A specific CBP/p300-dependent gene expression programme drives the metabolic remodelling in late stages of spermatogenesis.

Histone hyperacetylation is thought to drive the replacement of histones by transition proteins that occur in elongating spermatids (ElS) after a general shut down of transcription. The molecular machineries underlying this histone hyperacetylation remain still undefined. Here, we focused our attention on the role of Cbp and p300 in histone hyperacetylation and in the preceding late-gene transcriptional activity in ElS. A strategy was designed to partially deplete Cbp and p300 in ElS. These cells progressed normally through spermiogenesis and showed normal histone hyperacetylation and removal. However, a genome-wide transcriptomic analysis, performed in the round spermatids (RS) and ElS, revealed the existence of a gene regulatory circuit encompassing genes presenting high expression levels in pre-meiotic cells, undergoing a repressed state in spermatocytes and early post-meiotic cells, but becoming reactivated in ElS, just prior to the global shutdown of transcription. Interestingly, this group of genes was over-represented within the genes affected by Cbp/p300 knock down and were all involved in metabolic remodelling. This study revealed the occurrence of a tightly regulated Cbp/p300-dependent gene expression programme that drives a specific metabolic state both in progenitor spermatogenic cells and in late transcriptionally active spermatids and confirmed a special link between Cpb/p300 and cell metabolism programming previously shown in somatic cells.

The association of genetic variants in Krüppel-like factor 11 and Type 2 diabetes in the Japanese population.

AIMS: Krüppel-like factor 11 (KLF11) is a transcriptional factor of the zinc finger domain family that regulates the expression of insulin. In North European populations, its common functional variant Q62R (rs35927125) is a strong genetic factor for Type 2 diabetes (P = 0.00033, odds ratio for G allele = 1.29, 95% CI 1.12-1.49). We examined the contribution of KLF11 variants to the susceptibility to Type 2 diabetes in a Japanese population. METHODS: By re-sequencing Japanese individuals (n = 24, partly 96), we screened all four exons, exon/intron boundaries and flanking regions of KLF11. Verified single nucleotide polymorphisms (SNPs) were genotyped in 731 initial samples (369 control and 362 case subjects). Subsequently, we tested for association in 1087 samples (524 control and 563 case subjects), which were collected in different districts of Japan from the initial samples. RESULTS: We identified eight variants, including a novel A/C variant on intron 3, but no mis-sense mutations. In an association study, we failed to find any significant result of SNPs (minor allele frequency 8.2-46.2%) after correcting for multiple testing. Similarly, no haplotypes were associated with Type 2 diabetes. It is notable that the G allele in rs35927125 was completely absent in 1818 Japanese individuals. CONCLUSIONS: Genetic variants in KLF11 are unlikely to have a major effect of Type 2 diabetes in the Japanese population, although they were significantly associated in North European populations. These observations might help to determine the role of KLF11 variants in Type 2 diabetes in different populations.

Genetic association of single nucleotide polymorphisms in endonuclease G-like 1 gene with type 2 diabetes in a Japanese population.

AIMS/HYPOTHESIS: In order to identify type 2 diabetes disease susceptibility gene(s) in a Japanese population, we applied a region-wide case-control association test to the 20.4 Mb region between D3S1293 and D3S2319 on chromosome 3p24.3-22.1, supported by linkage to type 2 diabetes and its related traits in Japanese and multiple populations. MATERIALS AND METHODS: We performed a two-stage association test using 1,762 Japanese persons with 485 gene-centric, evenly spaced, common single nucleotide polymorphism (SNP) markers with minor allele frequency >0.1. For mouse studies, total RNA was extracted from various organs of BKS.Cg-+Lepr(db)/+Lepr(db) and control mice, and from MIN6, NIH3T3 and C2C12 cell lines. RESULTS: We detected a landmark SNP375 (A/G) (rs2051211, p = 0.000046, odds ratio = 1.33, 95% CI 1.16-1.53) in intron 5 of the endonuclease G-like 1 (ENDOGL1) gene. Systematic dense SNPs approach identified a susceptibility linkage disequilibrium (LD) block of 116.5 kb by |D'|, an LD units map and a critical region of 2.1 kb by r (2) in ENDOGL1. A haplotype-based association test showed that an at-risk haplotype is associated with disease status (p = 0.00001). The expression of ENDOGL1 was rather ubiquitous with relatively abundant expression in the brain and also in a pancreatic islet beta cell line. Mouse Endogl1 expression increased in pancreatic islets of hyperglycaemic BKS.Cg-+Lepr(db)/+Lepr(db) mice compared with that in control mice. CONCLUSIONS/INTERPRETATION: Based on the population genetics, fine mapping of LD block and haplotype analysis, we conclude that ENDOGL1 is a candidate disease-susceptibility gene for type 2 diabetes in a Japanese population. Further analysis in a larger sample size is required to substantiate this conclusion.

Evaluation of combination therapy using aciclovir and corticosteroid in adult patients with herpes simplex virus encephalitis.

OBJECTIVE: Herpes simplex virus encephalitis (HSVE) is associated with significant morbidity and mortality, even with appropriate antiviral therapy. In the present investigation, the first to assess efficacy of corticosteroid treatment with aciclovir therapy in HSVE, multiple logistic regression analysis was performed of predictors of outcome in adult patients with HSVE. METHODS: A non-randomised retrospective study of 45 patients with HSVE treated with aciclovir was conducted. The patients were divided into poor and good groups based on outcome at three months after completion of aciclovir treatment. The variables evaluated were: clinical variables (sex, age, days after onset at initiation of aciclovir, Glasgow Coma Scale (GCS) at initiation of aciclovir, initial and maximum values for the cell numbers and protein concentration in the cerebrospinal fluid, and corticosteroid administration); neuroradiological variables (detection of lesions by initial cranial computed tomography and by initial magnetic resonance imaging); and one neurophysiological variable (detection of periodic lateralised epileptiform discharges on the initial electroencephalogram). Single variable logistic regression analysis was performed followed by multiple logistic regression analysis. The best set of predictors for the outcome of HSVE was estimated by stepwise logistic regression analysis. RESULTS: A poor outcome was evident with older age, lower GCS score at initiation of aciclovir, and no administration of corticosteroid. Patient age, GCS at initiation of aciclovir, and corticosteroid administration were found to be significant independent predictors of outcome on multiple logistic regression analysis, and these three variables also formed the best set of predictors (R(2) = 0.594, p<0.0001). CONCLUSION: Combination therapy using both aciclovir and corticosteroid represents one of the predictors of outcome in HSVE.

Nested polymerase chain reaction for assessing the clinical course of tuberculous meningitis.

The authors examined the usefulness of nested PCR (N-PCR) to detect Mycobacterium tuberculosis (MTB) DNA in CSF for assessing the clinical course of tuberculous meningitis (TBM). N-PCR successfully detected MTB DNA in all nine CSF samples from patients with suspected TBM. During anti-tuberculosis treatments, N-PCR results converted from positive to negative, correlating with the improvement of the patient's clinical condition.

Microbial counts, fermentation products, and aerobic stability of whole crop corn and a total mixed ration ensiled with and without inoculation of Lactobacillus casei or Lactobacillus buchneri.

Whole crop corn (DM 29.2%) and a total mixed ration (TMR, DM 56.8%) containing wet brewers grains, alfalfa hay, dried beet pulp, cracked corn, soybean meal, and molasses at a ratio of 5:1:1:1:1:1 on fresh weight basis, were ensiled with and without Lactobacillus casei or Lactobacillus buchneri in laboratory silos. The effects of inoculation on microbial counts, fermentation products, and aerobic stability were determined after 10 and 60 d. Untreated corn silage was well preserved with high lactic acid content, whereas large numbers of remaining yeasts resulted in low stability on exposure to air. Inoculation with L. casei suppressed heterolactic fermentation, but no improvements were found in aerobic stability. The addition of L. buchneri markedly enhanced the aerobic stability, while not affecting the DM loss and NH3-N production. Large amounts of ethanol were found when the TMR was ensiled, and the content of ethanol overwhelmed that of lactic acid in untreated silage. This fermentation was related to high yeast populations and accounted for a large loss of DM found in the initial 10 d. The ethanol production decreased when inoculated with L. casei and L. buchneri, but the effects diminished at 60 d of ensiling. Inoculation with L. buchneri lowered the yeasts in TMR silage from the beginning of storage; however, the populations decreased to undetectable levels when stored for 60 d, regardless of inoculation. No heating was observed in TMR silage during aerobic deterioration test for 7 d. This stability was achieved even when a high population of yeasts remained and was not affected by either inoculation or ensiling period. The results indicate that inoculation with L. buchneri can inhibit yeast growth and improve aerobic stability of corn and TMR silage; however, high stability of TMR silage can be obtained even when no treatments were made and high population (>10(5) cfu/g) of yeasts were detected.

Accumulation of 1,2-propanediol and enhancement of aerobic stability in whole crop maize silage inoculated with Lactobacillus buchneri.

AIMS: To assess the effects of inoculation of Lactobacillus buchneri on the ensiling properties and aerobic stability of maize silage. METHODS AND RESULTS: Chopped whole crop maize was ensiled in 0.5 litre airtight polyethylene bottles (0.4 kg per bottle) and in double-layered, thin polyethylene bags (15 kg per bag), with or without inoculation of Lact. buchneri. The silos were stored for two to four months and the chemical composition, microbial numbers and aerobic stability were determined. Inoculation lowered lactic acid and yeasts, and increased acetic acid and pH value, resulting in improved aerobic stability of the silages. Inoculated silages produced 1,2-propanediol, the content of which increased as ensiling was prolonged, and nearly 50 g kg-1 dry matter had accumulated after four months of storage. The effects of inoculation, however, were much less pronounced in silages prepared in bags. Mannitol was found in all silages; the production was lowered by Lact. buchneri treatment and appeared to be unrelated to the accumulation of 1,2-propanediol. CONCLUSIONS: Inoculation of Lact. buchneri occasionally causes accumulation of 1,2-propanediol in silages without further degradation into propionic acid and 1-propanol. SIGNIFICANCE AND IMPACT OF THE STUDY: Substantial amounts of 1,2-propanediol could be consumed by ruminants when fed on silages inoculated with Lact. buchneri. In addition to increasing acetic acid, attention needs to be paid to 1,2-propanediol because the two fermentation products might affect the intake and utilization of silage-based diets.

Induction of gene into the rabbit eye by iontophoresis: preliminary report.

PURPOSE: To assess the transfer of 6-carboxyfluorescein (6-FAM)-labeled phosphorothioate oligonucleotides(S-ODNs) into the ocular tissues, their stability, and possibility of injury to the ocular tissues. METHODS: The S-ODNs(2 mL/eye)were transduced noninvasively into albino rabbit eyes.The iontophoresis group consisted of 6 rabbits (12 eyes); the control group consisted of 2 rabbits (4 eyes) given eye drops containing S-ODNs. Aqueous humor and vitreous humor were collected after iontophoresis, subjected to electrophoresis with a fluorescence DNA sequencer and analyzed by the Gene Scan program. Frozen sections, 10-microm thick, were prepared for observation under a fluorescence microscope. A plasmid 4.7 kbp in size that expresses green fluorescent protein (GFP) was induced into the 18 eyes of 9 rabbits by the same procedure. RESULTS: In the iontophoresis group, S-ODNs were detected in the anterior chamber 5 minutes after electrophoresis began and in the vitreous after 10 minutes. These S-ODNs maintained the same length as at the initial synthesis. The S-ODNs could also be detected in the posterior retina 20 minutes after electrophoresis. No evidence of degeneration or inflammation due to the above procedure was found in the ocular tissues. Fluorescence showing GFP gene expression was found in the cornea, the anterior chamber angle, and the ciliary subepithelial tissues. CONCLUSIONS: These findings show that iontophoresis is an effective method to induce genes into the rabbit eye.

[Cytokines in the vitreous fluid of patients with proliferative diabetic retinopathy--vascular endothelial growth factor and platelet-derived growth factor are elevated in proliferative diabetic retinopathy].

PURPOSE: To determine the relation between vascular endothelial growth factor (VEGF) and platlet-derived growth factor (PDGF) in the vitreous fluids from patients with proliferative diabetic retinopathy (PDR). SUBJECTS AND METHOD: Concentrations of VEGF and PDGF in the vitreous fluids from 53 eyes, 31 eyes in 30 PDR patients and 22 eyes in 22 non diabetes mellitus(DM) patients serving as controls, were measured by enzyme-linked immunosolvent assay. The levels of VEGF and PDGF were compared between PDR and non DM patients. The relationship between these factors and clinical characteristics such as age, sex, protein concentration in the aqueous humor, HbA1c and duration of diabetes were investigated. RESULTS: Levels of both VEGF and PDGF in the PDR group were significantly higher than in non DM group (p < 0.01). A positive correlation was observed between the levels of VEGF and protein concentration in the aqueous humor (p < 0.01). However, levels of both VEGF and PDGF did not correlate with age, sex, or HbA1c. CONCLUSION: Both VEGF and PDGF were shown to be correlated with the pathogenesis of PDR.

Absorption enhancement of a protein drug by nitric oxide donor: effect on nasal absorption of human granulocyte colony-stimulating factor.

The objective of this study was to evaluate the effect of a nitric oxide (NO) donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the nasal absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rabbits and to evaluate the irritation (cytotoxicity) potential of the NO donor on the mucosal membrane using a cultured cell system (strain KB, human epidermoid carcinoma of the floor of the mouth). Significantly higher serum G-CSF concentration and increased total leukocyte count in the peripheral blood were observed after coadministration of rhG-CSF (100 microg/kg) with SNAP at various doses (0.3-3.3 mg/kg). The serum G-CSF concentration and the increased total leukocyte count were markedly decreased by the presence of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazole-1-oxyl 3-oxide sodium salt (carboxy-PTIO), in combination with rhG-CSF and SNAP. However, no significant inhibitory effect of glutathione (peroxynitrite scavenger) on the absorption-enhancing effect of SNAP was observed. These results suggest that carboxy-PTIO inhibits the absorption-enhancing effect of NO released from SNAP. We found that SNAP has a very low potential for cytotoxicity, as evaluated by the cell detachment assay, release of lactate dehydrogenase (LDH) from cultured cells and morphological observations of nasal tissue of rabbits. It is concluded that a NO donor such as SNAP is a promising absorption enhancer for nasal protein-drug delivery.

Antiapoptotic activity of herpes simplex virus type 2: the role of US3 protein kinase gene.

In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.

Requirement for splenic CD4+ T cells in the immune privilege of the anterior chamber of the eye.

Injection of antigen into the anterior chamber of the eye induces suppression of antigen-specific DTH, called anterior chamber-associated immune deviation (ACAID). It has been shown that the spleen is required for the induction of ACAID and detecting the ACAID-inducing signal from the eye. To examine the in vivo role of spleen cells, fractions of spleen cells were adoptively transferred into splenectomized mice. The present study showed that DTH was not suppressed in splenectomized mice, but was inhibited in splenectomized mice transferred with a primed CD4+ T cell-containing fraction of spleen cells. This indicates that the splenic CD4+ T cells comprise the regulatory T cells for the DTH response. When we examined the cytokine profile of the infiltrating T cells in the eye of primed mice by reverse transcriptase-polymerase chain reaction (RT-PCR), we found that they expressed IL-4, IL-10 mRNA (Th2 type), but not IL-2 and interferon-gamma (IFN-gamma) mRNA (Th1 type). By contrast, T cells which can elicit normal DTH response expressed IL-2 and IFN-gamma mRNA. These results suggest that splenic CD4+ T cells comprising the regulatory phenotype are required for the induction of ACAID, and that a DTH response to the antigen may be prevented by Th2-dominant CD4+ T cells.

[Induction of genes into the rabbit eye by iontophoresis].

PURPOSE: After inducing 6-carboxyfluorescein (6-FAM)-labeled phosphorothioate oligonucleotides (S-ODNs) noninvasively into albino rabbit eyes by iontophoresis, we assessed the transfer of S-ODNs into the ocular tissues, their stability, and the possible presence of injury to the ocular tissues. METHODS: The iontophoresis group consisted of 12 eyes of 6 rabbits and the control group consisted of 4 eyes of 2 rabbits given eye drops containing S-ODNs. Aqueous humor and vitreous humor were collected after iontophoresis, subjected to electrophoresis with a fluorescent DNA sequencer and analyzed by the Gene Scan program. Frozen sections at 10 microns were prepared for observations under a fluorescent microscope. A plasmid 4.7 kbp in size that expresses green fluorescent protein (GFP) was induced into 18 eyes of 9 rabbits by the same procedure. RESULTS: In the iontophoresis group, S-ODNs were detected in the anterior chamber 5 minutes after electrophoresis and in the vitreous 10 minutes after. These S-ODNs maintained the same length as at the initial synthesis. S-ODNs could also be detected in the posterior retina 20 minutes after electrophoresis. No evidence of degeneration or inflammation due to the above procedure was found in the ocular tissues. Fluorescence showing GFP gene expressions were found in the cornea, the anterior chamber angle, and the ciliary subepithelial tissues. CONCLUSIONS: These findings show that iontophoresis is an effective method to induce gene into rabbit eyes.

Polymorphisms of thymidine kinase gene in herpes simplex virus type 1: analysis of clinical isolates from herpetic keratitis patients and laboratory strains.

Drug-resistance of herpes simplex virus (HSV) is caused most frequently by mutation of the viral thymidine kinase (TK) gene. To elucidate the significance of detecting nucleotide changes of the TK gene for screening drug-resistant viruses, the frequency and variation of the genetic polymorphisms in the whole coding region of the TK gene were studied in 14 acyclovir-susceptible HSV type 1 (HSV-1) clinical isolates from 14 patients with epithelial herpetic keratitis. Two reference HSV-1 laboratory strains, McKrae and PH, and two acyclovir-resistant variants of the PH strain were also studied as controls. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing detected nucleotide differences at 24 positions, and amino acid substitutions at 12 codons in the TK gene of the examined viruses. Nucleotide diversity of 0.0029 per base (the average number of nucleotide substitutions of 3.3 per 1,131 base pairs) in the TK gene in the clinical isolates was comparable to 0.0037 per base of the whole HSV-1 genome in Japanese isolates reported previously. PCR-SSCP analysis of the acyclovir-resistant strains easily detected aberrantly shifted bands by comparing them with those of the parental strain, followed by the quick determination of mutated sequences. These results suggest that detection of nucleotide changes of the TK gene is useful for serial observation of persistent or recurrent HSV infection as observed in immunocompromised hosts, but that it is not useful for screening drug-resistant viruses from nonepidemic clinical isolates because of the comparable genetic polymorphisms in the TK gene as in the whole HSV-1 genome.

Peripheral ulcerative keratitis associated with erythema elevatum diutinum and a positive rheumatoid factor: a report of three cases.

Peripheral ulcerative keratitis (PUK) is a complication of collagen-vascular diseases such as rheumatoid arthritis (RA) and other systemic vasculitides. We report three cases of erythema elevatum diutinum with PUK. These patients presented with nodules and plaques consistent with erythema elevatum diutinum on the extremities and crusted or ulcerated purpuric lesions on the soles. Histopathological examination of these lesions revealed a dense neutrophilic infiltrate with nuclear dust and fibrin around blood vessels. All the patients developed PUK concomitant with the development of the skin lesions. The rheumatoid factor was positive at high titre in all three patients; this was associated with probable RA in one. Cutaneous lesions were dramatically improved by administration of dapsone in all cases. Dapsone was also effective in treating the ocular lesions in two patients.

Patterns of expression of the genes for glutamine synthetase isoforms during somatic and zygotic embryogenesis in carrot.

Three cDNA clones encoding isoforms of carrot glutamine synthetase (GS) were isolated and used as probes for analysis of the patterns of expression of the genes for GS isoforms during somatic embryogenesis and seed development in carrot. Transcripts corresponding to two of the cDNAs, CGS102 and CGS201, accumulated in both somatic embryos and developing seeds in the same manner. Their levels were high at the early stage of embryogenesis but decreased at the late stage. This pattern of expression is similar to the pattern of changes in GS activity observed during somatic embryogenesis. In contrast, expression of the transcript for another GS isoform detected with CGS103 cDNA was observed at the late stage of seed development and in senesced leaves but not in somatic embryos or young leaves. We also analyzed the levels of the transcripts in somatic embryos that had been cultured in media with either ammonium ions or glutamine as the nitrogen source. The amounts of the CGS102 and CGS201 transcripts fell when glutamine was supplied in the medium. These results indicated that GS activity was regulated at the transcriptional level and that the pattern of expression of the genes for GS during somatic embryogenesis reflected that during zygotic embryogenesis. It is possible that somatic embryogenesis and zygotic embryogenesis have common regulatory systems with respect to nitrogen metabolism.

A chromo box gene from carrot (Daucus carota l.): its cDNA structure and expression during somatic and zygotic embryogenesis.

A cDNA clone, designated DcDB1, was isolated from a cDNA library prepared from embryogenic cell clusters of carrot (Daucus carota L.) and characterized. The cDNA (1416 bp) encoded for a protein of 392 amino acid residues that contained a conserved chromo domain. The chromo domain is a 37 aa region found in both the Polycomo gene product, which is a repressor of homeotic genes, and a heterochromatin protein 1 of Drosophila. This domain is postulated to function in the binding of proteins to chromatin. Genomic blot hybridization experiments suggested that the number of DcCB1 genes in the carrot genome is low. The level of DcCB1 mRNAs was high in somatic embryos at globular and heart-shaped stages but low in torpedo-shaped somatic embryos. The level of DcCB1 transcripts decreased during the formation of seeds. The existence of both homeo and chromo box genes in plants suggests that regulatory mechanisms of developmental genes in plants may resemble those in Drosophila.

C-ABI3, the carrot homologue of the Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in the regulation of embryo-specific ABA-inducible genes.

A carrot gene homologous to the ABI3 gene of Arabidopsis was isolated from a carrot somatic embryo cDNA library and designated C-ABI3. The sequence of C-ABI3 was very similar to those of ABI3 of Arabidopsis and VP1 of maize in certain conserved regions. The expression of C-ABI3 was detected specifically in embryogenic cells, somatic embryos and developing seeds. Thus, expression of C-ABI3 was limited to tissues that acquired desiccation tolerance in response to endogenous or exogenous abscisic acid (ABA). Endogenous levels of ABA in seeds increased transiently and then desiccation of seeds started. The expression of C-ABI3 in developing seeds was observed prior to the increase in levels of endogenous ABA that was followed by desiccation of seeds. In transgenic mature leaves in which C-ABI3 was ectopically expressed, expression of ECP31, ECP63 and ECP40 was induced by treatment with ABA, which indicates that the expression of ECP genes was controlled by the pathway(s) that involved C-ABI3 and ABA. This suggests that C-ABI3 has the same function as VP1/ABI3 factor in carrot somatic embryos.

An extracellular insoluble inhibitor of cysteine proteinases in cell cultures and seeds of carrot.

An 18 kDa extracellular insoluble protein (EIP18) was found previously in amorphous particles suspended in the culture medium and in the interspaces of cell clusters of carrot (Daucus carota L.) callus, as well as in the extracellular spaces of carrot seeds, being located both in the embryo and at the inner edge of the endosperm. We purified EIP18 by washing the amorphous particles with the mixture of Triton X-100, NaCl and ethylenediaminetetraacetic acid (EDTA). We determined several partial amino acid sequences, and then we cloned and sequenced a cDNA for EIP18. EIP18 was found to consist of 133 amino acid residues that included a signal sequence, but it did not contain cysteine, sites for N-linked glycosylation or hydrophobic regions. Since its sequence was found to be homologous to that of inhibitors of cysteine proteinases, namely cystatins, EIP18 was renamed EICC (extracellular insoluble cystatin of carrot). EICC expressed in yeast was also found in an insoluble form in yeast cell walls. EICC prepared from the culture medium of carrot cells inhibited commercial cysteine proteinases and a proteinase extracted from germinating carrot seeds. The expression of the gene for EICC was detected in developing seeds, and the level of its transcript was markedly enhanced upon treatment of somatic embryos with abscisic acid.