Identification and cloning of prs a 1, a 32-kDa endochitinase and major allergen of avocado, and its expression in the yeast Pichia pastoris.

Avocado, the fruit of the tropical tree Persea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals. We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocado- and/or latex-allergic patients. Natural Prs a 1 was purified, and its N-terminal and two tryptic peptide sequences were determined. We isolated the Prs a 1 encoding cDNA by PCR using degenerate primers and 5'-rapid amplification of cDNA ends. The Prs a 1 cDNA coded for an endochitinase of 326 amino acids with a leader peptide of 25 amino acids. We expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of culture medium. The recombinant Prs a 1 showed endochitinase activity, inhibited growth and branching of Fusarium oxysporum hyphae, and possessed IgE binding capacity. IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins. Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains. Characterization of chitinases as allergens has implications for engineering transgenic crops with increased levels of chitinases.

Cross-reacting allergens in tree pollen and pollen-related food allergy: implications for diagnosis of specific IgE.

BACKGROUND: A number of recombinant allergens are by now constituents of devices that can be routinely used for the detection of specific IgE. Therefore, the results of diagnostic procedures using conventional allergen extracts can be compared with those employing selected recombinant allergens. METHODS: Thirty-four sera from patients allergic to birch pollen were tested with the standard t3-CAP and rBet v 1a- and rBet v 2-CAP. cDNA was prepared by RT-PCR using primers according to the N terminus of purified allergens. Expression cDNA libraries were screened with IgE from selected patients. RESULTS: Twenty-four patients allergic to birch pollen showed the same RAST class with t3 as with rBet v 1a; 8 patients differed within 1 RAST class. In addition, 3 patients showed RAST class 3 with rBet v 2. Besides Bet v 1 and Bet v 2, 3 allergens from celery and avocado belonging to highly conserved protein families were cloned and sequenced. CONCLUSIONS: rBet v 1a can be expected to represent an excellent tool for the diagnosis of patients allergic to birch pollen in Central, Northern, and Eastern Europe. Still, a much higher number of patients has to be tested. For their high degree of conservation, further protein families have to be identified to explain cross-reactivities of birch pollen allergens other than Bet v 1 and Bet v 2 with, e.g., allergens from vegetable food.

Comparison of concentrations of Cry j 1 and Cry j 2 in diploid and triploid Japanese cedar (Cryptomeria japonica) pollen extracts.

Japanese cedar (Cryptomeria japonica) possibilities has been a serious allergic disease in Japan. There are two kinds of Japanese cedar trees; the popular one is diploid, the less popular is triploid. These trees are not very different morphologically. However, the relative allergenicity of their pollens is unknown, although both major allergens, Cry j 1 and Cry j 22, have been purified and cloned from the diploid line. Triploid trees are sterile and the allergenicity of their pollen may differ. Using Japanese-cedar-allergic patient sera, we compared the concentration of these two major allergens in both kinds of pollen. Pollen collected from different years and regions was also studied. The results indicate that triploid tree pollen extract has lower concentrations of both major allergens; therefore, the pollen may be less allergenic.

A novel acidic allergen, Hev b 5, in latex. Purification, cloning and characterization.

Latex allergy is recognized as a serious health problem among health care workers and children with spina bifida. A number of IgE-reactive proteins have been identified in natural and processed latex products. One of the most acidic proteins in the cytoplasm of lacticifer cells of rubber trees (Hevea brasiliensis) is demonstrated to be a potent allergen in eliciting allergic reactions in humans. This protein, with pI = 3.5, has a molecular mass of 16 kDa with a blocked N terminus and an unusual amino acid composition. This acidic protein was found in extracts prepared from latex gloves, which were shown to be allergenic. The purified protein elicits histamine release from human basophils passively sensitized with serum from latex-allergic individuals in a dose-dependent manner. From a latex cDNA library, the cDNA coding for this protein was isolated and sequenced. The deduced amino acid sequence shows a high degree of homology to another acidic protein identified in kiwifruit (Actinidia deliciosa var. deliciosa). The sequence homology (47% sequence identity) between these two acidic proteins suggests a molecular explanation for the high frequency of fruit hypersensitivity in latex-allergic patients.

Comparison of latex-specific IgE binding among nonammoniated latex, ammoniated latex, and latex glove allergenic extracts by ELISA and immunoblot inhibition.

BACKGROUND: Nonammoniated latex, ammoniated latex, and latex glove extracts have been used as source materials for the preparation of allergenic extracts for the diagnosis of latex allergy. These materials showed different patterns of protein bands and immunoreactive bands. However, their IgE-reactive repertoires were not compared. OBJECTIVE: The goals of this study were to compare the IgE reactivity and to define the common IgE-reactive epitopes among three latex allergenic extracts. METHODS: Two serum pools were obtained from adults and children with latex allergy to evaluate the IgE reactivity among three latex extracts. IgE reactivity and IgE-reactive proteins were compared by inhibition ELISA and inhibition immunoblot methods, respectively. RESULTS: In this study inhibition curves were similar for nonammoniated latex and ammoniated latex but were different when the latex glove extracts were used. Several protein bands of ammoniated latex and latex glove extracts could not be inhibited by the nonammoniated latex. The ammoniated latex and latex glove extracts were able to remove all the latex-specific IgE from the serum. CONCLUSION: The IgE-reactive proteins differ among different latex extracts. Ammoniated latex and latex glove extracts contain more complete immunoreactive repertoires for detecting IgE antibodies. Our study provides useful information for selecting the latex extract.

Characterization of apple 18 and 31 kd allergens by microsequencing and evaluation of their content during storage and ripening.

Patients with tree pollinosis frequently report allergic reactions after ingestion of apples. The severity of apple allergy has been related to the variety of apples and their degree of maturity. To generate a serum pool that is representative of various IgE-binding patterns of apple-allergic sera, serum samples from 34 patients allergic to tree pollens were screened. Only 24 serum samples reacted to the apple extract. Pooled serum was used to identify allergens in apples. An efficient and consistent extraction method for apple fruits was used to compare the immunoreactivities of extracts of different varieties (McIntosh, Red Delicious, Granny Smith, and Golden Delicious) of freshly picked and store-purchased apples. We found that Golden Delicious apples had the greatest amount of the 18 kd allergen, which has been reported to be a potent IgE-binding apple allergen. Store-purchased apples contained higher concentrations of the 18 kd allergen than freshly picked apples. In our study only 37.5% of sera reacted to the 18 kd protein, whereas 75% of the sera reacted to a 31 kd allergen. Other immunoreactive bands in apple extracts included proteins of 50, 38, 16, 14, and 13 kd. The amino-terminal amino acid sequences of the two major allergens, 18 kd and 31 kd, were determined. These sequences shared approximately 50% identity with disease resistance proteins of various plants or Bet v 1 in birch tree pollens. The appearance of various allergens was also investigated in mature apples during storage. The amount of 18 kd allergen increased significantly when apples were stored at 4 degrees C. However, under controlled atmospheric conditions in which oxygen- and carbon dioxide-induced ripening were regulated, the amount of 18 kd allergen remained unaffected. Because ripening and maturation were not associated with increases in 18 kd allergen content, the observed changes might be induced by factors related to disease resistance.

Serum reactivities to latex proteins (Hevea brasiliensis).

BACKGROUND: In recent years there has been an increasing incidence of allergy to latex among health care workers and children with spina bifida. The allergic response in these individuals can be severe and occasionally fatal. Several allergens have been identified with the use of sera from different patient groups. In our effort to identify reagents for in vitro testing and clinical use, we investigated the reactivities of latex proteins to sera collected from a wide range of patients with latex allergy. METHODS: Twenty-six serum samples were obtained from adult patients with latex allergy, both hospital workers and non-hospital workers. Serum pools were made either from sera of children with spina bifida or sera of adult patients with latex allergy. Proteins from C-serum and latex particles of latex sap (nonammoniated) were separated by different gel electrophoresis techniques and evaluated for specific IgE binding by immunoblotting. RESULTS: More than 50% of the sera tested reacted to an 18 kd protein, a 25.6 kd acidic protein with an isoelectric point of 3.5, or to both proteins; whereas only 23% of the individual serum samples tested reacted to the rubber elongation factor, which has been reported to be a major latex allergen. The immunoreactive patterns of children's and adults' serum pools were similar but not identical. CONCLUSIONS: With the use of gel electrophoresis and immunoblotting techniques, different immunoreactive proteins were identified in C-serum and particles of latex. Rubber elongation factor, which reacted to only 23% of sera tested, did not appear to cross-react immunologically with other latex allergens.

Bacterial DNA supercoiling and [ATP]/[ADP] ratio: changes associated with salt shock.

When Escherichia coli K-12 was shifted from a medium lacking salt to one containing 0.5 M NaCl, both the [ATP]/[ADP] ratio and negative supercoiling of plasmid DNA increased within a few minutes. After about 10 min both declined, eventually reaching a level slightly above that observed with cells growing exponentially in the absence of salt. Since in vitro the [ATP]/[ADP] ratio influences the level of supercoiling generated by gyrase (H. Westerhoff, M. O'Dea, A. Maxwell, and M. Gellert, Cell Biophys. 12:157-181, 1988), the physiological response of supercoiling to salt shock is most easily explained by the sensitivity of gyrase to changes in the intracellular [ATP]/[ADP] ratio. This raises the possibility that the [ATP]/[ADP] ratio is an important factor in the control of supercoiling.

Bacterial DNA supercoiling and [ATP]/[ADP]. Changes associated with a transition to anaerobic growth.

Shifting Escherichia coli from aerobic to anaerobic growth caused changes in the ratio of [ATP]/[ADP] and in negative supercoiling of chromosomal and plasmid DNA. Shortly after lowering oxygen tension, both [ATP]/[ADP] and supercoiling transiently decreased. Under conditions of exponential anaerobic growth, both were higher than under aerobic conditions. These correlations may reflect an effect of [ATP]/[ADP] on DNA gyrase, since in vitro [ATP]/[ADP] influences the level of plasmid supercoiling attained when gyrase is either introducing or removing supercoils. When the supercoiling activity of gyrase was perturbed by a mutation in gyrB, a shift to anaerobic conditions resulted in plasmid supercoil relaxation similar to that seen with wild-type. However, the low level of supercoiling in the mutant persisted during a time when supercoiling in wild-type recovered and then exceeded aerobic levels. Thus, changes in oxygen tension can alter DNA supercoiling through an effect on gyrase, and correlations exist between changes in supercoiling and changes in the intracellular ratio of [ATP]/[ADP].

Neurite extension factor induces rapid morphological differentiation of mouse neuroblastoma cells in defined medium.

The present study describes an in vitro bioassay for neurite-promoting factors using a mouse neuroblastoma cell line (Neuro-2A) in defined medium at low cell density. Neurite extension factor (NEF) is a disulfide-bonded dimer of a protein closely related to S100 beta, a calcium-binding protein made by glial cells. NEF induces process outgrowth from chick embryo cerebral cortical neurons. We now report that NEF induces rapid morphological differentiation of Neuro-2A cells. Within 4-6 h of addition of NEF, the cells elaborate multipolar neurites and the cell body becomes rounded. In the absence of NEF, the cells do not extend neurites and the cell body appears flattened. This response is dose-dependent, with half-maximal stimulation at a concentration of about 300 ng/ml (15 nM) of NEF. This completely defined system should be useful for characterizing NEF receptors and studying the mechanism of action of NEF.

Carcinogenicity of dietary aflatoxin M1 in male Fischer rats compared to aflatoxin B1.

Aflatoxin M1 (AFM), an hydroxy metabolite of the potent carcinogenic mycotoxin aflatoxin B1 (AFB) is frequently found in milk and other dairy products. Sufficient amounts of AFM were produced to study the carcinogenicity of this compound. AFM was fed to male Fischer rats starting at 7 weeks up to 21 months of age. Agar-based semisynthetic diets contained 0.0, 0.5, 5.0, and 50.0 micrograms/kg of AFM or 50 micrograms/kg of AFB. Hepatocellular carcinomas were detected in two of 37 rats and neoplastic nodules were found in six of 37 rats fed 50 micrograms/kg AFM between 19 and 21 months. No nodules or carcinomas were observed in the lower AFM dose groups. Nineteen of 20 rats fed a diet containing 50 micrograms/kg of AFB developed hepatocellular carcinomas by 19 months of age. Carcinogenic potency of the aflatoxins was reflected by morphometric quantitation of foci detected in hematoxylin and eosin stained sections. Three rats fed the diet containing 50 micrograms/kg AFM developed intestinal carcinomas. None were observed in other groups. Under the conditions of this experiment AFM was found to be a weak hepatic carcinogen compared to AFB and to possess intestinal carcinogenicity.

The effect of i-mu-ts'ao on a partially purified prostaglandin E 9-ketoreductase from swine kidney.

The enzyme system, prostaglandin E 9-ketoreductase, which catalyzes the reduction of prostaglandin E to form prostaglandin F has been partially purified from swine kidney. This NADPH-linked enzyme is studied spectrophotometrically. The KM of this enzyme for prostaglandin E2 was found to be 180 microM. Studies with the partially purified enzyme indicate that prostaglandin E 9-ketoreductase is affected by a Chinese herbal medicine i-mu-ts'ao (leonurus heterophyllus sweet). An increase in the concentration of i-mu-ts'ao aqueous extract may influence the conversion of prostaglandin E2 into prostaglandin F2 alpha. This finding offers a possible explanation for the physiological role of i-mu-ts'ao when it is treated as a medicine.

Cancer risks posed by aflatoxin M1.

The suspect milk-borne carcinogen, aflatoxin M1 (AFM), was produced and isolated from the rice culture of the fungus Aspergillus flavus NRRL3251 for confirmation and determination of the potency of its carcinogenicity in the male adult Fischer rat. The carcinogen was mixed into an agar-based, semisynthetic diet at 0, 0.5, 5, and 50 ppb (microgram/kg) and was fed to groups of animals continuously for 19-21 months. Aflatoxin B1 (AFB), of which AFM is a metabolite, at 50 ppb was used as a positive control. Hepatocarcinogenicity of AFM was detected at 50 ppb, but not at 5 or 0.5 ppb, with a potency of 2-10% that of AFB. A low incidence of intestinal adenocarcinomas was found in the AFM 50 ppb group, but not in any other groups. At 0.5 ppb, the action level enforced by the U.S.A. Food and Drug Administration, AFM induced no liver lesions in the rats but stimulated the animals' growth. On the average, the rats in the 0.5 ppb group weighed 11% (p less than 0.001) more than those in the control group. This increased growth was associated with increased feed intake. Based on the biological activity of AFM at the relevant low doses and the estimated level of human exposure to AFM through consumption of milk, the cancer risk posed by this contaminant for human adults is assessed to be very low. For infants, further studies are warranted because milk constitutes the major ingredient of the infant diet and because infant animals have been shown to be more sensitive to the carcinogenicity of AFB than adult animals.

Irreversibility of liver tumors in C3H mice.

The present study was designed to test the possibility that spontaneous regression of hepatocellular tumors might be observed in mice. This problem was studied by sequential liver biopsies in C3H male mice that had been treated with dieldrin (CAS: 60-57-1) as well as in animals treated with N-diethylnitrosamine (CAS: 55-18-5) and in untreated control mice. Adenomas were seen in some animals at the second laparotomy when there had been no tumor at the first laparotomy. In a few mice there was histologic progression from adenoma to carcinoma. A change in predominant cell type in adenomas from clear to basophilic or eosinophilic was also observed in some cases. Additional hepatocellular carcinomas were observed in some animals necropsied at 2 years of age. These observations suggest that spontaneous hepatic tumors and tumors in mice treated with either complete carcinogens or nongenotoxic compounds have a strong tendency to progress. Tumor regression in mice appears to be unusual. No consistent relationship of histologic grade of hepatocarcinoma to the type of chemical employed was observed.

Dieldrin-induced mallory bodies in hepatic tumors of mice of different strains.

Mallory bodies (MBs) were induced in hepatic tumors by administration for up to 85 weeks of a diet containing 10 ppm dieldrin to 50 C3H/He and 62 C57BL/6J x C3H/He B6C3F1 male mice. MBs were seen in 15 of 28 (54%) mice which developed benign hepatic tumors and 33 of 45 (73%) mice with hepatocellular carcinoma, but in only 3 of 39 (8%) mice without hepatic tumors. In mice with tumors, the MBs were predominantly confined to tumor tissue and persisted in a carcinoma transplanted into a nude mouse. MBs were not observed, however, in hepatic tumors of 67 C57BL/6J, 49 C3H/He, or 81 B6C3F1 mice given 12 micrograms diethylnitrosamine i.p. on Days 0, 3, 9, and 15. Thirty-one of 195 control mice of all three strains had hepatic tumors. Only one of the controls had a tumor with an MB, and no MBs were seen in nontumor-bearing livers of controls animals. These observations, coupled with the results of a previous study in which MBs were observed in hepatocytes of dieldrin-treated C57BL/6J mice, indicate that mice treated with dieldrin are a reliable animal model for the study of MBs.

Mallory body formation in hepatic nodules of mice ingesting dieldrin.

Mallory bodies (MBs) were observed in hepatic nodules induced by long-term administration of a diet containing 10 p.p.m. of dieldrin to C57BL/6 mice. MBs were first detected after 46 weeks and were seen in 26 of 41 mice which developed hepatic nodules. The MBs were limited to the nodules in 25 mice. Twenty-one of the nodules were carcinomas and 20 of those contained MBs. Our observations suggest that MBs may be a marker for neoplastic transformation and may prove useful in experimental studies of carcinogenesis.