Allergen concentration in natural rubber latex.

BACKGROUND: Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens. OBJECTIVE: This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy. METHODS: The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library. RESULTS: The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations. CONCLUSION: Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Appraisal of latex glove proteins in the induction of sensitivity to multiple latex allergens.

Six Hevea brasiliensis latex protein allergens, Hevb 1, Hev b 2, Hev b 3, Hev b 4, and two variants of Hev b 7 (7b and 7c), were purified from Hevea latex, while a seventh protein, Hev b 5, was prepared in recombinant form. The presence of these proteins in glove extracts was indicated by their respective antibodies in the serum of rabbits immunized against the extracts. The relative propensities of IgE binding to the individual latex allergens were compared using sera from latex-allergic patients. IgE recognition of Hev b 4, Hev b 7b, Hev b 5 and Hev b 2 was most frequently encountered, with 75, 61, 31 and 28%, respectively, of the patient sera reacting. Sensitivity to multiple latex proteins was common, and out of the 31 seropositive patients, 23 (74%/ ) had IgE against at least two latex allergens, while 12 (39%) had IgE specific for at least three allergens. Statistical analysis of the data suggested that many patients might have acquired sensitivity to Hev b 2, Hev b 4 and Hev b 7b from a common source. (e.g., from latex products). On the other hand, sensitivity to Hev b 5 and to Hev b 7c were interrelated. It is plausible that sensitivity to these two proteins might have been acquired from sources other than latex products (e.g., from certain foods).

Novel fabrication of open-pore chitin matrixes.

A novel method has been developed to produce open-pore chitin matrixes. Chitin solutions were loaded with calcium carbonate (CaCO3) crystals and the mixture cast to form gels. The CaCO3-chitin gels were submerged in 1 N HCl solution to produce highly porous matrixes with good water vapor permeability, water uptake profile, and enhanced mechanical properties. The open-pore system is obtainable because CaCO3 loaded into the chitin gel reacts with 1 N HCl solution to produce gaseous carbon dioxide. Evolution of carbon dioxide during the reaction results in continuous pore structures from the matrix' bulk to surface. When the concentration of CaCO3 loaded into the chitin gel is controlled, defined homogeneous pores measuring 100-500 and 500-1000 microns, with porosities of approximately 76% and 81%, respectively, can be produced.

Genes expressed in the latex of Hevea brasiliensis.

Rubber (cis-1,4-polyisoprene), an important raw material for many industrial uses, is synthesized in the latex of Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. We postulated that the genes uniquely or preferentially expressed in the latex may be important for rubber biosynthesis. We constructed cDNA libraries from the latex of H. brasiliensis to investigate the genes expressed in the latex by single-run partial sequencing of the cDNA clones. Sequence analyses identified 245 expressed sequence tags (ESTs), of which 57% showed homology to previously described sequences in public databases. About 16% of the database-matched ESTs encode rubber biosynthesis-related proteins such as rubber elongation factor (REF) and small rubber particle protein (SRPP). The second most frequent transcripts next to rubber biosynthesis-related genes were defense genes and protein metabolism-related genes (12.6% each). About 27% of the database-matched ESTs had sequence homology with genes of unknown function. Among the redundantly expressed genes, REF was the most frequently expressed (6.1%), followed by SRPP (3.7%) and HbLAR (2.9%). Northern blot analyses showed that ten (71%) of the 14 ESTs studied were expressed at a higher level in latex than in leaves.

Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis.

Biochemical evidence reported so far suggests that rubber synthesis takes place on the surface of rubber particles suspended in the latex of Hevea brasiliensis. We have isolated and characterized a cDNA clone that encodes a protein tightly bound on a small rubber particle. We named this protein small rubber particle protein (SRPP). Prior to this study, this protein was known as a latex allergen, and only its partial amino acid sequence was reported. Sequence analysis revealed that this protein is highly homologous to the rubber elongation factor and the Phaseolus vulgaris stress-related protein. Southern and Northern analyses indicate that the protein is encoded by a single gene and highly expressed in latex. An allergenicity test using the recombinant protein confirmed that the cloned cDNA encodes the known 24-kDa latex allergen. Neither ethylene stimulation nor wounding changed the transcript level of the SRPP gene in H. brasiliensis. An in vitro rubber assay showed that the protein plays a positive role in rubber biosynthesis. Therefore, it is likely that SRPP is a part of the rubber biosynthesis machinery, if not the rubber polymerase, along with the rubber elongation factor.

Two different genes encode ferrochelatase in Arabidopsis: mapping, expression and subcellular targeting of the precursor proteins.

Ferrochelatase is the last enzyme of haem biosynthesis. We have isolated 27 independent ferrochelatase cDNAs from Arabidopsis thaliana by functional complementation of a yeast mutant. Twenty-two of these cDNAs were similar to a previously isolated clone, AF3, and although they varied in length at the 5' and 3' ends, their nucleotide sequences were identical, indicating that they were derived from the same gene (ferrochelatase-I). The remaining five cDNAs all encoded a separate ferrochelatase isoform (ferrochelatase-II), which was 69% identical at the amino acid level to ferrochelatase-I. Using RFLP analysis in recombinant inbred lines, the ferrochelatase-I gene was mapped to chromosome V and that for ferrochelatase-II to chromosome II. Northern analysis showed that both ferrochelatase genes are expressed in leaves, stems and flowers, and expression in the leaves is higher in the light than in the dark. However, in roots only ferrochelatase-I transcripts were detected. High levels of sucrose stimulated expression of ferrochelatase-I, but had no effect, or repressed slightly, the expression of the ferrochelatase-II isoform. Import experiments into isolated chloroplasts and mitochondria showed that the ferrochelatase-II gene encodes a precursor which is imported solely into the chloroplast, in contrast to ferrochelatase-I which is targeted to both organelles. The significance of these results for haem biosynthesis and the production of haemoproteins, both within the plant cell and in different plant tissues, is discussed.

A single precursor protein for ferrochelatase-I from Arabidopsis is imported in vitro into both chloroplasts and mitochondria.

Ferrochelatase is the last enzyme of heme biosynthesis and in higher plants is found in both chloroplasts and mitochondria. We have isolated cDNAs for two isoforms of ferrochelatase from Arabidopsis thaliana, both of which are imported into isolated chloroplasts. In this paper we show that ferrochelatase-I is also imported into isolated pea mitochondria with approximately the same efficiency as into chloroplasts. Processing of the precursor was observed with both chloroplast stroma and mitochondrial matrix extracts. This was inhibited by EDTA, indicating it was due to the specific processing proteases. The specificity of import was verified by the fact that the mitochondrial preparation did not import the precursor of the light-harvesting chlorophyll a/b protein precursor or the precursor of porphobilinogen deaminase, an earlier enzyme of tetrapyrrole biosynthesis, both of which are exclusively chloroplast-located. Furthermore, import of ferrochelatase-I precursor into mitochondria was inhibited by valinomycin, but this had no effect on its import into chloroplasts. Thus a single precursor molecule is recognized by the import machinery of the two organelles. The implications for the targeting of ferrochelatase in a possible protective role against photooxidative stress are discussed.

[Primary hypoparathyroidism with basal ganglia calcification: report of a case].

A 3 years old boy was admitted due to recurrent attacks of tetany and carpopedal spasm since one and a half years of age. The tetany lasting for 1-2 minutes in each episode was often preceded by an upper respiratory tract infection and occurred 2-3 times a month. Both birth and family history were unremarkable. Physical findings showed mild psychomotor retardation with positive Chvostek sign. Laboratory examination revealed hypocalcemia, hyperphosphatemia, and low serum parathyroid hormone level. EEG showed abnormal tracing with increased slow waves. Head CT Scan demonstrated symmetrical calcification in the basal ganglia region. The clinical features and laboratory findings were consistent with hypoparathyroidism. The mechanism of calcium deposit in the basal ganglia still remains unclear. Tetany, muscle cramping and seizures secondary to hypocalcemia are the most common neurologic signs which respond quickly to calcium replacement. Subsequent supplemental therapy resolved movement disorders and mental retardation. If early treatment prior to the tetanic episodes is instituted in a patient with hypoparathyroidism, it may prevent the development of complications such as intracranial calcifications, cataract and permanent retardation.