Induction of direct somatic embryogenesis in garlic (Allium sativum)

An efficient novel method of direct somatic embryogenesis from basal tissue of garlic clove was developed. The influence of plant growth regulators, basal medium and explant type on somatic embryo induction was examined. The best plant growth regulator combination was, 2,4-D and kinetin at 1.0 mg/L and 0.5 mg/L respectively, inducing direct somatic embryogenesis in 60% of explants. White's medium was used as basal medium and somatic embryos developed on explants after six weeks. The technique has potential applicability for problems associated with plant regeneration and virus elimination in garlic.     

Systemic production of IFN-alpha by garlic (Allium sativum) in humans.

The effect of foods on the production of interferon-alpha (IFN-alpha) is currently unknown. Garlic (Allium sativum) used as a folk medicine is reported to stimulate nitric oxide (NO) production. We investigated the systemic increase of NO due to the ingestion of garlic on the plasma IFN-alpha level in normal volunteers. Normal volunteers (10 groups, 10 in each group) ate 2 g fresh garlic, and plasma NO and IFN-alpha levels were determined after 2 and 4 h. The participants were also asked to eat garlic for various periods of time, and plasma NO and IFN-alpha were similarly assayed. Ingestion of 2 g fresh, but not boiled, garlic was found to increase the basal plasma level of NO from 2.7 +/- 0.1 microM to 8.76 +/- 0.21 microM at 2 and 4 h, respectively. The basal plasma IFN-alpha level increased from 9.51 +/- 0.26 nM to 46.3 +/- 1.2 nM in normal volunteers (n = 10) at the same time. The chronic eating of garlic was found to maintain IFN-alpha at high levels for at least 7 days. The exposure of neutrophils to garlic in vivo or in vitro, which also stimulated synthesis of NO in these cells, was found to stimulate IFN-alpha synthesis as measured by the stimulation of IFN-alpha mRNA synthesis. Thus, consumption of garlic resulted in stimulated synthesis of NO and, in turn, IFN-alpha in humans, which could be beneficial in viral or proliferative diseases.     

Effects of garlic (Allium sativum) and its chief compound, allicin, on acute lethality of cyanide in rats

Cyanide, known as a potent suicidal, homicidal and chemical warfare agent, is widely found in plants and used in industry. Cytochrome oxidase, the main enzyme in cell respiration, is inhibited by cyanide resulting to acute or chronic toxicity. The effects of garlic (Allium sativum) and its chief compound, allicin, on the acute lethality of cyanide were studied in rats. Three groups of rats were fed a diet containing 10%, 20% and 30% garlic powder for 48 h and then were challenged with 10 mg/kg cyanide. The lethality of cyanide intoxication was markedly reduced in rats which received garlic by diet and the rate of protection was dose-dependent. The effect of 1,000 ppm of allicin on cyanide lethality was equal to 20% of garlic in diet. These results suggest that sulfur compounds of garlic may have a protective effect against cyanide intoxication.     

Identification of Dioscorea batatas (sanyak) allergen as an inhalant and oral allergen

Dioscorea batatas is widely used in Asia as a herbal medicine or food product with potential health benefits. There have been several reports of occupational asthma caused by inhalation of D. batatas dust. However, there has been no report of systemic allergic reactions after oral administration of D. batatas. Two patients with D. batatas allergy were enrolled. One had experienced severe urticaria and angioedema after indigestion, and the other had been exposed to D. batatas dust and was diagnosed as having occupational asthma. Both patients had high serum-specific IgE and IgG4 antibodies to D. batatas. And IgE immunoblot demonstrated that both sera bound to a 27-kDa protein with an IgE-binding motif, which was revealed by 2-D-electrophoresis to have the sequence Asn-Val-Glu-Asp-Glu-Phe-Ser-X-Ile- Glu-Gly-Asn-Pro-X-X-Pro-Glu-Asn-X-Gly (pI 6.40, 6.04). In conclusion, discorin from D. batatas (DB3S) was identified as the major allergen of D. batatas in patients sensitized via an oral or inhalant route.     

Effects of aqueous garlic (Allium sativum) extract on testicular morphology and function in lead nitrate (Pb(NO3)2)-treated albino rats

This study investigated the effects of aqueous garlic extract (Allium sativum) on testicular morphology and function in lead nitrate (Pb(NO₃)₂)-treated albino rats. Twenty four male albino rats, divided randomly into four groups of six rats per group, were used. Group A rats served as the control and received neither Pb(NO₃)₂ nor aqueous garlic extract (AGE) treatment. The treatments to the remaining three rat groups were as follows: group B, 300 mg/kg body weight of AGE; group C, 2 mg/kg body weight of Pb(NO₃)₂; and group D, 2 mg/kg body weight of Pb(NO₃)₂, and 300 mg/kg body weight of AGE 2 h later. Both the AGE and Pb(NO₃)₂ were orally given to these rats every 48 h for a period of 6 weeks. The testicular and epididymal (left and right sides) sperm reserves and the histomorphological features of the testes of the rats in the treatment groups were compared to the control rats. Results showed that testicular and epididymal sperm reserves were significantly (P?<?0.05) reduced in rats that received only Pb(NO₃)₂ treatment. AGE ameliorated the changes in testicular morphology and function associated with Pb(NO₃)₂ treatment in group D rats. Garlic in this study enhanced spermatogenesis as evidenced from the significant (P?<?0.05) increase in the epididymal (left and right sides) sperm reserves of the group B rats. This implies that garlic may serve as an agent that could be used in improving male fertility.     

Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3)

BACKGROUND: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. It is important to understand the allergenic composition of this ubiquitous fungal species. OBJECTIVE: The complementary DNA (cDNA) clone of an allergen from P citrinum was isolated and expressed in Escherichia coli as a fusion protein. mAbs were prepared with the recombinant protein as antigen. The corresponding natural allergen in the fungal extracts was identified with the mAbs. METHODS: A Uni-Zap XR P citrinum cDNA library was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and expressed as a glutathione-S-transferase fusion protein. The frequency of IgE binding to the expressed protein was analyzed by immunoblotting. Spleen cells from BALB/c mice immunized with the recombinant protein were fused with NS-1 cells for mAb generation. RESULTS: A P citrinum cDNA library was screened with a mixture of serum samples from 4 asthmatic patients. An IgE-binding cDNA clone was obtained and designated as PCE2. PCE2 has a 694-bp insert that contains a 167 amino acids open reading frame. The deduced amino acid sequence of the encoded protein has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxisomal membrane protein allergen (Asp f 3). PCE2 was expressed in E coli as a fusion protein and designated as Pen c 3. Sera from 13 (46%) of the 28 Penicillium-sensitized asthmatic patients demonstrated IgE binding to Pen c 3. In addition, 11 of the 13 Pen c 3-positive serum samples have IgE immunoblot reactivity to recombinant Asp f 3. The presence of IgE cross-reactivity between Pen c 3 and Asp f 3 was also detected by immunoblot inhibition. Four of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3. The presence of the corresponding 18-k natural allergens in the crude extracts of P citrinum and A fumigatus were detected by immunoblot with use of the mAbs and sera from asthmatic patients. CONCLUSION: Results obtained suggest that the peroxisomal membrane protein (Pen c 3) is an important allergen of P citrinum. PCE2 is a full-length cDNA clone encoding this allergen. In addition, the mAbs generated may be useful in standardizing the diagnostic allergenic extracts.     

Effect of a garlic derivative (alliin) on peripheral blood cell immune responses.

The in vitro effect of the garlic derivative alliin, on the mitogen induced peripheral blood mononuclear cell (PBMC) proliferation and cytokine production was examined. In addition, its effect on the engulfing capacity of phagocyting cells was evaluated. The results showed an increase in pokeweed mitogen (PWM) induced cell proliferation, IL-1beta and TNF-alpha production, as well as in the engulfing capacity of both percentage of phagocyting cells and number of latex particles phagocytized by each individual cell. The Con-A induced cell proliferation and IL-6 production decreased following incubation with alliin, whereas PHA-induced cell proliferation, IL-2 and superoxide anion generation remained unchanged. It is concluded that alliin in vitro exerts an immunomodulatory effect on certain functions of the peripheral blood cells.     

Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1)

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.     

cDNA cloning and immunologic characterization of a novel EF-1beta allergen from Penicillium citrinum

BACKGROUND: We have identified previously that Penicillium citrinum is the most prevalent Penicillium species in the Taipei area. It is important to delineate the whole spectrum of allergenic components of this prevalent airborne fungus. The purpose of this study was to identify novel P. citrinum allergens through molecular cloning of allergen genes using a cDNA library of P. citrinum and sera from patients with bronchial asthma. METHODS: A lambda-Uni-ZAP XR-based cDNA library of P. citrinum was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and heterologously expressed in Escherichia coli. The frequency of IgE-binding to the expressed protein and the IgE reactivity to allergen subunits were analyzed by immunoblotting. RESULTS: An IgE-reactive cDNA clone (clone B) was isolated by plaque immunoassay. The cDNA insert is 876-bp long and encodes a 228-amino acid polypeptide with a calculated molecular mass of 25 035 Da. Protein database search with the deduced clone B sequence revealed that 121 (53%) and 82 (36%) of the 228 amino acids were identical to those of the elongation factor 1-beta (EF-1beta) proteins from the yeast Saccharomyces cerevisiae and the parasite Echinococcus granulosus, respectively. His-tagged recombinant clone B proteins were constructed and expressed in E. coli. Seven (8%) of the 92 serum samples from patients with bronchial asthma showed IgE-binding to the recombinant clone B protein. Among these seven positive sera, five demonstrated IgE-binding to the C-terminal fragment (aa 119-228) while the other two sera showed IgE reactivity to the N-terminal fragment (aa 1-118) of this newly identified EF-1betaPenicillium allergen. CONCLUSIONS: A novel P. citrinum allergen (Pen c 24) was identified and characterized in the present study. Results obtained provide more information about allergens of prevalent airborne fungi and a basis to understand more about the IgE responses in human atopic disorders and in parasitic infections.     

Purification and characterization of an allergen from celery immunochemically related to an allergen present in several other plant species. Identification as a profilin

The purification of a 15 kD allergen from celery was obtained by a four step procedure. Evidence for at least two isoallergenic forms was obtained after analysis by two-dimensional-electrophoresis. A rabbit polyclonal antibody raised against this purified allergen allowed us to confirm our precedent results on the occurrence of allergenically and molecular mass-related components in celery and birch and mugwort pollens. In addition such components were also present in numerous other species like Cynodon dactylon, Sorghum halopense, Poa pratensis, Ambrosia elatior and in apple and carrot. The 15 kD allergen was identified as profilin by use of a specific rabbit polyclonal antibody that recognized a recombinant birch profilin. In addition, the purified celery allergen binds IgE from sera of patients allergic to birch profilin. These results reinforce the concept of profilin as a panallergen responsible in some patients for cross-allergic manifestations to various and unrelated species of grasses, weeds, trees, vegetables and fruits.     

Identification and characterization of Che a 1 allergen from Chenopodium album pollen

BACKGROUND: Pollinosis to Chenopodium album has been reported, but no data are available on its allergenic proteins. METHODS: An allergen from C. album pollen has been isolated by means of gel permeation and reverse-phase high-performance liquid chromatography. Molecular characterization was achieved by concanavalin A reaction, mass spectrometry, Edman degradation and cDNA sequence. Antigenic analyses were performed by immunoblotting, ELISA, and ELISA inhibition, using sera from allergic patients, two Ole e 1-specific monoclonal antibodies and an Ole e 1-specific polyclonal antiserum. RESULTS: The isolated allergen, Che a 1, is a glycoprotein of molecular mass 17.088 kD and 143 amino acid residues, whose sequence exhibits 27-45% identity with known members of the Ole e 1-like protein family. 77% of sera from patients allergic to chenopod pollen were reactive to Che a 1. No correlation was found between the IgE reactivities to Che a 1 and Ole e 1, the major allergens from olive pollen, and both allergens display low, although detectable, IgE and IgG cross-reactivities. CONCLUSIONS: Che a 1, a relevant allergen from chenopod pollen, is structurally related to the Ole e 1-like protein family, but exhibits significant differences on its polypeptide sequence that could explain its different antigenic behavior and limited cross-reactivity.     

Identification of a novel cofilin-related molecule (Der f 31) as an allergen from Dermatophagoides farinae

House dust mite (HDM) allergen is a major cause of allergic disease. In this study, two-dimensional immunoblot and Matrix-Assisted Laser Desorption Ionization tandem Time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify Der f 31. After Der f 31 was cloned, expressed and purified, skin prick test (SPT), Immune inhibitory assays, Western blot, ELISA and asthmatic mouse model were employed to examine the allergenicity of recombinant Der f 31. The gene of Der f 31 includes 447 bps, and encoded 148 amino acids. Positive responses of SPT to r-Der f 31 were 32.5% in 43 HDM-allergic patients. r-Der f 31 can induce allergic pulmonary inflammation in the mouse model. In conclusion, Der f 31 is a novel subtype of dust mite allergens.     

Identification and characterization of a 30 kd major allergen from Parapenaeus fissurus

The allergenic components of the shrimp (Parapenaeus fissurus) were identified by immunoblotting, with sera from 10 allergic patients. Six components, ranging in molecular weight from about 86 to 39 kd, showed IgE-binding activity and were identified as allergens of the shrimp. The component with a molecular weight of about 39 kd showed the highest frequency of IgE binding (70%) and was considered to be one of its major allergens. Two monoclonal antibodies against this 39 kd component were generated, and their antigenic cross-reactivity with five different kinds of seafood, shrimp, crab, cuttlefish, oyster, and pomfret was analyzed. Monoclonal antibody 1-6-10B reacted with the 39 kd component from shrimp only, but monoclonal antibody 2-7-IE also reacted with the 39 kd component from crab. By extraction with 0.5% sodium dodecylsulfate and ethanol precipitation, a highly purified shrimp 39 kd component was obtained. In two-dimensional gel electrophoresis six isoforms of this purified 39 kd component, with isoelectric point values from purified 39 kd component, with isoloelectric point values from 5.1 to 5.6, were identified. No marked difference was observed when the amino acid composition of this purified 39 kd allergen was compared with those of serum albumin from different animals. They all contain a high proportion of acidic amino acids. There was also a 62% to 83% sequence homology among three different pairs of peptide fragments of purified 39 kd components of shrimp and crab. In conclusion, a 39 kd major allergen from the shrimp has been identified and characterized in the present study. According to the suggestions of the International Union of Immunological Societies, this allergen is designated as Par f I.     

Identification and characterization of a novel allergen from Blomia tropicalis: Blo t 21

BACKGROUND: Allergenic components from Blomia tropicalis are important triggers of allergies in the tropics. OBJECTIVE: We sought to identify and characterize a novel allergen, Blo t 21, from B tropicalis. METHODS: Blo t 21 was initially identified from an expressed sequence tag database generated from a B tropicalis cDNA library. Allergenicity of this antigen was examined by means of skin prick testing, ELISA, and IgE immuno-dot blotting. We evaluated whether Blo t 21 and Blo t 5 were cross-reactive by using IgE inhibition ELISAs. RESULTS: Blo t 21, a 129-amino-acid protein sharing 39% identity with Blo t 5, is a product of a single-copy gene. It has an alpha-helical secondary structure and localizes to midgut and hindgut contents of B tropicalis, as well as fecal particles. Positive responses to Blo t 21 were shown in 93% (40/43) by means of ELISA and 95% (41/43) by means of skin prick testing when assayed in 43 adult patients with ongoing persistent allergic rhinitis. However, sera of 494 consecutive individuals attending outpatient allergy clinics over 1(1/2) years showed 57.9% (286/494) had positive responses to Blo t 21. Although the majority (>75%) of sensitized individuals were cosensitized to both Blo t 5 and Blo t 21, these 2 allergens had a low-to-moderate degree of cross-reactivity. CONCLUSION: Blo t 21 is a major allergen in B tropicalis that is not highly cross-reactive to Blo t 5, despite sharing some sequence and structural identity. CLINICAL IMPLICATIONS: Blo t 21, representing a new group of allergens, is an important B tropicalis allergen.     

Cloning, expression, and characterization of Der f 7, an allergen of Dermatophagoides farinae from China

A full-length cDNA encoding house dust mite allergen Der f 7 from Dermatophagoides farina (Acari: Pyroglyphidae) from China was cloned, sequenced, and successfully expressed. A reference sequence (GenBank accession AY283292) was used to design polymerase chain reaction primers. Analysis revealed eight mismatched nucleotides in five Der f 7 cDNA clones, and the projected amino acid sequence contained six incompatible residues. These results suggest that the sequence of Der f 7 may be polymorphic. Further bioinformatic analysis revealed that the mature Der f 7 allergen had a molecular mass of approximately 21.88 kDa and a theoretical isoelectric point of 4.90. Der f 7 protein secondary structure was composed of a helix (56.63%), extended strand (5.10%), and random coil (38.27%). Group 7 allergens are present in Pyroglyphidae, Acaridae, and Glycyphagidae families, and homology analysis revealed a 86% similarity between Der f 7 and Der p 7. Furthermore, a phylogenetic tree constructed of group 7 allergens from different mite species revealed that Der f 7 and Der p 7 clustered with 100% bootstrap support. Bioinformatics-driven characterization of Der f 7 allergen as conducted in this study may contribute to diagnostic and therapeutic applications for dust mite allergies.     

Identification and purification of a novel fish allergen from largemouth bass (Micropterus salmoides)

The prevalence of fish allergies has become a serious health problem and has increased alarmingly over the past few years. To contain this problem, we would need to identify all commonly consumed fish allergens. To date, however, no report has identified largemouth bass allergens. This study attempted to identify and purify the major allergen implicated in the allergic response to largemouth bass (Micropterus salmoides), a freshwater fish widely consumed in China. Fifteen patients with a positive history of type I allergy to fish were recruited from skin-prick test and the allergy screen. Total protein extracts and purified allergenic protein from bass were tested for their immunoglobulin E–binding properties. Immunoblot assay resulted in strong reactivity with the 17-kDa protein in all patients. In summary, nucleoside diphosphate kinase B was identified as a novel fish allergen in largemouth bass. This finding is important for allergy diagnoses and the treatment of freshwater fish–allergic disorders.