Relaxation techniques for acute pain management: a systematic review

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863-871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.).     

Factors related to the development of sensitization to green coffee and castor bean allergens among coffee workers

BACKGROUND: Occupational allergic respiratory symptoms in coffee workers have been frequently reported, but the ultimate cause of sensitization is still debated, castor bean being considered besides green coffee beans. Atopy and cigarette smoking have been suggested as promoting factors of sensitization for several occupational allergens. OBJECTIVE: This study was carried out to assess the prevalence of allergic respiratory symptoms and of sensitization to both green coffee beans and castor bean in the whole workforce of a coffee manufacturing plant. Furthermore we wanted to ascertain both the presence of castor bean antigens in the settled dust of the green coffee beans warehouse and the possible crossreactivity between the two beans. Meanwhile, the effect of smoking and atopy was considered. METHOD: Two-hundred and eleven workers were examined. A questionnaire on oculorhinitis and asthma was administered and skin-prick tests for green coffee beans, castor bean and 15 common inhalant allergens were carried out. Isoelectric focusing, isoelectric focusing immunoblot and radioallergosorbent assay (RAST) inhibition were performed on samples of settled environmental dust from the green coffee area, as well as on castor bean and green coffee beans. RESULTS: Ten per cent of the workers complained of oculorhinitis alone and 16% of asthma (nearly always associated with oculorhinitis). The overall prevalence of skin-sensitization was: 15% for green coffee beans, 22% for castor bean, 22% for common allergens. Evidence of sensitization to occupational allergens was more common in smokers, with a more than twofold increase in relative risk. The strong association between skin positivity to common and occupational allergens suggests that atopy acts as an enhancing host factor towards occupational sensitization. The analysis of the dust confirmed the presence of castor bean antigens. CONCLUSION: Our findings indicate that castor bean is the major cause of occupational sensitization among coffee workers, whereas smoking and atopy act as enhancing factors.     

Engineering the healing of the rabbit medial collateral ligament

A phospholipase D gene (CaPLD) has been cloned from the Candida albicans genomic DNA library. The CaPLD is a member of a highly conserved gene family of PLD and has the highest homology to Saccharomyces cerevisiae PLD (SPO14) with an overall homology of 42%. Phylogenetic analysis indicated that fungus PLDs including CaPLD composed one of the three clusters of PLD genes.     

Gene sequence tags from Plasmodium falciparum genomic DNA fragments prepared by the "genease" activity of mung bean nuclease

A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.     

Apolipoprotein E allelic influence on human cerebrospinal fluid apolipoproteins

A full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator. This construct was found infectious when inoculated to lettuce plants. The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity. The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E. coli. Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out. The symptoms induced in lettuce by either the intron-less or the intro-containing constructs were identical to those caused by the wild-type virus. However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA.     

The nucleotide sequence of the 3'-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production

A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA. The sequence of the 3'-terminal 3158 nucleotides, which consisted of the 3'-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57-68% similar at the nucleotide level and 72-82% similar at the amino acid level when compared with other potyviruses. Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus. A recombinant DsMV-Ch CP (approximately 39 kDa) expressed in E. coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA.     

Purification and immunological analysis of phospholipase D from castor bean endosperm

Phospholipase D (EC 3.1.4.4) has been implicated in diverse cellular processes, but its physiological role is not well established in plants. In order to develop immunological and molecular biology approaches to address the problem, we report here the immunological analysis and N-terminal amino acid sequence of a cytosolic phospholipase D from castor bean (Ricinus communis L.). The enzyme was purified to apparent homogeneity from germinating castor bean endosperm. The specific activity of the purified enzyme was enhanced by approximately 670-fold with an overall yield of 4%. Its molecular mass was estimated at 92 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this enzyme was KLVENIEETVGFGKG. Polyclonal antibodies were raised against the purified enzyme. The antibodies inhibited the activity of transphosphatidylation more than that of hydrolysis of phospholipase D. The differential effect on the two activities of this enzyme implies that different active sites on this enzyme may be involved in the two reactions. Immunoblot analyses showed that the amounts of phospholipase D protein relative to the total endosperm proteins increased during the first 5 days of germination. The antibodies cross-reacted to proteins from several tested plant species, and those proteins had molecular masses similar to that of castor bean phospholipase D. These results indicate that the expression of phospholipase D in castor bean changes according to growth stages and that phospholipase D enzymes of different plant species are structurally related.     

Targeting of castor bean glyoxysomal isocitrate lyase to tobacco leaf peroxisomes

The cDNA encoding castor bean endosperm isocitrate lyase (ICL) was expressed under the control of the promoter of the small subunit of pea ribulose bisphosphate carboxylase in transformed tobacco. ICL protein was detected using anti-ICL antibodies on immunoblots of total leaf protein extracts. Nycodenz density gradient separation of the extracts from the transgenic tobacco leaves showed ICL co-fractionated with hydroxypyruvate reductase, a peroxisomal matrix marker protein, and away from lactate dehydrogenase, a cytosolic marker protein. Immunoelectron microscopy of ultrathin leaf sections demonstrated the location of ICL within the matrix of the leaf peroxisomes of the transgenic plants. In vitro transcribed and translated ICL was also imported into leaf peroxisomes isolated from germinating sunflower seeds. The in vivo and in vitro import of this protein into leaf peroxisomes provides strong support for the notion that the import machinery of glyoxysomes and peroxisomes is very similar.     

Decreased release of glutathione into the systemic circulation of patients with HIV infection

The human DDX6 gene (alias RCK) at chromosome 11 band q23 was identified through the study of the breakpoint of t(11;14)(q23;q32) translocation in a B-cell lymphoma cell line, RC-K8. DDX6 encodes a DEAD box protein/RNA helicase. Positive mouse genomic and cDNA recombinant clones were obtained by screening mouse B-cell genomic and cDNA libraries with a human DDX6 cDNA probe. The deduced amino acid sequence of an open reading frame from a cDNA clone revealed a protein with 92.5% identity to human ddx6/p54. All positive mouse genomic recombinant clones, and cDNA clones containing mouse Ddx6 (previous gene symbol: Rck), were localized by fluorescent in situ hybridization to band B of mouse Chromosome 9, a region showing conserved linkage homology to human chromosome 11 band q23. Mouse Ddx6 was localized to the region between Ncam and D9Mit45 by molecular linkage analysis. A 7.5-kb mRNA and a 54-kDa protein were identified as mouse Ddx6 gene products which are similar in size to products of the human DDX6 gene, as shown by Northern and Western blot analyses.     

Analysis of genes encoding highly conserved lysine-rich proteins in Aplysia californica and Saccharomyces cerevisiae

To isolate a gene that can be used as an internal control in studies on gene expression in Aplysia californica neurons, we have characterized a cDNA clone (pKRP-A) isolated on the basis of its high expression in A. californica neurons. This cDNA is of 850 nucleotides and codes for a putative 29-kDa lysine-rich protein. Blotting experiments revealed that the gene is expressed in all tested A. californica tissues, and in individually identified neurons of the abdominal ganglion, suggesting that this gene can be efficiently used as internal control in studies of gene expression. We have also isolated one cDNA and two different genomic clones from yeast libraries that show 59% identity with pKRP-A. Sequence comparison of genomic clones, as well as PCR and Southern blotting experiments, revealed that at least two homologous genes are present in yeast. Northern blotting experiments revealed that the expression of the gene is strongly repressed at 39 degrees C.     

Cloning and nucleotide sequence of the alkaline protease gene from Fusarium sp. S-19-5 and expression in Saccharomyces cerevisiae

We have cloned a genomic DNA encoding the alkaline protease (Alp) of Fusarium sp. S-19-5 from a genomic DNA library and sequenced the nucleotides. Complementary DNA encoding Alp was also isolated from the cDNA library after amplifying the gene by PCR using partial sequences of the Alp genomic DNA as primers. The Alp gene has an open reading frame of 1137 nucleotides containing three introns. A TATA box (TAAATA) was observed 112 base pairs upstream from the translation initiation codon in the 5'-non coding region. The Alp protein has a pre region consisting of 14 amino acids and a pro region of 85 amino acids preceding the mature region, which consists of 280 amino acids. The amino acid sequence of Fusarium Alp has 52% homology with that of Aspergillus oryzae and 51% homology with that of Acremonium chrysogenum. The entire cDNA encoding Fusarium Alp was introduced into Saccharomyces cerevisiae, which then secreted enzymatically active Alp into the culture medium.     

Gene structure of human indoleamine 2,3-dioxygenase

Two genomic DNA clones that encode human indoleamine 2,3-dioxygenase (IDO) were isolated from the human genomic DNA library using the IDO cDNA as a probe, and their restriction maps and partial nucleotide sequences were determined. The human IDO gene spanned 15 kilobase pairs with ten exons. The 5' terminus of the IDO mRNA was 33 nucleotides upstream of the translation initiation codon ATG. The 5' flanking region contained ISRE, X-box, and Y-box like sequences. Southern blot analysis of the human genomic DNA indicated that the human IDO gene was present in a single copy in the genome.     

Expression of superoxide dismutase following axotomy

Genomic clones for a chitinolytic enzyme were isolated from a library of Sau 3A digested DNA from the tobacco hornworm, Manduca sexta, using a previously isolated chitinase cDNA clone as a probe [Kramer et al., Insect Biochem. Molec. Biol. 23, 691-701 (1993)]. Restriction enzyme mapping and Southern blot analysis of four genomic clones suggested that these are overlapping clones. Sequence analysis of the genomic clones and Southern blot analysis of total genomic DNA also suggest that the M. sexta genome has only one chitinase gene detectable by the cDNA probe. This gene is organized into at least 11 exons in a region spanning > 11 kb. The sequenced M. sexta chitinase gene has a series of exons corresponding to identifiable structural/functional regions of the protein. Similarities in structure and organization between the M. sexta chitinase gene and chitinase genes from other sources are described.