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.

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.

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.

Clinical evaluation of acyclovir in the treatment of ulcerative herpetic keratitis.

For treatment of herpetic keratitis, 5-iodo-2'-deoxyuridine (IUdR) has been the only drug available in Japan. However, IUdR is not completely satisfactory, and alternative antivirals are required. The Association for Research in Infectious Diseases of the Eye, Japan, carried out a clinical trial in 92 cases of ulcerative herpetic keratitis using 3 percent acyclovir ophthalmic ointment to determine its efficacy and safety. In 63 eyes, it had an excellent effect and ulcers healed within seven days. In 26 eyes, it had a good effect and ulcers healed within 14 days or became half-size within seven days. Therefore, the response to acyclovir in 96.8 percent of cases was excellent or good. The average healing time in 81 eyes in which ulcers disappeared within 14 days was 5.8 +/- 3.4 days. Including the five eyes in which ulcers took more than 14 days to heal, the average healing time of ulcers was 6.6 +/- 4.8 days. Of the 92 patients treated with acyclovir ointment, one had contact blepharoconjunctivitis and another complained of mild irritation. Mild punctate keratitis, as another side effect of acyclovir ointment, was observed in 22 eyes (23.9 percent) by photo-slit lamp examination, but in these patients it was possible to continue treatment without complaint.

Efficacy of acycloguanosine against herpetic ulcers in rabbit cornea.

The effect of a new antiherpetic compound of very low toxicity called acycloguanosine (Wellcome 248U) on herpetic ulcers in rabbit cornea was studied by the Corneal Epithelial Lesion Therapeutic Assay (CELTA). The therapeutic effect of 3% acycloguanosine ointment on dendritic ulcers was equal to that of 0.5% 5-iodo-2'-deoxyuridine (IDU) ointment. No toxic symptoms could be detected by slit-lamp on 4 days' treatment with this concentration of acycloguanosine. Because of its selective action on virus only, its extremely low toxicity in animals, and its availability for systemic administration, acycloguanosine seems to be an ideal antiviral compound for use in the treatment not only of herpetic keratitis but also of other herpetic diseases in man.

Clinical evaluation of carbocyclic oxetanocin G eyedrops in the treatment of herpes simplex corneal ulcers.

BACKGROUND: Acyclovir (ACV) ophthalmic ointment is effective in the treatment of herpetic keratitis. However, when applied, the ointment has an unpleasant feeling and some cases are resistant to ACV. A new antiviral compound, carbocyclic oxetanocin G (C.OXT-G) has potent anti-herpes simplex virus activity and high water solubility, so the clinical effect of C.OXT-G eyedrops on ulcerative herpetic keratitis was evaluated. METHODS: Studies were conducted on the corneal ulcers in 37 eyes of 27 patients. Patients with typical dendritic or geographic corneal ulcers were treated with 0.1% C.OXT-G eyedrops, applied five times a day, together with eyedrops of an antibiotic applied four times a day. The eyes were examined at least twice a week until the ulcers healed, and thereafter at intervals for up to 3 months. RESULTS: All of the ulcers healed, their average healing time being 4.9 (SD 2.2) (range 2 to 9) days. The ulcers in 20 of the 37 eyes were induced by the use of corticosteroid or immunosuppressive drugs, and their average healing time was 4.8 (2.3) days. No adverse drug reactions were seen during the observation period in this trial. CONCLUSION: Eyedrops containing 0.1% C.OXT-G are excellent and safe for treatment of herpes simplex corneal ulcers in humans.

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.

Side effects in the treatment of herpetic keratitis.

Various side effects due to antiherpetic drugs observed in the last ten years in our department were studied. A total of 132 patients were treated with 5-iodo-2'-deoxyuridine (IDU), 69 with trifluorothymidine (F3T), 58 with acyclovir (ACV) and 33 with adenine arabinoside (ara-A). Patch tests were routinely done when patients exhibited contact dermatitis. Of the patients treated with IDU, 3 (2.3%) showed contact dermatitis, 2 (1.5%) follicular conjunctivitis and 1 (0.8%) punctate keratopathy. Of the patients treated with F3T, 7 (10.1%) exhibited contact dermatitis and 1 (1.4%) follicular conjunctivitis. In the group treated with ACV, 2 (3.4%) patients showed punctate keratopathy. The patients who received ara-A did not show any side effects. We found that F3T caused contact dermatitis more frequently in Japanese people than Europeans. These side effects were resolved by switching to another anti-herpetic drug without the occurrence of cross-allergy. Therefore, switching to another drug is strongly recommended when patients exhibit side effects in the treatment of herpetic keratitis. Other complications were allergy to atropine and to drug preservative.

Anti-herpes simplex virus (HSV) effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) in rabbit cornea.

The anti-herpes simplex virus (HSV) effect and cytotoxicity of a new nucleoside analogue, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) in rabbit cornea were studied. In tests of the anti-HSV effect of DHPG, even 0.03% ointment, given 5 times per day for 2 days, prevented lesion formation. The preventive effect of DHPG was much stronger than that of acyclovir (ACV) or 5-iodo-2'-deoxyuridine (IDU). In tests on the therapeutic effect of DHPG against dendritic ulcers, 0.3% ointment, given 5 times per day for 4 days, had a dramatic therapeutic effect. The effect was stronger than that of 3% ACV or 0.5% IDU ointment. Application of 0.3%, 1% or 3% DHPG ointment to normal rabbit corneas, 5 times per day for 2 weeks resulted in no histopathological abnormalities. The above results show that DHPG is superior to ACV or IDU for treatment of HSV infections.

Effects of 9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG) eye drops and cyclosporine eye drops in the treatment of herpetic stromal keratitis in rabbits.

The effect of 9-(1,3-dihydroxy-2-propoxymethyl) guanine (ganciclovir, DHPG) eye drops and the anti-inflammatory effect of cyclosporine (CYA) eye drops on experimental herpetic stromal keratitis in rabbits with herpes simplex virus (HSV) were studied. Ten rabbits were presensitized by subcutaneous injection of HSV, type 1, on days 14 and 7 before intrastromal inoculation of the same virus. These rabbits were divided into 2 groups and treatment was started 48 hours after virus inoculation. The eye drops used were 0.1% DHPG, 0.1% CYA or the vehicles of these eye drops. Eye drops were given 5 times a day and eyes were scored every day for 3 weeks after inoculation. The right eyes of Group 1 were treated with 0.1% DHPG and 0.1% CYA eye drops, and the left eyes with 0.1% DHPG and the vehicle of CYA eye drops. The right eyes of Group 2 were treated with 0.1% CYA and the vehicle of DHPG eye drops, and the left eyes with the vehicles of CYA and DHPG eye drops. The combination of DHPG and CYA was the most effective in decreasing the severity of herpetic stromal keratitis. Treatment with DHPG alone was also effective, but CYA alone was less effective.

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.

Effects on herpes simplex virus type 1 of combined use of interferon-beta and anti-herpetic agents in vitro.

It has been shown that the use of interferon (IFN) alone has little or no therapeutic effect in the treatment of herpetic keratitis. However, IFN used in combination with anti-herpetic agents has high therapeutic efficacy. This interaction between IFN and anti-herpetic agents has not been elucidated clearly. In this study, the interaction between human fibroblast interferon (HuIFN-beta) and anti-herpetic agents, 5-iodo-2'-deoxyuridine (IDU), aciclovir (ACV) and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), was studied in vitro using VERO cells. After the formation of a monolayer of VERO cells, HuIFN-beta of various concentrations was added to the maintenance medium. After 24 hours incubation, the medium was removed and herpes simplex virus (HSV) type 1 was inoculated on the VERO cells. Then, anti-herpetic agents were added to the medium in various concentrations. After 48 hours incubation, plaques were counted and the reduction rate in numbers of plaque was calculated. It was found that the combination of HuIFN-beta with IDU, ACV or DHPG worked synergistically in the inhibition of HSV replication.

Detection of herpes simplex virus DNA in tear fluid of stromal herpetic keratitis patients by nested polymerase chain reaction.

The value of the polymerase chain reaction (PCR) in the diagnosis of stromal herpetic keratitis was examined using tear fluid specimens; the sensitivity of the nested PCR method in detecting the herpes simplex virus (HSV) genome was 1 plaque-forming unit/mL. PCR assay of 72 tear samples from the eyes of 15 patients with stromal herpetic keratitis detected the HSV genome in 5 (33.3%). No positive band was detected in 20 tear samples from both eyes of 10 healthy volunteers. Seven of 38 tear samples (18.4%) and 1 of 34 samples (2.9%) collected from the diseased eye and the contralateral normal eye, respectively, produced positive results; the difference in rates was statistically significant (P < 0.05). The positive rate of samples collected from the diseased eye during an active phase was 16.0% (4 of 25 samples); in the quiescent phase, 23.1% (3 of 13 samples); the difference was not significant (P = 0.45). In HSV genome-positive cases, the average number of tear collections needed to detect the HSV genome was 3.3. Results indicate that PCR assay of tear fluid provides valuable information for the diagnosis of stromal herpetic keratitis, and that repeated tear samples should be collected regardless of the phase of the phase of disease activity.

Histopathological studies of a case of cytomegalovirus retinochoroiditis.

A case of cytomegalovirus (CMV) retinochoroiditis initially misdiagnosed as fungal endophthalmitis is reported. An 83-year-old man who was suspected of having cholangiocarcinoma presented uveitis in both eyes. Candida endophthalmitis was suspected on the basis of ophthalmic findings and past history, which included systemic corticosteroid administration and intravenous hyperalimentation. Intravenous treatment with miconazole was not effective. At autopsy, 3 months after the initial ophthalmological examination, the right eye was enucleated and examined histologically and histochemically. Light microscopic examination showed extensive retinal necrosis and numerous cytomegalic cells, so-called owl's eye cells, with intranuclear and intracytoplasmic inclusion bodies. CMV particles were seen by electron microscopy, and CMV-infected cells were observed by immunohistochemical staining by the direct method with fluorescein-labeled antibodies. These findings indicate that in suspected cases of fungal endophthalmitis various tests should also be carried out for CMV.

[Development of Wernicke's encephalopathy during the period of oral food intake after a subtotal colectomy for ulcerative colitis].

A 61-year-old woman was hospitalized because of ulcerative colitis which had caused fever, vomiting and diarrhea since June 16, 1992. Then she developed toxic megacolon, and was transferred to our hospital on the 1st of July and underwent subtotal colectomy the same day. After surgery, she received intravenous hyperalimentation (IVH) which contained 1,000 Kcal/day without vitamin supplementation. From the 8th to the 13th post-operative days, she took 3/4 or more of the liquid diet which contained 1,050 Kcal, protein 35 g, carbohydrate 166 g and vitamin B1 0.59 mg per day. From the 14th to the 23rd post-operative days, she ate 4/5 or more of the oral diet which contained 1,700 Kcal/day, protein 68 g, carbohydrate 236 g and vitamin B1 0.93 mg per day. During the 7th to the 23rd day, the IVH was reduced to 800 Kcal and then 500 Kcal per day. She talked less on the 19th post-operative day, and in a few days, her level of consciousness began to decline progressively. On the 27th post-operative day, neurological examinations revealed the following: semi-coma, almost fixed pupils which were 3 mm in diameter, absent doll's eye movement to all directions, flaccid extremities with abolished deep tendon reflexes. Pertinent abnormalities on laboratory data at that time consisted of hemoglobin 7.8 g/dl and serum total protein 5.4 g/dl. Lumbar puncture revealed normal cerebrospinal fluid under normal opening pressure. Cranial magnetic resonance imaging (MRI) taken on the 27th post-operative day demonstrated, on T2-weighted images, symmetrical high intensity lesions in the periventricular areas of the third and fourth ventricles, and periaqueductal area of the midbrain.(ABSTRACT TRUNCATED AT 250 WORDS)

[Sensitivities to other antiviral drugs and thymidine kinase activity of aciclovir-resistant herpes simplex virus type 1].

Sensitivity tests of various antiviral drugs on aciclovir-resistant strains of herpes simplex virus type 1 (HSV-1) were done in vitro. Activity of thymidine kinase (TK) of the aciclovir-resistant strains was also investigated. A strain of HSV-1 isolated from a patient with herpes labialis was grown in Eagle's minimum essential medium containing 10(-6)M aciclovir (ACV) and passaged 20 times. Then ACV-resistant strains of HSV-1 were obtained. Sensitivity tests of 5 anti-herpetic agents (ACV, ganciclovir, carbocyclic oxetanocin G, (S)-9- (3-hydroxy-2-phosphonylmethoxypropyl)adenine ((S)-HPMPA), and adenine arabinoside (ara-A)) were done on these ACV-resistant strains. The ACV-resistant strains were resistant to ganciclovir and carbocyclic oxetanocin G, but they were sensitive to (S)-HPMPA and ara-A. These ACV-resistant strains showed no detectable TK activity.

[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.

[A study of acute infection of herpes simplex virus in mouse trigeminal ganglia].

We investigated the shift to latency and protective reaction in mice trigeminal ganglia after inoculation of herpes simplex virus type 1 (HSV-1) onto the cornea. BALB/c mice were inoculated and the trigeminal ganglia were removed periodically. Lymphocytes in the ganglia were observed using immunocytochemical techniques. The results obtained were as follows: (1) HSV-1 positive neuronal cells were recognized at 3 days after inoculation but not at 14 days. (2) The relative proportion of T cells in lymphocytes was greater than that of B cells at 3 days, but B cells were more numerous at 5 days. Then T cells become more numerous again at 7 days. (3) Among the subsets in T cells, the ratio of CD4+ and CD8+ cells was almost equal at 3 days, but then CD4+ cells increased and CD8+ cells had disappeared at 14 days after inoculation. These results show that HSV-1 that reached the trigeminal ganglion from the cornea by axonal transport infected neuronal cells, multiplied there and then disappeared resulting in latency. Cellular immunity, especially the function of CD4+ cells, played a main role in this protective reaction by suppressing the viral growth.

[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.

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.