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Production of rubber-like polymers by microorganisms.

Biopolymers with true rubber properties are exceptionally rare in prokaryotic microorganisms. Only some polyhydroxyalkanoic acids are thermoplastic elastomers within a narrow temperature range that can be extended by crosslinking. Other polyhydroxyalkanoic acids are rigid and need specific thermal treatment during annealing to yield elastomeric materials. The most important elastomer is natural rubber (cis-1,4-polyisoprene). The rubber tree Hevea brasiliensis is so far the only relevant commercial source of this polymer, although many other plants are also capable of its synthesis. Recent advances in the analysis of isoprenoid and polyisoprene biosynthesis pathways have encouraged attempts to establish production of natural rubber in bacteria. Establishment of functional cis-1,4-polyisoprene biosynthesis pathways in bacteria depends firstly on the biosynthesis of isoprene moieties via the mevalonate or methylerythritol phosphate pathway and secondly on the polymerisation of isoprene moieties by a prenyltransferase yielding high-molecular-weight polyisoprenoids. The genes that encode prenyltransferases are as yet unknown and so the establishment of such a pathway and formation of rubber in recombinant bacteria will be a difficult task.

Bacteria↗

Degradation of vulcanized and nonvulcanized polyisoprene rubbers by lipid peroxidation catalyzed by oxidative enzymes and transition metals.

Despite numerous reports concerning the biodegradation of rubber materials, there has been no report of rubber degradation by fully characterized enzymes. In the present paper, we presented a new method to decompose nonvulcanized and vulcanized polyisoprene rubbers by controlling the free radical chain reactions of lipids using oxidative enzymes, manganese peroxidase (MnP), laccase (Lac), and horseradish peroxidase (HRP). Nonvulcanized synthetic polyisoprene (IR) was degraded by the free radicals from unsaturated fatty acids produced by MnP, HRP, and a combination of Lac/1-hydroxybenzotriazole. In contrast, lipoxygenase caused no apparent degradation. Degradation of IR was also observed in lipid peroxidation initiated by the Fenton reaction (FR) and Mn(III), an oxidation product produced by MnP. Vulcanized polyisoprene rubber sheets were degraded by the lipid peroxidation initiated by HRP, MnP, Mn(III), and FR. Pyrolysis GC-MS analysis demonstrated that the lipid peroxidation liberated isoprenoid fragments from the vulcanized rubbers.

Butadienes↗

Allergic reactions to rubber gloves in dental patients: report of three cases.

Three cases of allergy to rubber are described, in which the patients exhibited peri-oral rashes following dental treatment by personnel wearing latex rubber gloves. Two of the patients were aware of possible allergy to domestic rubber products but did not reveal this as part of their medical history. With the increase in numbers of dentists wearing rubber gloves it is probable that there will be many more such cases reported in the future. Rubber products must then be added to the list of potential allergens which may be of some import to the practice of dentistry.

Adult↗

Ocular rubber bullet injuries.

PURPOSE: To report the type and severity of ocular and orbital injuries caused by rubber bullets. METHODS: A total of 42 consecutive patients seen over a 3-month period with ocular and orbital rubber bullet injuries were assessed clinically and radiographically within 1 day of injury, and the findings were recorded. Clinical outcomes following treatment were also recorded up to 6 months postinjury. RESULTS: A total of 90% of the patients were male. The mean age of patients was 25.0 years (4-60). Of the patients, 54% had lid or skin lacerations, 40% hyphaema, 38% ruptured globe, 33% orbital fracture, 26% retinal damage, and 21% retained rubber bullet in or around the orbit. At follow-up, 53% of the patients had a visual acuity of less than 6/60, 7% less than 6/18 to 6/60, and 40% 6/18 or better. CONCLUSIONS: The term 'rubber bullet' is misleading. 'Rubber bullets' cause a wide variety of ocular and periocular injuries. Orbital fractures are common. The tissues of the orbit are easily penetrated. If the globe is hit, it is rarely salvageable.

Adolescent↗

Repeatability and reproducibility of thermal field-flow fractionation in molecular weight determination of processed natural rubber.

The purpose of this study is (1) to determine the repeatability and reproducibility of thermal field-flow fractionation (ThFFF) in measuring the molecular weight of compounded natural rubber, and (2) to examine the correlation between the molecular weights obtained from ThFFF and the rheological data. 8 batches of compounded natural rubber were obtained from a thermo-mechanical mastication process, and were analyzed by ThFFF in a designed testing sequence. ThFFF analysis showed the compounded natural rubbers range in weight-average molecular weight (M(w)) from 143,000 to 360,000. By taking into account both the short term variability (repeatability) as well as the long term variability (reproducibility) of the instrument, ThFFF was shown to be able to distinguish between samples differing by as little as 21,000 in M(w) and 15,600 in number-average molecular weight, M(n) (based on cis-polyisoprene calibration); and thus is a useful tool for the molecular weight analysis of natural rubber-related materials. It was also found that the rheological data (G' and tan delta) measured on both the virgin and the compounded natural rubber correlated well with the molecular weights obtained from ThFFF when normalized.

Chemical Fractionation↗

The major protein of guayule rubber particles is a cytochrome P450. Characterization based on cDNA cloning and spectroscopic analysis of the solubilized enzyme and its reaction products.

Guayule plants accumulate large quantities of rubber within parenchyma cells of their stembark tissues. This rubber is packed within discrete organelles called rubber particles composed primarily of a lipophilic, cis-polyisoprene core, small amounts of lipids, and several proteins, the most abundant of which is the M(r) 53,000 rubber particle protein (RPP). We have cloned and sequenced a full-length cDNA for RPP and show that it has 65% amino acid identity and 85% similarity to a cytochrome P450 known as allene oxide synthase (AOS), recently identified from flaxseed. RPP contains the same unusual heme-binding region and possesses a similar defective I-helix region as AOS, suggesting an equivalent biochemical function. Spectral analysis of solubilized RPP verifies it as a P450, and enzymatic assays reveal that it also metabolizes 13(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid into the expected ketol fatty acids at rates comparable with flaxseed AOS. RPP is unusual in that it lacks the amino-terminal membrane anchor and the established organelle targeting sequences found on other conventional P450s. Together, these factors place RPP in the CYP74 family of P450s and establish it as the first P450 localized in rubber particles and the first eukaryotic P450 to be identified outside endoplasmic reticulum, mitochondria, or plastids.

Amino Acid Sequence↗

New phenylenediamine antiozonants for commodities based on natural and synthetic rubber.

For protection of elastomeric materials against ageing, antioxidants such as UV-stabilizers and antiozonants are used. Although historically N-phenyl-N'-(1,3-dimethylbutyl)-p-phenylenediamine (6PPD) was the only approved antiozonant in Germany, a range of other phenylene diamine antiozonants (excluding 6PPD) are permitted for use in rubber articles intended for repeat food-contact use in the US (FDA regulations chapter 21 Part 177.2600). The biggest disadvantage of 6PPD is its partial decomposition during the vulcanization leading to the formation of toxic primary aromatic amines (PAA), such as aniline and secondary aromatic amines (SAA). A number of new PPDs have been developed and patented, that due to their chemical structures, are far less soluble in aqueous solutions but a lot more soluble within the rubber matrix. They therefore show significantly less migration of PAA and SAA. These new antiozonants were investigated and compared to 6PPD using commercial rubber materials with a certain content of antiozonant with regard to their migration of PAA and SAA into three different food simulants. The lowest concentration of PAA and SAA in all food simulants was measured in the RU 997 stabilized elastomer. Due to this fact RU 997 was permitted as a new antiozonant for commodities based on rubber according to the Recommendation XXI 'Articles based on natural and synthetic rubber' of the Federal Institute for Risk Assessment (BfR). RU 997 therefore represents an alternative for 6PPD with less migration of aromatic amines.

Amines↗

Economic status, smoking, occupational exposure to rubber, and lung cancer: a case-cohort study.

Recent studies tend consistently to confirm the presence of a moderate excess risk of lung cancer in the rubber industry. However, the agent responsible for the excess of lung cancer is still obscure. Also, analyses without regard to the modifying effects of sex, economic status, and smoking habit are less than satisfactory. To explore these questions, we have conducted a case-cohort study using the data of 51 lung cancer deaths in 1973-1997 and a random sample (sub-cohort) of 188 from among 1598 subjects in a rubber factory in Shanghai, China. We computed the risks of lung cancer by economic status, smoking habit, coal fumes in home, and year of first employment. We assessed lung cancer risks for occupational exposures, unadjusted and adjusted for economic status and smoking. After confounding effects of smoking and economic status were controlled, we found that rate ratios were 1.43 (95% confidence interval (CI) 0.43-4.69), 1.79 (95% CI 0.64-5.03), and 3.76 (95% CI 1.44-9.86) for 1-14, 15-29, and 30-45 exposure-years in curing department, respectively. The data showed significant trends in increased risk of lung cancer with duration of exposure in tire-curing department (score test for trend:, P = 0.004). However, in front rubber processing (weighing and mixing, calendering, extruding, and milling), no significant excess risk of lung cancer was found. If it can be confirmed that nitrosamines are mainly generated in back rubber processing (curing and vulcanizing), it would be reasonable to conclude that excess risk of lung cancer in rubber industry is attributable, at least partially, to exposure to nitrosamines.

Aged↗

Occupational nitrosamine exposure. 1. Rubber and tyre industry.

To determine the role of N-nitrosamines in the known increased cancer risk of rubber workers, air concentrations of such carcinogens were measured by area sampling or personal monitoring in 19 factories. N-Nitrosodimethylamine (NDMA) and N-nitrosomorpholine (NMOR) were found regularly, the air concentrations varying between 0.1 and 380 micrograms/m3 personal monitoring. The mean concentration was usually in the range of 1-10 micrograms/m3. Several other nitrosamines could be detected in certain production branches. In retail shops and storage rooms of tyres NDMA and NMOR were found. Most rubber chemicals based on amines are contaminated with N-nitrosamines, but this contamination cannot explain the air concentrations of nitrosamines found. The occurrence of nitrosamines mainly depends upon their formation during production of rubber and rubber products from used vulcanisation accelerators based on amines and the presence of nitrosating agents, such as diphenylnitrosamine (retarder A) and of nitrous gases, in products or production areas. Elimination of one or both precursors for nitrosamine formation resulted in significant reduction of airial contamination of nitrosamines. The results are discussed in regard to the mechanisms of nitrosamine formation during rubber production, as basis for future epidemiological studies and their potential for exposure prevention.

Air Pollutants↗

A cohort mortality and cancer incidence survey of recent entrants (1982-91) to the UK rubber industry: findings for 1983-2004.

AIM: To monitor the occurrence of cancer in a recently defined cohort of UK rubber workers. METHODS: A cohort of 8651 male and female workers from 41 UK rubber factories has been enumerated. All employees had a minimum of 12 months employment and were first employed at one of the participating factories in the period 1982-91. Mortality and cancer incidence data for the period 1983-2004 were compared with expected values based on appropriate national rates. RESULTS: Mortality from lung cancer was close to expectation for males [observed 22, standardized mortality ratio (SMR) 93] and females (observed 2, SMR 70). Mortality from stomach cancer was also unexceptional in males (observed 4, SMR 86) and females (observed 0, SMR 0). Although based on small numbers, significantly elevated mortality was shown for multiple myeloma in males (observed 5, SMR 385) and females (observed 2, SMR 952). All seven of these latter deaths occurred in workers from the general rubber goods (GRG) sector. CONCLUSIONS: The findings should be treated with caution as they relate to a relatively early period of follow-up. Nevertheless, they hold out the prospect that the elevated SMRs for stomach and lung cancers reported for historical cohorts of UK rubber workers will not be present in more recent cohorts. The elevated occurrence of multiple myeloma may represent no more than a chance finding. Alternatively, these findings may reflect the presence of an unrecognized occupational cancer hazard in parts of the GRG sector of the UK rubber industry.

Adult↗

Isolation of stress-related genes of rubber particles and latex in fig tree (Ficus carica) and their expressions by abiotic stress or plant hormone treatments.

Two rubber particle protein genes and one latex gene in fig tree (Ficus carica) have been isolated and their expression following various abiotic stress treatments have been investigated. The two major proteins that are tightly associated with the catalytically active rubber particles have been sequenced to be peroxidase (POX) and trypsin inhibitor (TRI). A cDNA encoding a basic class I chitinase (CHI) has also been isolated from the fig tree latex. Wounding treatment strongly induced the expression of the three stress-related genes. Among the abiotic stresses investigated, drought treatment greatly induced the expression of POX, whereas the expression of CHI and TRI decreased after the same treatment. Cold treatment reduced slightly the transcript levels of the thee genes, and NaCl reduced marginally the expression of CHI. The expression of POX, CHI, and TRI was induced by jasmonic acid and abscisic acid, by jasmonic acid, and by salicylic acid, respectively. Different expression of the stress-related genes following various abiotic stress or plant hormone treatments suggests that a crosstalk exists between the signal transduction pathways elicited by abiotic stresses and hormones in plants. Our present results showing the expression of stress-related proteins on the surface of rubber particles and latex in F. carica also imply the possible role of rubber particles and latex in defense in rubber-producing plant species.

Base Sequence↗

IgE-mediated anaphylactoid reactions to rubber.

Delayed hypersensitivity to rubber chemicals is well known, but there are only a limited number of case reports on an immediate type of hypersensitivity inducing contact urticaria. We have recently seen several patients with rubber glove-induced contact urticaria who have had both positive skin prick tests and positive RAST tests to natural latex from the rubber tree (Hevea brasiliensis). We now report on five patients who also developed systemic reactions. It is clear that sensitized subjects are at risk of developing anaphylaxis during medical examination or surgery when rubber gloves are used. The occurrence of IgE-mediated allergy to rubber is probably underestimated.

Adult↗

A gram-negative bacterium, identified as Pseudomonas aeruginosa AL98, is a potent degrader of natural rubber and synthetic cis-1, 4-polyisoprene.

A Gram-negative bacterium, strain AL98, was isolated from foul water inside of a deteriorated car tire on a farmer's field in Münster, Germany. The strain was able to considerably disintegrate natural rubber (NR), either in the raw state as NR latex concentrate or in the vulcanized state as NR latex glove, as well as raw synthetic cis-1,4-polyisoprene (IR). Determination of carbon dioxide evolution and living cell number during batch cultivation with each of the materials as sole source of carbon, revealed mineralization of the rubber polymer during biomass increase. Surface investigation by scanning electron microscopy gave evidence for an adhesive growth behavior of the strain proceeding by colonizing the rubber surface, merging into the rubber and forming a biofilm prior to disintegration of the material. Schiff's reagent staining performed with NR latex gloves indicated production and accumulation of aldehyde groups during colonization. The solid glove substrate disappeared completely after a prolonged cultivation period as a result of continuous degradation. Taxonomic analyses of the strain, which were also based on similarity examination of the complete 16S rRNA gene, revealed classification of strain AL98 as a strain of Pseudomonas aeruginosa. This is the first report about the isolation of a Gram-negative bacterium exhibiting strong rubber decomposing properties.

Automobiles↗

Identification of causative chemicals of allergic contact dermatitis using a combination of patch testing in patients and chemical analysis. Application to cases from rubber footwear.

5 cases of allergic contact dermatitis from rubber footwear were investigated by a combination of patch testing in patients and chemical analysis of causative rubber products. Our studies revealed 2-mercaptobenzothiazole (MBT) and benzothiazyl disulfide (MBTS) (typical allergenic accelerators) as causative chemicals in 3 cases from children's rubber shoes, ladies' rubber boots and ladies' canvas shoes. These 3 patients reacted to mercaptobenzothiazole-type accelerators including MBT and MBTS. MBT and MBTS were determined in each item of causative footwear by chemical analysis, including extraction by shaking with acetone-chloroform (1:1) mixture at room temperature and determination using reversed-phase high-performance liquid chromatography (RP-HPLC). Subsequently, we identified styrenated phenol (SP), a newly found allergenic antioxidant, as a causative chemical in a case from ladies' canvas shoes. The patient reacted to SP but not to MBT and MBTS, though SP, MBT and MBTS were determined in the causative shoes by gas chromatography (GC), GC-mass spectrometry (GC-MS) and HPLC. We also identified p-tert-butylphenol-formaldehyde resin (PTBP-F-R), (a known allergenic adhesive ingredient) as a causative chemical in a case from ladies' sneakers. The patient reacted to PTBP-F-R but not to p-tert-butylphenol (PTBP), MBT and MBTS. These 4 compounds were determined in the causative sneakers by GC, GC-MS and HPLC. Thus, our studies revealed that not only known allergens, such as MBT, MBTS and PTBP-F-R, but also a newly found one, such as SP, were important causes of allergic contact dermatitis from rubber footwear.

Adhesives↗

Identification of causative chemicals of allergic contact dermatitis using a combination of patch testing in patients and chemical analysis. Application to cases from rubber gloves.

5 cases of allergic contact dermatitis from rubber gloves were investigated by our recommended procedures using a combination of patch testing in patients and chemical analysis of causative rubber products by gas chromatography (GC) and high-performance liquid chromatography (HPLC). We previously confirmed that zinc ethylphenyldithiocarbamate (ZEPC), a dithiocarbamae-type accelerator (DTC), was causative in a case of allergic contact dermatitis from rubber work gloves. Subsequently, we have clarified that DTCs such as zinc dimethyldithiocarbamate (ZDMC), zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) and amines such as dimethylamine (DMA), diethylamine (DEA) and piperidine (PIP) were also causative in cases from surgical rubber gloves. Thus, our investigative studies revealed that, although thiurams have been taken much more notice of as allergenic compounds than their corresponding DTCs and amines, not only DTCs such as ZDMC, ZDEC, ZDBC and ZEPC, but also amines such as DMA, DEA and PIP were noteworthy causative candidates of allergic contact dermatitis from rubber gloves.

Adult↗

Contact dermatitis due to rubber boots worn by Japanese farmers, with special attention to 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline (ETMDQ) sensitivity.

An investigation was conducted as follows in 9 farmers with contact allergy due to rubber boots: (i) patch tests with 19 rubber additives; (ii) chemical analysis of additives in 6 pairs of rubber boots; (iii) use tests on a hypoallergenic trial product in 5 patients. The following results were obtained: (i) in the patch tests, all 9 patients showed positive reactions to 1 or more of the nitrogen(N)-containing antioxidants (IPPD, DMBPPD, ETMDQ); (ii) ETMDQ was detected in 1 pair of rubber boots, and IPPD and DMBPPD in another pair; (iii) no patient using hypoallergenic boots during rice-planting had recurrent dermatitis. N-containing antioxidants, such as IPPD, DMBPPD and ETMDQ, were thus considered as the main causative agents and the trial product was found useful for managing contact dermatitis. Contact allergy due to ETMDQ in rubber is reported here for the 1st time.

Aged↗

Anaphylaxis and angioedema due to rubber allergy in children.

Delayed hypersensitivity to rubber chemicals is well known but there are an increasing number of case reports on an immediate type of hypersensitivity inducing contact urticaria and anaphylaxis in adults. We now report on three atopic children who developed angioedema, which in two of them progressed to anaphylaxis after exposure to natural rubber products. All three patients showed positive skin prick tests and one had a positive RAST test to latex from the rubber tree (Hevea brasiliensis). It is clear that sensitized subjects are at risk of developing anaphylaxis when exposed to natural rubber products, e.g. during medical examination or surgery when rubber gloves are used.

Adolescent↗

Novel type of heme-dependent oxygenase catalyzes oxidative cleavage of rubber (poly-cis-1,4-isoprene).

An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latex-grown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH2--and--H2-COCH3. An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40 degrees C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly(cis-1,4-isoprene). Analysis of the amino acid sequence deduced from the cloned gene (roxA [rubber oxygenase]) revealed the presence of two heme-binding motifs (CXXCH) for covalent attachment of heme to the protein. Spectroscopic analysis confirmed the presence of heme, and approximately 2 mol of heme per mol of RoxA was found.

Amino Acid Sequence↗