Rainbow trout interleukin-2: cloning, expression and bioactivity analysis.

In this study the rainbow trout (Oncorhynchus mykiss) interleukin-2 (IL-2) cDNA has been cloned, and its expression and bioactivity analysed in head kidney leucocytes. The IL-2 precursor encoded an open reading frame of 429 bp, that translates into a predicted protein of 142 aa, with a 20 aa signal peptide. The trout IL-2 had moderate protein homology (30.9% identity/48.3% similarity) with Fugu IL-2, the only IL-2 homologue identified in fish to date, with lower homology to avian (17.8% identity/23.2% similarity) and mammalian (34.2 identity/46.5% similarity) IL-2s. IL-2 expression was induced by the T cell mitogen PHA and by the mixed leucocyte reaction, where leucocytes from pairs of fish were cultured together for four days. Expression was also induced in vivo during bacterial (Yersinia ruckeri) infection. The Escherichia coli produced recombinant IL-2 was shown to increase the expression of two transcription factors, STAT5 and Blimp-1, known to be involved in IL-2 signalling in mammals, as well as IFN-gamma, gIP and IL-2 itself. The potential signalling pathways involved and possible use as an adjuvant for fish vaccines are discussed.

Induction of mononuclear cell infiltration into liver by Japanese herbal medicine.

Juzen-Taiho-To (JTT) is a Japanese herbal medicine that has been administered mainly to patients weakened by long illness. Currently, it has also been used for cancer patients and showed antitumor effects that have been reported as phagocytosis enhancement, cytokine induction and antibody production. In this study, we examined the effect of oral administration of JTT in mice on the immunological restoration of the liver, especially focused on natural killer (NK) T-cell induction. Mice were grouped to receive JTT or placebo orally for a period of 1, 3 and 7 days. After sacrifice, the liver tissue was fixed, embedded and stained with hematoxylineosin and some antibodies by common staining methods. Transmission electron microscope (TEM) observation was also carried out. Although the JTT-treated mice had the same appearance as the non-JTT-treated mice, their livers were infiltrated by massive mononuclear cells, some of which were aggregated in clusters. Immunohistochemical staining revealed that there was abundant cytokine expression of interleukin (IL)-12 and massive infiltration of mononuclear cells with large granules in the liver of JTT-treated mice. Oral administration of JTT may induce the expression of IL-12 and be followed by immunological restoration such as NK T-cell induction in liver

Methotrexate-induced acute lung injury in a patient with rheumatoid arthritis.

Methotrexate (MTX)-induced acute lung injury developed in a female patient with rheumatoid arthritis. She was successfully treated with high-dose glucocorticoid therapy. During her hospital stay, the serum concentration of surfactant protein (SP)-D, which was markedly elevated on admission, was finally normalized and the disease resolved. However, the serum concentration of Klebs von den Lungen (KL)-6 remained high. Although the mechanisms of lung injury by MTX have not been well defined, serial measurements of serum SPD might be useful for the clinical evaluation of drug-induced acute lung injury.

Organization of the NKEF gene and its expression in the common carp (Cyprinus carpio).

Natural killer enhancing factor (NKEF) is a member of the newly defined peroxiredoxin (Prx) family. Its functions are to enhance the cytotoxic capacity of natural killer cells and to prevent DNA and protein from being damaged by oxidative stress in the presence of thiol compounds. However, little is known about the structure and function of NKEF in lower vertebrates. We have recently cloned a cDNA encoding NKEF from the common carp (Cyprinus carpio) by use of suppression subtractive hybridization (SSH). In the present study, we used PCR to obtain a genomic DNA which covers the entire coding region of carp NKEF. In the 3363bp-long genomic sequence, six exons and five introns were identified. The carp NKEF gene has splice donor/acceptor site sequences at the boundaries of exons and introns, and contains two Val-Cys-Pro (VCP) motifs. The exon/intron organization of the carp NKEF gene shows complete conservation with other members of the Prx family. Genomic Southern blotting analyses suggest that carp has multiple copies of the NKEF gene. RT-PCR analyses reveal that carp NKEF has very different expression levels not only in tissues but also from individuals.

Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization.

To develop tools for analysis of the acute phase response, we used suppression subtractive hybridization of cDNAs from the livers of trout in an unchallenged state and in the course of a response to injection with a Vibrio bacterin emulsified in Freund's Incomplete Adjuvant. The resulting cDNA library contains 300-600bp long fragments of 25 or more immune-relevant genes. Fifteen were previously unreported for salmonids, and 12 were not known from any fish species. Known acute phase proteins include serum amyloid A, transferrin and precerebellin-like protein; trout C-polysaccharide-binding protein 1 is probably also an acute phase protein. Components of both the complement system (n=5) and the clotting system (n=3), as well as lectins, various binding proteins, a putative antibacterial peptide, a chemotaxin, an anti-oxidant enzyme, as well as some likely cell-surface receptors and metabolic and lysosomal enzymes are represented in the library. One clone closely resembles a group of Toll-like receptors, including the human IL-1 receptor. Three cDNAs appear to represent complete open reading frames.

Prospective evaluation of a new anti-ulcer agent, ecabet sodium, for the treatment of Helicobacter pylori infection.

BACKGROUND: A new anti-ulcer agent, ecabet sodium, is active against Helicobacter pylori. AIM: To assess the efficacy of ecabet sodium for the eradication of H. pylori in patients with gastroduodenal diseases. METHODS: In a prospective, randomized and controlled study, patients infected with H. pylori were assigned to one of the following two groups: group LA, who received lansoprazole 30 mg o.d. + amoxycillin 500 mg q.d.s. after meals for 2 weeks, and group LAE, who received lansoprazole 30 mg o.d. + amoxycillin 500 mg q.d.s. + ecabet sodium 1000 mg b.d. after meals for 2 weeks. H. pylori status was determined before and at least 4 weeks after the therapy by rapid urease test, histology and a urea breath test. RESULTS: Of 101 patients (mean age 53 years, range 17-77 years, M/F: 68/33) enrolled in the study, 97 patients completed the protocol. Four patients were withdrawn because of diarrhoea (three from group LA) and skin rash (one from group LAE). The eradication of H. pylori was achieved in 28/48 (58%) patients in group LA and 38/49 (78%) patients in group LAE. The rate of eradication of H. pylori produced by the LAE treatment was significantly higher than that produced by the LA treatment. Side-effects appeared in two patients (malaise 1, skin rash 1) in group LAE and in seven patients (diarrhoea 6, dizziness 1) in group LA. These side effects disappeared spontaneously with cessation of the treatment. CONCLUSIONS: Ecabet sodium in combination with lansoprazole and amoxycillin increased the rate of eradication of H. pylori. Ecabet sodium appeared to reduce the incidence of diarrhoea as a side-effect of the dual LA therapy.

The terminal bases of ribonucleic acid from Caulobacter RNA phage phiCp2.

The terminal bases of the viral ribonucleic acid from the Caulobacter ribonucleic acid phage phiCp2 were examined. The viral ribonucleic acid contained guanosine triphosphate (pppG) at the 5'-terminus and adenosine (AOH) at the 3'-terminus.

Amino acid composition of peptidoglycan in Caulobacter crescentus.

Peptidoglycan of a gram-negative stalked bacterium, Caulobacter crescentus CB13, contained alanine, diaminopimelic acid, and glutamic acid, in molar ratios of 2 : 1 : 1. The amino acid compositions of peptidoglycans isolated from cultures enriched in swarmer and stalked cells, and from a stalk-less mutant were similar. This finding conflicts with a previous observation that swarmer peptidoglycan does not contain diaminopimelic acid (Goodwin and Shedlarski (1975) Arch. Biochem. Biophys. 170, 23-36). It appears that, despite the morphological differences, the Caulobacter cells all contain a similar peptidoglycan in the cell wall.

Freeze-fracture and immunohistochemical studies of gap junctions in human gastric mucosa with special reference to their relationship to gastric ulcer and gastric carcinoma.

Our aim was to determine whether the development of gap junctions in the human gastric mucosa has any relation to gastric ulcer and gastric carcinoma. Freeze-fracture replicas were prepared from the endoscopic biopsy specimens of 20 patients with gastric ulcer and 7 healthy volunteers. Large fractured areas of lateral cell membranes of surface mucous cells were examined randomly under an electron microscope. Small gap junctions were observed between gastric surface mucous cells in all healthy volunteers. Gap junctions in the patients with gastric ulcer were significantly fewer than in the healthy volunteers. In addition, gap junctions in patients with recurrent ulcer were significantly fewer than in those with first-onset ulcer. There was no obvious relationship between age and the development of gap junctions in patients with gastric ulcer or in healthy volunteers. In the areas of intestinal metaplasia, gap junctions were occasionally seen between absorptive cells of the villi, but not in the lateral membranes of goblet cells. Fresh frozen sections for indirect immunofluorescence were prepared from the endoscopic biopsy specimens of 19 patients with gastric ulcer and 5 patients with gastric cancer. Monoclonal antibody against liver gap junction protein (anti-connexin 32, 6-3G11) was used for the indirect immunofluorescence. On the border of gastric ulcer, fluorescent spots in the surface mucous cells were significantly fewer than in the surface mucous cells of the body and antrum which were distant from the ulcer area in the same patients. In gastric cancer tissue specimens, fluorescent spots were not observed at all.(ABSTRACT TRUNCATED AT 250 WORDS)

Birth order and parental age in microphthalmos and other ocular diseases.

We compared the distribution of birth order and maternal and paternal ages of blind school children throughout Japan with that of the total Japanese population of the corresponding age groups and with that of a subgroup of children with acquired blindness. The number of first-born children with microphthalmos was smaller, and the number of second-, third-, or fourth-born children was larger, as compared with the control groups. The differences were highly statistically significant by chi-square test. There was a less pronounced indication of birth order effect in amblyopia, congenital cataract, and optic nerve atrophy, which involved more first-borns than in the controls. The distribution of maternal age was also different from the control group in microphthalmos, congenital cataract, corneal opacity, and optic nerve atrophy. Less mothers in their 20s and more in their 30s produced children with these conditions. We believe this finding may be partly related to the rapid decline in infant mortality and in the incidence of congenital blindness in Japan.

Q118X mutation of M1S1 gene caused gelatinous drop-like corneal dystrophy: the P501T of BIGH3 gene found in a family with gelatinous drop-like corneal dystrophy.

PURPOSE: To analyze BIGH3 and M1S1 genes in two Japanese brothers with gelatinous drop-like corneal dystrophy and five unaffected family members. METHODS: DNA was extracted, and each part of the two genes was amplified and directly sequenced. RESULTS: On the BIGH3 gene, a heterozygous P501T mutation was found in the elder brother and three unaffected family members. On the M1S1 gene, both brothers with gelatinous drop-like corneal dystrophy showed a homozygous Q118X mutation, whereas all unaffected members were heterozygous. CONCLUSIONS: The Q118X mutation of M1S1 gene caused gelatinous drop-like corneal dystrophy. Although the P501T of the BIGH3 gene found in this pedigree was precisely the one reported for lattice corneal dystrophy IIIA, no clinical feature was shown, even in the 85-year-old father. This fact shows that the P501T mutation for LCDIIIA has low penetrance.

Clinical features of autosomal dominant retinitis pigmentosa with rhodopsin gene codon 17 mutation and retinal neovascularization in a Japanese patient.

A 49-year-old Japanese man had autosomal dominant retinitis pigmentosa with a point mutation in codon 17 of the rhodopsin gene, resulting in a threonine-to-methionine change, and retinal neovascularization in both eyes. Pigmentary degeneration mainly in the inferior area of the fundus, and severe loss in the upper portion of the visual field were observed. Moderately preserved rod and cone functions were demonstrated by electroretinograms. These findings differed from those of Japanese and white patients with autosomal dominant retinitis pigmentosa with a codon 347 mutation and were almost the same as those of white patients with the codon 17 mutation. Our study indicates that phenotypic similarities exist among patients with the same mutation, but of different racial backgrounds. The neovascularization in the right eye diminished over a two-year period in conjunction with the progression of retinal degeneration.

Leu518Pro mutation of the beta ig-h3 gene causes lattice corneal dystrophy type I.

PURPOSE: To describe a Japanese family with lattice corneal dystrophy type I, which segregates with a novel mutation, Leu518Pro of the beta ig-h3 gene. METHODS: DNA was extracted from leukocytes in four members (three affected and one unaffected) of a Japanese family with lattice corneal dystrophy type I. Exon 12 of the beta ig-h3 gene was amplified and analyzed with a molecular biologic method. Clinical data were also collected. RESULTS: Three generations of this family have been positively diagnosed with lattice corneal dystrophy, indicating autosomal dominant inheritance. We found a heterozygous point mutation that segregates with the disease phenotype. It was a single base-pair transition (CTG to CCG, Leu to Pro). CONCLUSION: Although it is extremely rare compared with the Arg124Cys mutation of the beta ig-h3 gene, Leu518Pro mutation of the beta ig-h3 also causes lattice corneal dystrophy type I.

Secondary mutations of mitochondrial DNA in Japanese patients with Leber's hereditary optic neuropathy.

PURPOSE: Based on studies on the pathogenesis of Leber's hereditary optic neuropathy (LHON), mitochondrial DNA (mtDNA) mutations have been divided into two types: primary and secondary. Primary mutations at nucleotide positions (nt) 11778, 3460, and 14484 can each cause LHON. Secondary mutations may be simultaneously found in LHON patients with a primary mutation, may occur at higher frequency in LHON patients than in normal controls, and may play an additional role in the pathogenesis of LHON. We examined the frequencies of secondary mutations of mtDNA at nt3394, 7444, 9438, 9804, 13708, and 15257 in 19 Japanese patients with LHON associated with primary mutations and 108 normal controls. METHODS: Mutations were determined by restriction enzyme analysis or DNA sequencing using polymerase chain reaction (PCR) products. RESULTS: One patient with an nt11778 mutation also had an nt13708 mutation. Another patient with an nt3460 mutation also had an nt7444 mutation. During DNA sequencing of the PCR fragment harboring nt3394, three novel mutations in the ND1 gene (nt3316, 3496, and 3497 mutations) were found in three patients with an nt11778 mutation. The frequency of these mutations in 108 control subjects was studied further: one (0.9%) had an nt3394 mutation, none (0%) had an nt9804 mutation, one (0.9%) had an nt13708 mutation, two (1.9%) had nt3316 mutations, one (0.9%) had an nt3496 mutation, and two (1.9%) had nt3497 mutations. CONCLUSION: It is unlikely that the frequencies of secondary mutations in Japanese patients with LHON are higher than those in normal Japanese controls. It is possible that the mutations at nt3316, 3496, and 3497 are secondary mutations of LHON.

Analysis of phosducin as a candidate gene for retinopathies.

Phosducin, a retina-expressed gene mapped to chromosome 1q25-32.1, was analyzed as a candidate gene for retinopathies. The phosducin gene was cloned and characterized, and PCR primers were designed. Eighty-three patients with various retinopathies and 45 control subjects (24 American, 21 Japanese) were analyzed for mutations in the phosducin gene by PCR, denaturing gradient gel electrophoresis (DGGE), and sequencing. A heterozygous sequence variant changing a glycine to arginine at codon 178 was found in one Usher syndrome type II (USH2) patient, while the other USH2 patients did not show any coding sequence variant. A heterozygous sequence variant changing an asparagine to lysine at codon 174 was found in a patient with a severe retinal degeneration in the category of diseases known as acute zonal occult outer retinopathy (AZOOR). Three non-coding sequence variants were found. Two of these were always present together and found in 20.8% of American and 2.4% of Japanese control subjects, reflecting a difference in population pools. In conclusion, the phosducin gene did not show mutations consistent with it being the causative gene for USH2, but its possible pathogenicity in AZOOR or other retinopathies remains an open question which may be answered by further analysis.

Visual impairment and REP-1 gene mutations in Japanese choroideremia patients.

Choroideremia (CHM), an X-linked recessive hereditary disease, is an intractable chorioretinal dystrophy. The rate of disease progression of CHM reportedly shows considerable variability. A number of mutations involving the gene that codes for Rab escort protein-1 (REP-1) have been detected in CHM patients. We have analyzed REP-1 gene mutations of Japanese CHM patients. The present study was designed to investigate the clinical variability and the genotype to phenotype relationship in 15 Japanese CHM patients referred to the Department of Ophthalmology of Juntendo University Hospital. The clinical investigation of visual acuity, visual field, color vision and refraction revealed inter-individual variability. Mutation analyses of the REP-1 gene revealed 10 types of mutations in 13 patients from 11 families, including an insertion, small deletions, nonsense mutations and an A to CC mutation. In 13 CHM patients with detectable REP-1 gene mutations, no relationship of genotype to phenotype was detected. At present, we consider the REP-1 genotype to be an unreliable prognostic factor for counseling of CHM patients. In two patients from one family, no mutations were detected in coding regions of the REP-1 gene. These patients may have intron mutations of the REP-1 gene, not detectable by the techniques employed in this study, or other causative genes. Both were observed to have somewhat slower disease progression than the other 13 patients. More advanced analyses are necessary to answer questions regarding the genotype-phenotype relationship in CHM patients.

Corneal amyloidosis caused by Leu518Pro mutation of betaig-h3 gene.

AIM: To report a Japanese family diagnosed clinically as having lattice corneal dystrophy type I (LCDI) in which a Leu518Pro mutation in the betaig-h3 gene and not the R124C mutation reported previously was found. METHODS: Molecular genetic analysis was performed on DNA extracted from peripheral leucocytes from four members (three affected and one unaffected) of a family. Exon 4 of the betaig-h3 gene was amplified by PCR and directly sequenced. Histopathological study was performed on the corneal tissue from the proband obtained during deep lamellar keratoplasty. RESULTS: All the affected members were clinically diagnosed as having LCDI, and the pedigree indicated an autosomal dominant inheritance. A heterozygous single base pair transition (CTG to CCG, leucine to proline) was detected in codon 518 of the betaig-h3 gene in the three affected members, and not in the unaffected member. No mutation was found in codon 124. Amyloid deposits were observed between the collagen bundles of the corneal stroma and were seen to extend deep into the stroma. CONCLUSION: The Leu518Pro mutated betaig-h3 forms amyloidogeneic intermediates which precipitate in the cornea and gives rise to a clinical appearance of LCDI.

H626R and R124C mutations of the TGFBI (BIGH3) gene caused lattice corneal dystrophy in Vietnamese people.

BACKGROUND/AIMS: Mutations of the human transforming growth factor beta induced gene (TGFBI) were reported to cause lattice corneal dystrophy (LCD) in various nationalities. This study analysed the TGFBI gene in Vietnamese people with LCD. METHODS: 13 unrelated families, including 34 patients and 21 unaffected members were examined. 50 normal Vietnamese people served as controls. Blood samples were collected. Genomic DNA was extracted from leucocytes. Analysis of TGFBI gene was performed using the polymerase chain reaction and direct sequencing. Corneal buttons were studied histopathologically. RESULTS: Two clinically distinguishable forms of LCD were revealed: one was typical of LCDI; the other was characterised by the late onset, thick lattice lines, and asymmetry between two eyes. Sequencing of the TGFBI gene revealed R124C mutation in three families and H626R mutation in 10 families. Congo red staining of the H626R-LCD cornea showed amyloid deposits in the subepithelial and stromal layers. CONCLUSIONS: R124C and H626R mutations of TGFBI gene caused LCD in Vietnamese people. R124C, a common cause of LCDI in many nationalities, was relatively rare, whereas H626R reported in several white people but not yet in Asians was most common (>75%) in Vietnamese people. Since the phenotype caused by H626R represents a new variant intermediate between LCDI and LCDIIIA, we proposed to consider it as LCD type IIIB.

Homozygotic patient with betaig-h3 gene mutation in granular dystrophy.

PURPOSE: This study investigated patients with granular dystrophy and identified a homozygotic patient and his family with a mutation in the betaig-h3 gene. METHODS: Genomic DNAs were extracted from leukocytes of the peripheral blood of the proband, his parents, and his grandmother. All had granular dystrophy. Genomic DNAs from 50 unrelated normal volunteers were used as controls. Exon 4 of betaig-h3 gene was amplified and analyzed by direct sequence. Clinical data were collected. RESULTS: A single-base-pair transition was detected. This was a substitution of G to A of the second nucleotide position of codon 124 in the betaig-h3 gene that led to a replacement of histidine for arginine (Arg124His, CGC-->CAC). This mutation was the precise one previously reported for Avellino dystrophy. Although the proband was homozygotic for the mutant alleles, his grandmother, and parents were heterozygotic for these alleles. No sequence modification in the codon 124 from 50 nonaffected control individuals was detected. Clinical findings of the proband were severe. Keratectomies were performed for both his eyes 5 times for a 24-year period. His grandmother and parents showed mild clinical symptoms, had a few annular granules in the subepithelial stroma, and maintained good visual acuities. CONCLUSION: Arg124His mutation of the betaig-h3 gene was found in a pedigree with granular dystrophy. This mutation was the precise one previously reported for Avellino dystrophy. This fact shows an existence of Avellino form in Japanese. Homozygotic patient for mutant gene showed severe symptoms and an early onset.

Large-scale sequencing of the rabbit corneal endothelial cDNA library.

PURPOSE: This study sought to identify novel or active genes in corneal endothelial cells with description of the gene-expression profile. METHODS: We performed the single-pass sequencing of 1,000 clones from a rabbit corneal endothelial cDNA library. Inserts of the library were amplified by polymerase chain reaction (PCR), sequenced, and compared with several databases. We used four database similarity search programs: FASTA, BLASTN, TBLASTX, and BLASTX. RESULTS: Of the sequences generated, 618 (61.8%) showed sequence homology with known genes, whereas 192 (19.2%) matched previous reported expression sequence tags (ESTs) and 174 (17.4%) showed no sequence similarity. Among the homologous clones to known genes are collagen type VIII, secreted protein acidic and rich in cysteine (SPARC), lysyl oxidase, phosphatidylcholine-2-acylhydrolase, and thrombospondin. Several matched ESTs, and no matched clones that showed high frequency were also detected. CONCLUSION: Large-scale sequencing can be useful in obtaining a profile of the active genes. Several ESTs showed relatively frequent expression, suggesting that these genes may have important functions in the corneal endothelium.