The prevalence of immunoglobulin E antibodies to the proteins of rubber (Hevea brasiliensis) latex and grass (Phleum pratense) pollen in sera of British blood donors.

BACKGROUND: Although there have been many studies of the prevalence of latex allergy in populations deemed to be at risk, little is known of the potential allergic susceptibility to latex products prevailing in the general population. OBJECTIVE: To assess the possible prevalence of allergy to latex goods in a population of blood donors by measurement of specific antilatex immunoglobulin (Ig) E in blood, to relate this to prevalence of antigrass IgE in the blood donations, and to assess the prevalence of antibodies to grass, house dust mite and cat allergens in those donors having antilatex IgE antibodies. METHODS: Sera from two groups of donations obtained in the English West Midlands were assayed. A group of 2000 donations obtained in midwinter was assayed for antilatex and antigrass pollen IgE. A group of 5000 midsummer donations was assayed for total IgE, and antilatex IgE and the sera giving a positive reaction, assayed for antigrass pollen, antihouse dust mite and anticat IgE. The nature of the principal latex and grass pollen polypeptides reacting with IgE in the sera was assessed by immunoblotting. RESULTS: Anti-latex IgE was detected in approximately 4% of the winter and 7% of the summer donations. The prevalence of antigrass IgE in the winter donations was approximately 20% and amongst the latex-positive sera approximately 84% contained antigrass IgE. Of the summer donations of latex-positive sera, 96% contained antigrass, 48.6% antimite IgE and 34% anticat IgE. The prevalence of both antilatex and antigrass IgE was age and sex related. Inhibition studies indicated cross-reactivity of IgE with latex and grass pollen proteins. CONCLUSIONS: Whilst 4-7% of the population may have serum IgE reacting with latex, the levels are low compared with those reacting with the aeroallergens studied. The apparent cross-reactivity of some serum IgE with both latex and grass pollen taken with other evidence suggests that, in some individuals, allergy to latex may arise from an initial sensitization by grass pollen.

Identification, cloning, and sequence of a major allergen (Hev b 5) from natural rubber latex (Hevea brasiliensis).

Proteins in commercial latex products, derived from the rubber tree Hevea brasiliensis, cause anaphylaxis in susceptible individuals, especially health care workers and children with spina bifida. To identify latex allergens, we utilized IgE from the serum of a latex-allergic health care worker to screen a cDNA library from Hevea latex. The identified cDNA clone, cDNA Hev b 5, encodes an open reading frame of 163 peptide residues. Hybridization analysis of cDNA Hev b 5 with RNA extracted from Hevea tissue indicates that the full-length transcript is about 1000 bases. The nucleotide and deduced protein sequences have significant homology to sequences from kiwi and potato, which are known to cause allergic reactions in some latex-allergic patients. Fifty-six percent of spina bifida patients and 92% of health care workers with latex allergy have IgE specific to the protein encoded by cDNA Hev b 5. A monoclonal antibody raised from a mouse immunized with Hev b 5 binds to a protein in Hevea latex with an Mr identical to that of the expressed and cleaved recombinant protein. Taken together, these results establish that the antigen Hev b 5 contains a major epitope for IgE-mediated reactions to H. brasiliensis latex products.

The allergenic properties of fresh and preserved Hevea brasiliensis latex protein preparations.

The allergenic properties of the proteins of two lyophilized fractions of fresh natural rubber latex obtained by ultracentrifugation, the C serum and the sedimented bottom or lutoid fraction, have been compared with those of the serum proteins of two samples of high ammonia latex (HAL) [A]HALS obtained from HAL stored for more than 1 year, and [M]HALS derived from HAL stored for 6 weeks before ultracentrifugation and lyophilization. The most potent source of allergenic polypeptides both for skin prick testing of latex-sensitive patients and for immunoblots of their blood serum was the lutoid fraction of fresh latex. Skin prick tests and immunoblots of patients' sera showed that the allergenicity of the ammoniated latex decreased during storage. Skin prick tests using fractions of [A]HALS, C serum and lutoid proteins obtained after passage through a Sephacryl S300 column showed that the components of all three preparations which eluted in the largest volumes were almost equally effective in provoking the largest number of responses. Immunoblots of the sera of 43 latex-sensitive individuals showed that the majority (66%) of sera of the adult allergic patients reacted with a polypeptide of 19 kD. No characteristic pattern of binding latex polypeptides could be recognized in the sera from patients who were also asthmatic or from those who had an anaphylactic response to latex proteins.

Molecular cloning of higher-plant 3-oxoacyl-(acyl carrier protein) reductase. Sequence identities with the nodG-gene product of the nitrogen-fixing soil bacterium Rhizobium meliloti.

cDNA clones encoding the fatty-acid- biosynthetic enzyme NADPH-linked 3-oxoacyl-(acyl carrier protein) (ACP) reductase were isolated from a Brassica napus (rape) developing seed library and from an Arabidopsis thaliana (thale cress) leaf library. The N-terminal end of the coding region shows features typical of a stromal-targeting plastid-transit peptide. The deduced amino acid sequences have 41% and 55% identity respectively with the nodG-gene product of Rhizobium meliloti, one of the host-specific genes that restrict infectivity of this bacterium to a small range of host plants. The probability that the nodG-gene product is a oxoreductase strengthens the hypothesis that some of the host-specific nod-gene products are enzymes which synthesize polyketides that uniquely modify the Rhizobium nodulation signal molecule.

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

3-Oxoacyl-(acyl-carrier protein) reductase from avocado (Persea americana) fruit mesocarp.

The NADPH-linked 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (EC 1.1.1.100), also known as 'beta-ketoacyl-ACP reductase', has been purified from the mesocarp of mature avocado pears (Persea americana). The enzyme is inactivated by low ionic strength and low temperature. On SDS/PAGE under reducing conditions, purified 3-oxoacyl-ACP reductase migrated as a single polypeptide giving a molecular mass of 28 kDa. Gel-filtration chromatography gave an apparent native molecular mass of 130 kDa, suggesting that the enzyme is tetrameric. The enzyme is inactivated by dilution, but some protection is afforded by the presence of NADPH. Kinetic constants have been determined using synthetic analogues as well as the natural ACP substrate. It exhibits a broad pH optimum around neutrality. Phenylglyoxal inactivates the enzyme, and partial protection is given by 1 mM-NADPH. Antibodies have been raised against the protein, which were used to localize it using immunogold electron microscopy. It is localized in plastids. N-Terminal amino-acid-sequence analysis was performed on the enzyme, and it shows close structural similarity with cytochrome f. Internal amino-acid-sequence data, derived from tryptic peptides, shows similarity with the putative gene products encoded by the nodG gene from the nitrogen-fixing bacterium Rhizobium meliloti and the gra III act III genes from Streptomyces spp.

The characteristics of some components of the fatty acid synthetase system in the plastids from the mesocarp of avocado (Persea americana) fruit.

Preparations of NADH-specific and NADPH-specific 3-oxoacyl-[acyl-carrier-protein] reductase enzymes (EC 1.1.1.100), enoyl-[acyl-carrier-protein] reductase (EC 1.3.1.9) and [acyl-carrier-protein] malonyltransferase (EC 2.3.1.39) have been purified from preparations of avocado mesocarp plastids and characterised. The enzymes are quite similar in molecular and kinetic characteristics to analogous enzymes known in Escherichia coli and Euglena and are clearly components of a type-II fatty acid synthetase system.

Acetyl-coenzyme A carboxylase from avocado (Persea americana) plastids and spinach (Spinacia oleracea) chloroplasts.

Preparations of acetyl-CoA carboxylase [acetyl-CoA-carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] have been obtained from the plastids of avocado (Persea americana) fruit mesocarp and from spinach (Spinacia oleracea) chloroplasts. Both preparations required bovine serum albumin, HCO3-, citrate and glycerol for stabilization. The molecular weight of the avocado enzyme was about 6.5 X 10(5) on the basis of 1 mol of biotin/mol of enzyme, the behaviour of both enzymes on gel filtration being in accord with such a value. Removal of the stabilizing bovine serum albumin resulted in the loss of a biotin-containing fragment from the avocado enzyme. Citrate stabilized the enzyme at 10 mM and activated it optimally at 3.0 mM, effecting an approx. 2-fold increase in Vmax. It is suggested that in vivo the enzyme may be located within the chloroplast lamellae.

The synthesis of fatty acids in avocado mesocarp and cauliflower bud tissue.

1. Plastid and mitochondrial preparations were obtained by density-gradient centrifugation of homogenates made by gentle disintergration of avocado fruit mesocarp and cauliflower bud tissue. 2. The mitochondrial preparations had respiratory activity but did not incorporate [1-14C]acetate into fatty acids. 3. The plastid preparations incorporated [1--14C]acetate into the range of fatty acids found in the parent tissue. No fatty acid synthetase activity could be detected in the 12000g supernatant of these homogenates. 4. Homogenates produced by rupture of the tissue in an Ato-Mix blender and plastid preparations disintegrated by ultrasonic treatment both had fatty acid synthetase activity which did not sediment at 105000g and which formed mainly [14-C]stearate from [2-14C]malonyl-CoA. 5. It is concluded that the plastids are the principal site of fatty acid biosynthesis in the tissues studied.

The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids.

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.