined effort will soon provide a detailed, unified view of an entire response system.

Evidence of an age-related decrease in intestinal responsiveness to vitamin D: relationship between serum 1,25-dihydroxyvitamin D3 and intestinal vitamin D receptor concentrations in normal women.

Although aged rats reportedly have reduced intestinal vitamin D receptor (VDR) concentrations, it is unclear whether an analogous age-related defect occurs in man. Thus, we assessed the interrelationship among serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], calcium absorption and intestinal VDR in 44 healthy, ambulatory women, ages 20-87 yr. Fractional calcium absorption was measured after oral administration of 45Ca (20 mg CaCl2 as carrier); serum 1,25-(OH)2D3, by the calf thymus binding assay; and serum intact PTH, by a two-site immunochemiluminometric assay. Vitamin D receptor concentration was measured, by a new immunoradiometric assay, in biopsy specimens taken from the second part of the duodenum during gastroduodenoscopy in 35 of the women. Despite an age-related increase in serum PTH (r = 0.48; P less than 0.001) and in serum 1,25-(OH)2D3 concentration (r = 0.32; P less than 0.05), intestinal VDR concentration decreased with age (r = -0.38; P = 0.03) and fractional calcium absorption did not change with age. Although a contribution of decreased 25-hydroxyvitamin D 1 alpha-hydroxylase activity to the blunting of the increase in serum 1,25-(OH)2D3 concentration late in life is not excluded, the data are far more consistent with impaired intestinal responsiveness to 1,25-(OH)2D3 action. This defect could lead to compensatory increases in PTH secretion and 1,25-(OH)2D3 production which maintain calcium absorption and serum ionic calcium, but at the expense of increased bone loss.

Tissue distribution of the 1,25-dihydroxyvitamin D3 receptor in the male rat.

Tissue distribution of 1,25-dihydroxyvitamin D3 receptors was studied in male rats using a quantitative immunoradiometric assay. Extracts were prepared from 16 different rat tissues and assayed for 1,25-dihydroxyvitamin D3 receptor. Measurable levels of receptor were detected in intestine, stomach, kidney, bone thyroid/parathyroid, skin, liver, spleen, heart and lung. The highest levels were found in the proximal small intestine and colon, containing over 1000 fmol/mg total protein, while ileum and kidney contained one-half and one-fourth of this amount, respectively. Other parts of the vitamin D endocrine system, including bone, thyroid/parathyroid and skin, contained moderate levels of receptor of 40 to 80 fmol/mg, while lung, heart, stomach, spleen and liver had levels at or below 20 fmol/mg. No 1,25-dihydroxyvitamin D3 receptor was detected in cerebrum, cerebellum or skeletal muscle. The data support a wide-spread role for 1,25-dihydroxyvitamin D3 on cellular processes and suggest a more important role for vitamin D in colon.

1,25-Dihydroxyvitamin D3 receptors in human carcinomas: a pilot study.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] receptor concentration was measured by an accurate immunoradiometric assay in primary tumors from 10 patients with colorectal carcinoma and 11 patients with non-small cell carcinoma of the lung. Measurements were also performed on a noncancerous sample of the same origin as the tumor from each patient. All of the tumors contained receptor with a mean concentration of 123.4 fmol/mg protein for colorectal and 75.1 fmol/mg protein for lung carcinoma. Compared to normal tissue from the same patient, 100% of the lung tumors and 70% of the colorectal tumors had significantly higher levels of the 1,25-dihydroxyvitamin D3 receptor. A correlation was found between well-differentiated colorectal tumors with no or few metastases and high levels of 1,25-dihydroxyvitamin D3 receptor. Receptor concentration was also assayed in metastatic lesions of malignant melanoma from 7 patients. 1,25-Dihydroxyvitamin D3 receptor was present in 85% of the metastases at a mean level of 26 fmol/mg protein. For these patients an inverse correlation was found between receptor level and age. The results obtained in this pilot study suggest that an alteration in 1,25-dihydroxyvitamin D3 receptor regulation may occur in vivo when a cell undergoes malignant transformation.

Serum calcium and vitamin D regulate 1,25-dihydroxyvitamin D3 receptor concentration in rat kidney in vivo.

The effects of vitamin D status, serum calcium, and serum phosphorus levels on 1,25-dihydroxyvitamin D receptor levels in kidney were investigated. Weanling rats were fed for 4 weeks on a diet with various levels of calcium and phosphorus with or without vitamin D. The 1,25-dihydroxyvitamin D3 receptor concentration in kidney was determined by an immunoradiometric assay. In the absence of vitamin D, total receptor concentration is increased 2-fold by an increase in serum calcium concentration. At normal serum calcium levels, the administration of vitamin D resulted in a 5-fold increase in receptor concentration. In hypocalcemic animals, however, vitamin D did not change receptor levels. Serum phosphorus levels could not be linked to any changes in 1,25-dihydroxyvitamin D3 receptor concentration. This study demonstrates that serum calcium levels and vitamin D regulate 1,25-dihydroxyvitamin D3 receptor concentration in vivo in kidney. On the other hand, vitamin D is unable to exert control of receptor levels in kidney under hypocalcemic conditions.

1,25-Dihydroxyvitamin D3 up-regulates the 1,25-dihydroxyvitamin D3 receptor in vivo.

The level of mRNA encoding the 1,25-dihydroxyvitamin D3 receptor in the intestine of vitamin D-deficient rats given 1,25-dihydroxyvitamin D3 was determined by Northern blot analysis using a 32P-labeled cDNA probe to the 1,25-dihydroxyvitamin D3 receptor. mRNA levels increased 10-fold above deficiency levels at 6 and 12 hr after an intravenous dose of 1,25-dihydroxyvitamin D3, returning to predosing levels at 24 hr. Total receptor protein level determined by an immunoradiometric assay was increased 2-fold at 12 hr. No change in unoccupied receptor levels determined by ligand-binding assay was observed during this period. These results suggest that 1,25-dihydroxyvitamin D3 increases receptor mRNA and total receptor level to maintain constant levels of unoccupied receptor.

An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor.

A ligand-independent, quantitative assay has been developed for the measurement of 1,25-dihydroxyvitamin D receptor utilizing purified receptor from pig intestine as a standard and two high affinity monoclonal antibodies directed to two different epitopes on the receptor. In this assay a fixed amount of 125I-labeled antibody is incubated with a fixed amount of a second antireceptor antibody linked to biotin and increasing amounts of purified receptor protein or sample. Antibody-receptor complexes can then be immunoprecipitated with avidin-Sepharose beads and counted. This method is highly reproducible and can detect 150 pg of 1,25-dihydroxyvitamin D3 receptor in crude extracts with intra- and interassay coefficients of variation of 8.6 and 18.2%. The monoclonal antibodies used recognize both native and denatured receptors from several different species, including human. This immunoradiometric assay should prove useful for studies of receptor regulation, occupancy, distribution, and turnover.