Vanadium containing bromoperoxidase: An example of an oxidoreductase with high operational stability in aqueous and organic media

The conversion is described of phenolsulphonephtalein (phenol red) to 3,3',5,5'-tetrabromophenolsulphonephthalein (bromophenol blue) by bromoper-oxidase from the brown alga Ascophyllum nodosum. This reaction provides a convenient assay for the detection of bromoperoxidase activity in vitro. Bromoperoxidase was shown to be stable under turnover conditions for three weeks at room temperature, catalyzing the bromination of phenol red into bromophenol blue. When stored at room temperature in organic sol vents such as acetone, methanol, ethanol [present up to 60% (v/v)], and 1-propanol [40% (v/v)], bromoperoxidase was stable for more than one month. As far as we know this is the first example of an oxidoreductase which displays such great stability. This enhances the applicability of the enzyme in organic synthesis.     

The variability of the hepatitis B virus genome: statistical analysis and biological implications

A statistical analysis of the nucleotide sequence variability in 14 published hepatitis B virus (HBV) genomes was carried out using parametric and nonparametric methods. A parametric statistical model revealed that the different regions of the genome differed significantly in their variability. The conclusion was supported by a nonparametric kernel-density model of the HBV genome. Genes S, C, and P, region X, the precore region, and the pre-S2/pre-S1 regions were ranked in order of increasing variability. In many instances, conserved regions of the genome identified with sequences of known function in HBV biology. However, other characterized regions (such as pre-S) showed much variability despite the involvement of their encoded peptides in specific functions. Point mutations that may result in the formation of stop codons and amino acid changes may affect the clinical picture of HBV infection and may be reflected in atypical serological patterns.      

Studies on the Stability of Lipase from Candida Cylindracea, Free and Incorporated in Polymerisable Zwitterionic Surfactant Vesicles

Lipase from Candida cylindracea (CCL) was incorporated into vesicles of a polymerisable zwitterionic surfactant: bis[2-(pentacosa-10,12-diynoyloxy)ethyl]-2-aminoethanesulfonic acid (BPAS). Vesicle systems of BPAS were characterised in terms of morphology (Electron Microscopy) and stability. Polymerisation of BPAS vesicles did not alter the morphology and polymeric vesicles were considerably more stable than the monomeric analogues. CCL was incorporated into the vesicle membrane by spontaneous insertion. The enzyme remained fully active after incorporation into the vesicle bilayer; especially in homogeneous assay mixtures the vesicle incorporated enzyme showed an increased activity when compared to the free lipase. The stability of free and incorporated lipase was determined by measuring the residual activity of the various systems when mixed with ethanol (50% v/v) or 2-(n-butoxy)ethanol (37.5% v/v), at 50°C and 60°C and in the presence of the proteolytic enzyme trypsin. In all cases the vesicle incorporated enzyme showed an increased stability against the denaturating conditions.     

Phase I/II study of recombinant interferon α and γ in advanced progressive renal-cell carcinoma

Summary In vitro studies have documented the synergistic antiviral and antiproliferative activity of recombinant interferon (rIFN) and rIFN. Furthermore, rIFN is a strong immunomodulator with optimal effects at a relative low dose (0.1 mg/m²). On the basis of these observations, we began a phase I/II study with the combination of rIFN at 100 µg/m² (2 × 10⁶ IU/m²) and rIFN2c 6 µg/m² (2 × 10⁶ IU/m²), injected twice a week subcutaneously. In cases of stable or progressive disease we increased the dose of rIFN2c every 2 weeks by 6 µg/m² until the maximum tolerated dose was reached. A total of 32 patients with proven progressive renal-cell carcinoma were included. Of the 31 eligible patients, 21 were male and 10 female, their average age was 57.2 years (range 35–72), 28 had had nephrectomy, their median Karnofsky performance status was 90% (70%–100%), and their tumors were localized predominantly to visceral tissue. In 2, response was complete and in 6 it was partial, for a response rate of 25%. The disease had stabilized in 5 patients and progressed in 16. The median duration of partial response was 14 months (8–16 months); of 2 cases of complete response, 1 persists (23+ months), and the other suffered a relapse after 22 months. The median time to response was 24 weeks (18–24 weeks). The maximum tolerated dose of rIFN was 30 µg/m² (range of 6–36 µg/m²). Side-effects included those known to be associated with interferon treatment. One patient developed septicemia during a period with grade 4 leukopenia. Our study permits no conclusion regarding the additional value of rIFN.     

Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses

Computer analyses have revealed sequence homology between two non-structural proteins encoded by cowpea mosaic virus (CPMV), and corresponding proteins encoded by two picornaviruses, poliovirus and foot-and-mouth disease virus. A region of 535 amino acids in the 87-K polypeptide from CPMV was found to be homologous to the RNA-dependent RNA polymerases from both picornaviruses, the best matches being found where the picornaviral proteins most resemble each other. Additionally, the 58-K polypeptide from CPMV and polypeptide P2-X from poliovirus contain a conserved region of 143 amino acids. Based on the homology observed, a genetic map of the CPMV genome has been constructed in which the 87-K polypeptide represents the core polymerase domain of the CPMV replicase. These results have implications for the evolution of RNA viruses, and mechanisms are discussed which may explain the existence of homology between picornaviruses (animal viruses with single genomic RNAs) and comoviruses (plant viruses with two genomic RNAs).     

Expression of Middle-Component RNA of Cowpea Mosaic Virus: In Vitro Generation of a Precursor to Both Capsid Proteins by a Bottom-Component RNA-Encoded Protease from Infected Cells

The internal organization of endogenous xenotropic murine leukemia virus proviruses was determined in a series of blot hybridization experiments in which DNA from several different inbred mouse strains, digested with restriction enzymes known to cleave xenotropic proviral DNAs at least twice, was annealed to generalized murine leukemia virus or xenotropic env-specific DNA probes. Comigrating bands of variable intensity which hybridized to the xenotropic env probe were identified in all inbred mouse DNA preparations. At least seven classes of endogenous xenotropic proviral DNA with respect to SacI cleavage maps were detected in mouse DNA. Two of the seven classes were indistinguishable from proviruses associated with known infectious xenotropic murine leukemia viruses. These results are consistent with the existence of related but organizationally distinct families of endogenous xenotropic proviral DNA that are present in different relative abundances in mouse genomic DNA.     

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.      

The root epidermis-specific pea gene RH2 is homologous to a pathogenesis-related gene

Two-dimensional gel electrophoresis of pea root and root hair proteins revealed the existence of at least 10 proteins present at elevated levels in root hairs. One of these, named RH2, was isolated and a partial amino acid sequence was determined from two tryptic peptides. Using this sequence information oligonucleotides were designed to isolate by PCR an RH2 cDNA clone. In situ hybridization studies with this cDNA clone showed that rh2 is not only expressed in root hairs, but also in root epidermal cells lacking these tubular outgrowths. During post-embryonic development the gene is switched on after the transition of protoderm into epidermis and since rh2 is already expressed in a globular pea embryo in the protoderm at the side attached to the suspensor, we conclude that the expression of rh2 is developmentally regulated. At the amino acid level RH2 is 95% homologous to the pea PR protein I49a. These gene encoding I49a is induced in pea pods upon inoculation with the pathogen Fusarium solani [12]. We postulate that rh2 contributes to a constitutive defence barrier in the root epidermis. A similar role has been proposed for chalcone synthase (CHS) and chitinase, pathogenesis-related protein that are also constitutively present in certain epidermal tissues.     

Comparison of soybean and pea ENOD40 cDNA clones representing genes expressed during both early and late stages of nodule development

A pea cDNA clone representing the homologue of the soybean pGmENOD40-1 was isolated and characterized. At the nucleotide level both clones share 55% homology. Strikingly, the homology between the polypeptides derived from the pea and soybean ENOD40 cDNA sequences is only 14%. Despite this low homology Southern analyses revealed that the isolated pea cDNA clone represents the single pea ENOD40. In situ hybridizations showed that at early stages of nodule development and in mature nodules the expression pattern of pea ENOD40 is comparable to that of soybean ENOD40. Although ENOD40 show similar expression patterns in these two nodules, it is questionable whether the putative polypeptides have a similar function, since the homology is very low.