A single U/C nucleotide substitution changing alanine to valine in the beet necrotic yellow vein virus P25 protein promotes increased virus accumulation in roots of mechanically inoculated, partially resistant sugar beet seedlings.

Beet necrotic yellow vein virus (BNYVV) A type isolates E12 and S8, originating from areas where resistance-breaking had or had not been observed, respectively, served as starting material for studying the influence of sequence variations in BNYVV RNA 3 on virus accumulation in partially resistant sugar beet varieties. Sub-isolates containing only RNAs 1 and 2 were obtained by serial local lesion passages; biologically active cDNA clones were prepared for RNAs 3 which differed in their coding sequences for P25 aa 67, 68 and 129. Sugar beet seedlings were mechanically inoculated with RNA 1+2/RNA 3 pseudorecombinants. The origin of RNAs 1+2 had little influence on virus accumulation in rootlets. E12 RNA 3 coding for V(67)C(68)Y(129) P25, however, enabled a much higher virus accumulation than S8 RNA 3 coding for A(67)H(68)H(129) P25. Mutants revealed that this was due only to the V(67) 'GUU' codon as opposed to the A(67) 'GCU' codon.

NMR in the SPINE Structural Proteomics project.

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

Zygocactus virus X-based expression vectors and formation of rod-shaped virus-like particles in plants by the expressed coat proteins of Beet necrotic yellow vein virus and Soil-borne cereal mosaic virus.

Expression vectors were constructed from 35S promoter-containing full-length cDNA clones of Zygocactus virus X (ZVX). The expression of foreign genes was driven by the ZVX coat protein (cp) subgenomic promoter. It was successful only when the variable region downstream of the conserved putative promoter region GSTTAAGTT(X(12-13))GAA was retained. Most of the ZVX cp gene, except for a short 3' part, was replaced by the corresponding sequence of the related Schlumbergera virus X (SVX) and its cp subgenomic promoter to enable encapsidation of the transcribed RNA by an SVX/ZVX hybrid cp. Vector-expressed cp of Beet necrotic yellow vein virus (BNYVV) assembled in Chenopodium quinoa, Tetragonia expansa and Beta vulgaris leaves into particles resembling true BNYVV particles. The virus produced from these constructs retained its ability to express BNYVV cp in local infections during successive passages on C. quinoa. This ability was lost, however, in the rarely occurring systemic infections.

Molecular characterization of isolates of anagyris vein yellowing virus, plantago mottle virus and scrophularia mottle virus -- comparison of various approaches for tymovirus classification.

The complete nucleotide sequences were determined for the genomic RNAs of three tymoviruses, i.e. isolates of anagyris vein yellowing virus (AVYV), plantago mottle virus (PlMoV) and scrophularia mottle virus (SrMV) which are all serologically closely related to ononis yellow mosaic virus (ibid) and to Nemesia ring necrosis virus (NeRNV), a recently described recombinant virus which is widely spread in commercially grown ornamental plant species belonging to the Scrophulariaceae. Total nucleotide and coat protein amino acid sequence identities revealed similar groupings in the genus tymovirus as serological studies did. The latter, however, tended to suggest much closer relationships than the molecular data and may fail to recognise the distinctiveness of new tymovirus species. The usefulness of various species demarcation criteria for the classification of tymoviruses is discussed.

Nemesia ring necrosis virus: a new tymovirus with a genomic RNA having a histidylatable tobamovirus-like 3' end.

The complete nucleotide sequence of the genomic RNA of the new virus Nemesia ring necrosis virus (NeRNV), which is widespread in various ornamental plant species belonging to the Scrophulariaceae and Verbenaceae, has been determined. Based on its gene content, the folding properties of its 5'-untranslated region and in vitro translation experiments, NeRNV RNA is a typical tymovirus RNA. Its 3' end, however, differs greatly from those of the valine-specific tymoviral RNAs that have been analysed previously. It can be folded into an upstream pseudoknot domain and a histidine-specific tRNA-like structure, a combination that, so far, has been found only in tobamoviral RNAs. The identity elements found in NeRNV RNA for recognition by yeast histidyl-tRNA synthetase are more similar to those of yeast tRNAHis than the ones found in tobacco mosaic virus RNA. As a result NeRNV RNA can be charged with histidine even more efficiently than tobacco mosaic virus RNA.

Molecular characterisation of potexviruses isolated from three different genera in the family Cactaceae.

The genome properties of three potexviruses which previously had been isolated from different genera in the family Cactaceae and had been found to be only distantly related serologically have been studied. The sequence of the 3040 3' terminal nucleotides of the genomic RNA of isolate K11 from Schlumbergera bridgesii and the complete RNA sequences of isolates B1 and CC10 from Zygocactus sp. and Opuntia sp., respectively, were determined. Starting sequences were obtained by means of immunocapture reverse transcription PCR using primers derived from highly conserved sequences in other potexviral RNAs. The known parts of the sequences were extended by means of random-primed cDNAs and specific primers derived from the known parts of the sequences. The genome structure of the three viruses resembles that of other potexviruses. The conserved motifs typical for replication-associated proteins, triple gene block (TGB) proteins and coat proteins of potexviruses were readily identified in the translation products of the five open reading frames. The 3' untranslated regions of the three RNAs are folded into secondary structures containing three characteristic hairpins. Rather low percentages of amino acid sequence identities ranging from 62% to 76% for the coat proteins and 41% to 49% for TGB proteins 3 suggest that these viruses should be regarded as distinct virus species for which the names Zygocactus virus X, Schlumbergera virus X and Opuntia virus X are proposed. It is also suggested that the name Cactus virus X which originally was coined for all three virus isolates should no longer be used.

A New York isolate of Soil-borne wheat mosaic virus differs considerably from the Nebraska type strain in the nucleotide sequences of various coding regions but not in the deduced amino acid sequences.

A wheat-infecting furovirus found in Tompkins County, New York, U.S.A. was identified as a strain of Soil-borne wheat mosaic virus (SBWMV) by means of sequence analyses of portions of its RNA 1 and 2. The nucleotide sequences of several of its genes differed by c. 9 to 12% from those of the corresponding genome regions of the Nebraska type strain of SBWMV. The deduced amino acid sequences of the putative translation products, however, suggested much closer relationships. Thus, the amino acid sequences of the coat proteins of the two virus strains were 100% identical despite the fact that their coding regions differed in as many as 68 nucleotide positions. The New York (NY) strain of SBWMV is possibly closely related to an isolate from Illinois for which so far only the nucleotide sequences of its coat protein gene and the 5' untranslated region of its RNA 2 are known.

Strong P=P pi Bonds: The First Synthesis of a Stable Phosphanyl Phosphenium Ion.

A 35-fold excess of methyl triflate (2) is required to quantitively prepare 3, the first phosphanyl phosphenium ion, from diphosphene 1. Experimental data and calculations indicate that the P=P bond becomes stronger upon alkylation.

Beet soil-borne virus RNA 1: genetic analysis enabled by a starting sequence generated with primers to highly conserved helicase-encoding domains.

The complete sequence of the 5834 nucleotides of RNA 1 of beet soil-borne furovirus (BSBV, Ahlum isolate) was determined using a PCR product obtained with primers to highly conserved coding regions for helicase-like proteins in RNA 1 of furo-, hordei- and tobraviruses as a starting sequence. Unknown parts of the sequence upstream and downstream of this starting sequence were amplified by means of RT-PCR techniques using combinations of specific and random primers. BSBV RNA 1 contains one large ORF for a readthrough protein with a molecular mass of 204 kDa (204K protein) which is interrupted internally by a UAA stop codon terminating the coding region for a protein of 145 kDa (145K protein). The N- and C-terminal parts of the 145K protein and the readthrough domain of the 204K protein contain methyltransferase, helicase and RNA-dependent RNA polymerase motifs, respectively. Unlike other furo- and tobraviruses BSBV contains no further genes on its RNA 1.

Beet soil-borne virus RNA 2: similarities and dissimilarities to the coat protein gene-carrying RNAs of other furoviruses.

The complete sequence of the 3454 nt of RNA 2 of the Ahlum isolate of beet soil-borne furovirus (BSBV) has been determined starting with two short stretches of cloned cDNA. Unknown parts of the sequence were amplified by means of RT-PCR techniques using combinations of specific and random primers. BSBV RNA 2 is more similar in its genetic organization to potato mop top virus (PMTV) RNA 3 than to any other furoviral RNA, although it is more than 1100 nt longer. Its 3'-end, unlike that of PMTV RNA 3, has the potential to fold into a tRNA-like structure. It contains one large open reading frame for a readthrough protein with a molecular mass of 104 kDa (104K protein) which is interrupted internally by an amber stop codon terminating the coding region for a protein of 19 kDa (19K), most likely the viral coat protein (CP). The readthrough domain of the 104K protein is much larger than that of PMTV, but the N- and C-proximal portions of these domains are similar for the two viruses. No serological relationships were found between the particles of the two viruses, although more than 50% of the amino acid sequences of the putative CPs are identical.

Clarithromycin-induced mania in two patients with AIDS.

Acute psychosis was observed in two patients with AIDS who were treated with clarithromycin for disseminated Mycobacterium avium complex infection. The psychosis resolved when treatment with clarithromycin was discontinued and recurred when it was resumed. An adverse response to clarithromycin therapy is a rare but curable cause of acute psychosis in patients with AIDS.

Nutrient uptake and organic acid anion metabolism in lupins and peas supplied with nitrate.

Unlike many plants reported in the literature, lupins do not excrete OH(-) in amounts equivalent to the net excess of inorganic anion uptake over inorganic cation uptake. To investigate the mechanisms involved in the maintenance of charge balance, nutrient uptake and organic anion accumulation of lupins and peas supplied with a range of NO(3)(-) concentrations, were compared. Lupins absorbed less NO(3)(-) than peas on a dry weight basis, which largely ACCOUNTED for the smaller excess of anion uptake over cation uptake in lupins than in peas at the same NO(3)(-) supply. When anion uptake exceeded cation uptake, peas excreted an equivalent charge of OH(-), whereas lupins excreted much smaller amounts of OH(-) than the excess of anion over cation uptake. It was calculated that lupins excreted significant amounts of organic anions when anion uptake exceeded cation uptake, whereas organic anion excretion from peas was negligible, regardless of their NO(3)(-) supply and cation-anion balance. In this study, organic anion excretion was measured from lupin roots grown in near-sterile conditions while supplied with NO(3)(-) at 0, 500 and 2000 microM. Although complete sterility was not achieved, there was close agreement between the organic anion excreted and the excess anion over cation uptake.

Logarithmic relationship of the CD4 count to survival in patients with human immunodeficiency virus infection.

BACKGROUND: Survival, and the incidence of events that define the acquired immunodeficiency syndrome (AIDS), are known to be inversely related to the CD4 count in patients with human immunodeficiency virus infection. We wished to quantify this relationship more precisely, particularly for patients with CD4 counts of less than 50/mm3. METHODS: Prospective surveillance for survival and for all AIDS-defining events was performed on all 2682 patients with human immunodeficiency virus infection who had at least one CD4 count performed at a large urban public hospital during a 3-year period. Product-limit survival and incidence of AIDS-defining events were calculated as a function of baseline CD4 count. RESULTS: The 1-year product-limit survival was 17% +/- 6% for patients after a baseline CD4 count of 1 to 4/mm3; 44% +/- 6% after a count of 5 to 9/mm3; 48% +/- 5% after a count of 10 to 19/mm3; 51% +/- 4% after a count of 20 to 39/mm3; 62% +/- 5% after a count of 40 to 59/mm3; 71% +/- 4% after a count of 60 to 99/mm3; 79% +/- 3% after a count of 100 to 199/mm3; and 92% +/- 2% after a count of 200 to 499/mm3. One-year survival and baseline CD4 count were related by the following formula: percent 1-year survival = 10 + 32(log10 CD4 count) (R2 = .97; P < .001). The 1-year incidence of a first AIDS-defining event and baseline CD4 count were related by the following formula: percent developing AIDS in 1 year = 104-36(log10 CD4 count) (R2 = .89; P < .001). Similar relationships were calculated between the logarithm of the baseline CD4 count and the 1-year incidence of most AIDS-defining events. These relationships were linear over the CD4 range of 1 to 499/mm3 and over follow-up periods of 6 months to 2 years. CONCLUSIONS: The relationship of the CD4 count to survival, and to the incidence of AIDS-defining events, is logarithmic. This relationship helps explain the substantial differences in 1-year survival associated with baseline CD4 counts in the range below 50/mm3.

Primary prophylaxis with fluconazole against systemic fungal infections in HIV-positive patients.

OBJECTIVE: To investigate the efficacy of fluconazole prophylaxis against systemic fungal infections in HIV-positive patients. DESIGN: Open label treatment compared with historical controls. SETTING: Patients were seen at the Parkland Memorial Hospital HIV Clinic, Dallas, Texas, USA between 1 March 1990 and 28 February 1991. PATIENTS, PARTICIPANTS: Three hundred and thirty-seven historical controls were followed for 157 patient-years, and 329 fluconazole-treated patients for 145 patient-years. INTERVENTIONS: Fluconazole (100 mg daily) was administered to all patients with CD4 lymphocyte counts less than 68 x 10(6)/l seen at our HIV clinic after 1 March 1990. MAIN OUTCOME MEASURES: Lysis-centrifugation blood cultures were recorded monthly for all patients during both study periods. RESULTS: Twenty infections (16 cryptococcosis, four histoplasmosis) occurred in 337 historical reference control patients (product-limit 1-year incidence, 7.5 +/- 2.0/year). Four infections (one cryptococcosis, three histoplasmosis) occurred in the treated patient group (product-limit 1-year incidence, 1.8 +/- 0.9/year). CONCLUSIONS: Fluconazole warrants further evaluation for prophylaxis against systemic fungal infections in HIV-positive patients.

Follow-up of nosocomial infections on nursing units: an approach to infection control.

The infection control department at Niagara Falls Memorial Medical Center in Niagara Falls, New York, has developed an innovative approach to decreasing nosocomial infections. By following up on the infections that occur on nursing units, nurses actively work to prevent their occurrence. The approach utilizes nursing accountability, continuing staff education, and documentation, with positive results.