Sugar-sweetened beverage and diet soda consumption and the 7-year risk for type 2 diabetes mellitus in middle-aged Japanese men.

PURPOSE: This cohort study investigated the association between sugar-sweetened beverage (SSB) and diet soda consumption and the incidence of type 2 diabetes in Japanese men. METHODS: The participants were 2,037 employees of a factory in Japan. We measured consumption of SSB and diet soda using a self-administered diet history questionnaire. The incidence of diabetes was determined in annual medical examinations over a 7-year period. Hazard ratios (HRs) with 95 % confidence intervals (CIs) for diabetes were estimated after adjusting for age, body mass index, family history, and dietary and other lifestyle factors. RESULTS: During the study, 170 participants developed diabetes. The crude incidence rates (/1,000 person-years) across participants who were rare/never SSB consumers, <1 serving/week, ≥ 1 serving/week and <1 serving/day, and ≥ 1 serving/day were 15.5, 12.7, 14.9, and 17.4, respectively. The multivariate-adjusted HR compared to rare/never SSB consumers was 1.35 (95 % CI 0.80-2.27) for participants who consumed ≥ 1 serving/day SSB. Diet soda consumption was significantly associated with the incident risk of diabetes (P for trend = 0.013), and multivariate-adjusted HRs compared to rare/never diet soda consumers were 1.05 (0.62-1.78) and 1.70 (1.13-2.55), respectively, for participants who consumed <1 serving/week and ≥ 1 serving/week. CONCLUSIONS: Consumption of diet soda was significantly associated with an increased risk for diabetes in Japanese men. Diet soda is not always effective at preventing type 2 diabetes even though it is a zero-calorie drink.

Serum gamma-glutamyltransferase and the risk of hyperuricemia: a 6-year prospective study in Japanese men.

We conducted a longitudinal study to investigate whether increased serum gamma-glutamyltransferase independently predicts subsequent development of hyperuricemia. The study participants included 3,310 Japanese men without hyperuricemia, aged 20-54 years. The participants had annual heath examinations for 6 years to assess incident hyperuricemia (defined as serum uric acid>416.4 µmol/l and/or taking medication for hyperuricemia). The risk of incident hyperuricemia was compared in participants grouped according to their baseline serum gamma-glutamyltransferase level. During follow-up, there were 529 incident cases of hyperuricemia. A positive, dose-response relationship was observed between serum gamma-glutamyltransferase and the risk of incident hyperuricemia. The hazard ratios (95% confidence intervals) for hyperuricemia, compared with a serum gamma-glutamyltransferase level ≤19 U/l, were 1.32 (1.05-1.67) for 20-39 U/l, 1.28 (0.90-1.83) for 40-59 U/l, 1.56 (0.98-2.47) for 60-79 U/l, and 1.57 (1.02-2.41) for ≥80 U/l after adjustment for baseline serum uric acid, creatinine, total cholesterol, and glycated hemoglobin levels, ln(serum alanine aminotransferase), age, systolic blood pressure, medications for hypertension, hypercholesterolemia, and diabetes, body mass index, and smoking and exercise habits. A similar positive relationship was observed regardless of the presence or absence of alcohol drinking, obesity, metabolic disorders (any combination of hypertension, hypercholesterolemia and/or diabetes), or clinically high serum aminotransferases, without evidence of a significant interaction between increased serum gamma-glutamyltransferase and risk factors for incident hyperuricemia. These findings indicate that increased serum gamma-glutamyltransferase is an independent predictor of subsequent development of hyperuricemia.

Alcohol intake and the risk of hyperuricaemia: a 6-year prospective study in Japanese men.

BACKGROUND AND AIMS: Since there is little information derived from prospective studies on the amount of alcohol drinking required to induce hyperuricaemia, we attempted to address this issue in a Japanese population. METHODS AND RESULTS: A total of 3310 Japanese men aged 20-54 years that were free of hyperuricaemia were classified according to their alcohol intake per week at baseline. Incident hyperuricaemia, defined as >7.0 mg/dl and/or taking medication for hyperuricaemia, was assessed through annual heath examinations for 6 years after the baseline examination. During follow-up, 529 incident cases of hyperuricaemia occurred. There was a positive, dose-response relationship between alcohol intake and the risk of incident hyperuricaemia. The hazard ratio (95% confidence interval) for hyperuricaemia in drinkers compared with non-drinkers was 1.10 (0.85-1.42) for <10.0 drinks/week, 1.40 (1.07-1.84) for 10.0-19.9 drinks/week, 1.64 (1.23-2.21) for 20.0-29.9 drinks/week and 1.98 (1.40-2.80) for ≥30.0 drinks/week (one drink contained 11.5 g of ethanol) after adjusting for age, baseline serum uric acid, body mass index, smoking habits, exercise habits, serum creatinine, blood pressure, serum cholesterol and blood glucose. The fraction of hyperuricaemia in the population attributable to alcohol intake was 21.6%. A clear dose-response pattern was observed for both beer and sake, when the consumption of these two beverages was analysed separately. CONCLUSION: Habitual alcohol intake significantly contributed to the development of hyperuricaemia in Japanese men, regardless of type of alcoholic beverage consumed. Therefore, it is essential to reduce excessive alcohol intake to prevent and manage hyperuricaemia.

Homeostasis model assessment of insulin resistance and the risk of cardiovascular events in middle-aged non-diabetic Japanese men.

AIMS/HYPOTHESIS: Little is known about the relationship between the HOMA of insulin resistance (HOMA-IR) and the risk of cardiovascular events in Asian populations, which have lower levels of HOMA-IR than Western populations. Accordingly, we determined the predictive value of HOMA-IR for cardiovascular risk in a Japanese population that was apparently free of diabetes, addressing whether insulin resistance itself increases cardiovascular risk independently of other relevant metabolic disorders. METHODS: We followed 2,548 non-diabetic men aged 35 to 59 years for 11 years. The hazard ratios for the incidence of cardiovascular events due to increased HOMA-IR were estimated using a Cox proportional hazards model that was adjusted for potential confounding factors. RESULTS: The multivariate-adjusted hazard ratio for cardiovascular events compared with the first quartile of HOMA-IR (or=1.52). The hazard ratio associated with a one SD (0.61) increment in log-transformed HOMA-IR was 1.51 (1.13-2.02). A similar positive relationship was observed for coronary events and stroke. In addition, the relationship between HOMA-IR and cardiovascular risk was broadly similar in participants with and without hypertension, dyslipidaemia (elevated triacylglycerol and/or reduced HDL-cholesterol), abdominal obesity and current smoking. CONCLUSIONS/INTERPRETATION: Increased HOMA-IR predicted subsequent cardiovascular events in non-diabetic Japanese men. The association was independent of traditional cardiovascular risk factors and other relevant metabolic disorders.

J-shaped relationship between waist circumference and subsequent risk for Type 2 diabetes: an 8-year follow-up of relatively lean Japanese individuals.

AIMS: This study investigated the relationship between waist circumference and the subsequent incidence of Type 2 diabetes and the association with insulin resistance and pancreatic B-cell function in relatively lean Japanese individuals. METHODS: The study participants were 3992 employees (2533 men and 1459 women, aged 35-55 years) of a metal-products factory in Japan. The incidence of diabetes was determined in annual medical examinations during an 8-year follow-up. We calculated age- and sex-adjusted hazard ratios (HRs) according to the sex-specific quintile of waist circumference at baseline. Differences in baseline insulin resistance [homeostatis model assessment (HOMA)-IR] and pancreatic B-cell function (HOMA-B) were compared between participants who developed diabetes and those who did not. RESULTS: During the follow-up, 218 participants developed diabetes. Age- and sex-adjusted HRs across the quintiles of waist circumference were 1.78, 1.00 (reference), 1.59, 3.11 and 3.30, respectively (P for trend, < 0.0001). The HR for the lowest quintile was significantly higher than that for the second quintile. Among participants with waist circumference of the lowest quintile, HOMA-B was lower in those who developed diabetes than in those who did not [33.1 (24.1-45.0) vs. 54.3 (37.9-74.6) median (interquartile range), P < 0.0001], but HOMA-IR did not differ between these groups. CONCLUSIONS: There was a J-shaped relationship between waist circumference and subsequent risk for Type 2 diabetes in relatively lean Japanese individuals; lower pancreatic B-cell function may also increase the risk of diabetes in very lean Japanese people. Diabet. Med. 26, 753-759 (2009).

Correlation of autophagy type in podocytes with histopathological diagnosis of IgA nephropathy.

OBJECTIVE: IgA nephropathy (IgA-N) frequently leads to progressive renal failure, thus estimation of the degree of progression is important for patient management. Autophagy is a mechanism that facilitates clearance of waste products to preserve renal function. The aim of this study was to assess autophagy in podocytes in children with progressive IgA-N at initial diagnosis by electron microscopy and investigate the relationship between the types of autophagy and severity of the disease. METHODS: Renal biopsies from 16 children with established progressive IgA-N were examined by light and transmission electron microscopy with reference to autophagy types in the podocytes and histopathological diagnosis of IgA-N. RESULTS: Two autophagy types were found. Type I rarely transformed to autophagic vacuoles and did not dissolve, thus possibly impairing cell function. However, type II frequently transformed to autophagosomes and autophagic vacuoles thus facilitating protein and lipid clearance. Of the 16 children studied, 8 (50%) with type I autophagy at initial diagnosis showed focal proliferative glomerulosclerosis (GN) of mild type (3 cases, 37.5%), mild/moderate type (2 cases, 25%) and moderate type (3 cases, 37.5%). In contrast, the remaining 8 children with type II autophagy at initial diagnosis showed focal proliferative GN of mild type in 7 (87.5%) and mild/moderate type in 1 (12.5%) case. CONCLUSION: In IgA-N children, the occurrence of type I autophagy is correlated with histopathologically more progressive disease, possibly reflecting a tendency to a poorer prognosis.

Combination of the C1429T polymorphism in the G-protein beta-3 subunit gene and the A1330V polymorphism in the low-density lipoprotein receptor-related protein 5 gene is a risk factor for hypercholesterolaemia.

The present study examined the relationship between genetic combinations of the C1429T polymorphism in the G-protein beta-3 subunit (GNB3) gene and the A1330V polymorphism in the low-density lipoprotein receptor-related protein 5 (LRP5) gene and the risk of hypercholesterolaemia in Japanese workers. The present study included observations from 1997 to 2002 in 927 males and 662 females who were not hypercholesterolaemic on entry. The endpoint was the development of hypercholesterolaemia, defined as a total cholesterol level > or =240 mg/dl. The odds ratios for the combination of polymorphisms were calculated using pooled logistic regression analyses that incorporated other potential factors into the model. The odds ratios in males and females with GNB3/1429TT and LRP5/1330VV or AV genotypes were 4.17 compared to males with the 1429CT or TT and 1330AA genotypes and 3.53 compared to females with the 1429CC, CT or TT and 1330AA genotypes. Assuming these effects were a mere addition of two independent effects, the odds ratios for both GNB3/1429TT and LRP5/1330VV or AV were estimated to be 3.27 for males and 1.42 for females. Therefore, the synergic effects were shown to be 1.28 times in males (not significant) and 2.49 times in females (P<0.05 by bootstrap method). These results provide clear evidence that the genetic combination has a synergic effect. This study indicates that the combination of GNB3/C1429T and LRP5/A1330V is a very useful marker for predicting the development of hypercholesterolaemia in the general Japanese population.

Locomotion ability variation among instars of the bean bug, Riptortus pedestris (Fabricius) (Heteroptera: Alydidae) nymphs.

The nymphal locomotion ability (walking distance) of the stenophagous bean bug Riptortus pedestris (Fabricius) was studied in each instar. We measured the walking distance using two systems. The walking distance in photophase was measured for 6 h using a tracking system with a charge coupled device (CCD) camera and computer software. The daily activity of nymphs was measured by an actograph system counting the number of infrared beam intercepts. The actograph data were converted to distance using a linear regression against the data of the tracking system. The longevity of nymphs without food was also studied to estimate the potential walking distance. Using both the tracking and actograph systems, it was determined that first instars walked less than the other instars (only 10.7 m within 6 h). The second to fifth instars could move 20-25 m within 6 h, and this distance did not differ among instar. This indicates that first instars seldom move after hatching in the field. The walking distance for 24 h varied and was greatest for the third instars (80.8 m). The potential longevity of nymphs was found to increase with instar age. Potential locomotion ability (walking distance for 24 hxpotential longevity) was high in the third to fifth instars (approximately 340 m). The potential locomotion ability for the second instars was relatively low compared with the elder instars (approximately 180 m). From these results, nymphs of R. pedestris seem to adapt by identifying feeding site locations after hatching and elder instars may be able to find a novel feeding site after the degradation of previous habitat.

Skipping breakfast and 5-year changes in body mass index and waist circumference in Japanese men and women.

OBJECTIVE: This study investigated the relationship between frequency of skipping breakfast and annual changes in body mass index (BMI) and waist circumference (WC). METHODS: The participants were 4,430 factory employees. BMI and WC were measured repeatedly at annual medical examinations over a 5-year period. The association between frequency of skipping breakfast at the baseline examination and annual changes in anthropometric indices was evaluated using the generalized estimating equation method. RESULTS: The mean (standard deviation) BMI was 23.3 (3.0) kg m-2 for men and 21.9 (3.6) kg m-2 for women; and the mean WC was 82.6 (8.7) cm for men and 77.8 (9.8) cm for women. During the follow-up period, mean BMI increased by 0.2 kg m-2 for men and women, and mean WC increased by 1.1 cm for men and 1.0 cm for women. The annual change in the BMI of men who skipped breakfast four to six times per week was 0.061 kg m-2 higher, and that of those who skipped breakfast seven times per week was 0.046 kg m-2 higher, compared with those who did not skip breakfast. Annual changes in the WC of male participants who skipped breakfast seven times per week was 0.248 cm higher than that of those who did not skip breakfast. Skipping breakfast was not associated with changes in BMI or WC in women. CONCLUSIONS: Skipping breakfast was closely associated with annual changes in BMI and WC among men, and eating breakfast more than four times per week may prevent the excessive body weight gain associated with skipping breakfast.

Evaluation of sentinel node identification with isosulfan blue in gastric cancer.

AIMS: The aim of this study was to clarify whether the metastasis of gastric cancer can be detected by identifying the sentinel lymph nodes (SNs) using only lymphatic dye. METHOD: The study was based on 101 patients clinically diagnosed with T1 and T2 gastric cancer. Isosulfan blue was intraoperatively injected within the submucosal layer around the lesion through an endoscope and blue stained nodes (BNs) were identified as SNs and harvested. Standard radical gastrectomy with D2 lymphadenectomy was performed on all patients and SNs and other nodes were evaluated. RESULTS: Out of 101 patients, 21 had lymph-node metastases. The accuracy of SN identification was 97.0% (98/101) and the metastasis detection rate was 85.7% (18/21). With improvement of the manoeuvre in the latter 84 cases, the detection rate was elevated up to 100.0% (18/18), as was the accuracy (84/84). CONCLUSION: The sentinel concept using lymphatic dye is applicable to gastric cancer, but the identification of SNs with lymphatic dye requires some practice. Furthermore, the issue of intraoperative metastasis detection remains to be resolved.

Expression of nestin in rat and human glomerular podocytes.

Nestin is a neuroepithelial precursor cell marker expressed in a variety of human cell types during development. However, no information exists on the expression of nestin in mature glomeruli as well as during the glomerular development. Here, we examined nestin expression in rat and human glomerular tissues in quiescent states using RT-PCR and immunohistochemical methods. Nestin mRNA was detected in the rat glomeruli in parallel with its expression in developing rat brains. In the normal mature rat glomeruli, WT-1 positive cells expressed nestin. Co-expression of nestin and vimentin was observed in mature rat podocytes. Immunoelectron microscopy revealed nestin localization in the cell bodies and primary processes of podocytes. A similar expression pattern was observed for vimentin. In matured glomeruli, nestin was not expressed by mesangial and endothelial cells. In the newborn rat, early developing glomeruli (metanephric cap, metanephric vesicle, comma-shaped vesicle and S-shaped body phases) expressed nestin. In the capillary loop stage, Bowman's capsules also expressed nestin. Immunoelectron microscopy demonstrated that developing podocytes and endothelial cells in S-shaped phase glomeruli expressed nestin. Additionally, in immature glomeruli, the mesangial cells in capillary stage of glomerulus also expressed nexin. As in the rat, WT-1 positive cells in human glomeruli also expressed nestin and immunoelectron microscopy confirmed nestin expression in human glomerular podocytes. These results reveal that in normal condition nestin is expressed in several glomerular cell types at early stage of development and becomes confined to podocytes in mature glomeruli, thus implicating nestin in podocyte functions.

Reduplicated basal lamina of the peritubular capillaries in renal biopsy specimens.

Reduplicated basal lamina of the peritubular capillaries (PTC) is usually found in kidney allografts in association with chronic transplant nephropathy and sometimes in native renal biopsies. In order to assess the incidence of this phenomenon in native renal biopsy specimens, we have carried out a retrospective review of the diagnostic ultrastructural pathology records of 80 consecutive renal biopsies excluding renal allografts and children with clinical signs of heavy proteinuria. Reduplicated basal lamina of the PTC was found in 19 out of the 80 cases (23.8%) with renal diseases. It was frequently seen in lupus nephritis, IgA nephropathy, and membranoproliferative glomerulonephropathy, being the subtypes of mesangial proliferative lesions. In a few cases it was also found in anti-neutrophil cytoplasmic autoantibody (ANCA) associated glomerulonephritis and benign nephrosclerosis renal biopsies. Reduplicated basal lamina of the PTC was strongly associated with glomerular and peritubular inflammation, and tubular necrosis. Peritubular interstitial edema, slight to moderately increased collagen fibrils, many spiraled collagen fibrils (indicative of degeneration), and collagen fibrils drawing from basal lamina were found around the reduplicated basal lamina of the PTC but not in normal basal lamina. These results indicate that in native renal biopsy specimens, reduplication of the basal lamina of the PTC is associated with endothelial cell injury and capillary permeability abnormality.

Alcohol, high blood pressure, and serum gamma-glutamyl transpeptidase level.

The influence of the level of serum gamma-glutamyl transpeptidase, a biological marker of alcohol consumption, on elevations of blood pressure and on the development of hypertension related to increases in alcohol consumption was determined in a cross-sectional study of 1,492 middle-aged male workers and in a subsequent 5-year follow-up study of 1,393 workers. Blood pressure levels, as well as the prevalence and incidence of hypertension, were higher in the subjects with serum gamma-glutamyl transpeptidase levels above 50 units/l than in those with normal levels. These differences were more marked in drinkers who consumed 30 ml or more of alcohol per day. Thus, elevated serum gamma-glutamyl transpeptidase activity may identify drinkers at higher risk for the development of alcohol-related hypertension.

The relationship between employment grade and plasma fibrinogen level among Japanese male employees. YKKJ Research Group.

Plasma fibrinogen is an important risk factor for cardiovascular disease, and is associated with socioeconomic status in Europe and the United States. We evaluated whether the relationship between socioeconomic status and plasma fibrinogen level exists in Japanese male employees, and whether this relationship is independent of other correlates of plasma fibrinogen. This cross-sectional study was conducted on full-time male employees aged 20-58 in a metal-products factory between April 1996 and March 1997. Altogether 4375 employees (92.9%) participated. Low employment grade and low educational background were associated with increased age-adjusted plasma fibrinogen level. Adjusting for body mass index, waist to hip ratio, height, smoking habit, alcohol consumption, physical activity at leisure and systolic blood pressure did not attenuate these associations much. Adjusting for white blood cell count and hemoglobin Alc reduced the associations of both employment grade and educational background with plasma fibrinogen level, nevertheless these relationships remained significant.

Clinical study of niceritrol on serum lipids in the treatment of hyperlipidemia.

The effects of niceritrol, a nicotinic acid derivative, on the levels of HDL-cholesterol (HDL-Ch) and a mixture of VLDL- and LDL-Ch (VLDL- + LDL-Ch) were studied in hyperlipidemic patients. Serum total cholesterol (sTC) and serum triglyceride (sTG) were significantly reduced during niceritrol administration. Lipoprotein electrophoresis showed that niceritrol increased the alpha:beta ratio. HDL-Ch showed a significant increase of 12.5% by the 16th week of therapy. This increase was more marked in patients with lower pre-treatment HDL-Ch levels and significant in patients whose pre-treatment sTG levels were in excess of 200 mg/dl. Females displayed higher pre-treatment HDL-Ch levels (38.5 mg/dl) than males (30.6 mg/dl). However, niceritrol increased HDL-Ch significantly in both groups. At 16 weeks, the VLDL- + LDL-Ch level showed a significant decrease of 9.2%; the HDL-Ch:VLDL + LDL-Ch and HDL-CH:sTC ratios were significantly increased throughout niceritrol administration. Niceritrol is thought to be effective in preventing the development and progression of atherosclerosis because it raises the level of anti-atherogenic HDL-Ch and lowers the level of atherogenic VLDL- + LDL-Ch.

Plasma insulin and blood pressure in normotensive Japanese men with normal glucose tolerance.

OBJECTIVE: To determine whether blood pressure within the normal range is associated with plasma insulin concentration in a Japanese population with normal glucose tolerance. METHODS: We studied 491 normotensive Japanese men with normal glucose tolerance, who had never been treated with antihypertensive medications. Plasma glucose and insulin response during a 75-g oral glucose-tolerance test, blood pressure, body mass index, serum lipids, alcohol consumption and smoking status were obtained. The subjects were divided into quartiles according to mean blood pressure level (< 79, 79-84, 85-91 and > or = 92 mmHg). To eliminate possible confounding factors, analysis of covariance was also performed. RESULTS: In univariate analyses the plasma insulin response after an oral glucose load, especially after 60 min, was significantly related to blood pressure level. Plasma glucose was related to blood pressure level only at fasting. When adjusted for age, body mass index, plasma glucose response and alcohol consumption, plasma insulin concentrations after 30 and 60 min in the highest quartile were significantly higher than those in the lowest quartile. CONCLUSIONS: Blood pressure was significantly and independently related to plasma insulin level after an oral glucose load in normotensive Japanese men with normal glucose tolerance. The results suggest that the relationship between blood pressure and insulin resistance already exists in a prehypertensive state even in a lean Asian population.

Expression of collagen I, smooth muscle alpha-actin, and vimentin during the healing of alkali-burned and lacerated corneas.

PURPOSES: Alkali-burned corneas can seldom heal properly to restore corneal transparency. To provide a better understanding of this devastating corneal injury, we compared the expression of collagen I, smooth muscle alpha-actin (alpha-SMA), and vimentin in lacerated and alkali-burned rabbit corneas. METHODS: A radiolabeled cDNA probe of alpha 1(I) chain was used in slot-blot hybridization to determine the levels of alpha 1(I) mRNA in alkali-burned corneas. In situ hybridization was used to identify the cell types that express the alpha 1(I) chain. Antibodies against collagen I, alpha-SMA, and vimentin were used in immunohistochemical studies to determine the tissue distribution of collagen I and to identify cells expressing alpha-SMA and vimentin. RESULTS: The levels of alpha 1(I) mRNA in alkali-burned corneas increased steadily after the alkali burn and reached a plateau within 2 weeks. One day after alkali burn, specific in situ hybridization signals were detected in stromal cells immediately surrounding the edge of the corneal injury. As the healing proceeded, the fibroblastic cells migrated into the injured stroma, and they showed positive reactions by in situ hybridization and by immunostaining with anti-collagen I probes. In alkali-burned corneas, retrocorneal membranes were formed 1 week after injury. This fibrillar membrane was stained by anti-collagen I antibody, and the fibroblastic cells in the membrane were hybridized by the 3H-labeled alpha 1(I) cDNA probe. No retrocorneal membrane was formed in the lacerated corneas, even after the injured corneas were allowed to heal for 3 weeks. The epithelial cells in the epithelial plug of lacerated corneas were positive by in situ hybridization, whereas the epithelial cells in the regenerated epithelium of alkali-burned cornea was not. Antibodies against alpha-SMA reacted with the migrating fibroblastic cells but did not react with epithelial cells or endothelial cells in the injured corneas. Anti-vimentin antibody reacted with fibroblastic cells, endothelial cells, and keratocytes in normal and injured corneas, and with the basal epithelial cells of injured corneas. CONCLUSIONS: During wound healing, the keratocytes that migrate to injured stroma transform into myofibroblasts. These myofibroblasts express high levels of alpha 1(I) mRNA, alpha-SMA, and vimentin. The healing of alkali-burned corneas differ from that of lacerated corneas in that the retrocorneal membranes are formed in the former but not in the latter. In addition, the epithelial cells of alkali-burned corneas lack alpha 1(I) mRNA, whereas it is found in the epithelium of lacerated corneas. These differences may result from the persistence of inflammatory cells in the alkali-burned corneas.

Distribution of collagen IV in human ocular tissues.

PURPOSE: To examine the distribution of the collagen alpha 1(IV) chain and a novel collagen alpha(IV)-related chain in human ocular tissue. METHODS: Two monoclonal antibodies, JK199 and M3F7, against the alpha 1(IV) chain, and one monoclonal antibody, JK132, against a novel alpha(IV)-related chain were used in the avidin biotin peroxidase complex procedure of immunohistochemical studies. In situ hybridization and reverse transcription polymerase chain reaction were used to examine the presence of alpha 1(IV) messenger RNA in corneal epithelium. RESULTS: Our data indicate that monoclonal antibodies JK199 and M3F7 react with most ocular basement membranes, but not with those of corneal epithelium. Similarly, monoclonal antibody JK132 reacts with most basement membranes of ocular tissues, with the exception of the inner limiting membrane of neural retina, Bruch's membrane, and corneal epithelial basement membrane. To examine if the epitopes recognized by the monoclonal antibodies were masked in corneal epithelium, the tissue sections were subjected to limited enzyme digestion, that is, pepsin, hyaluronidase, trypsin, and pronase E, or chemical treatments such as 0.1 N NaOH or 6 M urea. Proteinase treatment removed the JK132 epitope from all ocular basement membranes examined. Despite the pretreatment, corneal epithelial basement membrane was not stained by any of the monoclonal antibodies. However, the alpha 1(IV) messenger RNA was detected in corneal epithelium by in situ hybridization and reverse transcription polymerase chain reaction. Western immunoblotting indicates the presence of the alpha 1(IV) and the novel alpha(IV)-related chain in the basal lamella of corneal epithelium. CONCLUSIONS: The epitopes recognized by JK199, M3F7, and JK132 are masked in basement membrane of corneal epithelium. Based on the tissue distribution and partial amino acid sequences of peptides recognized by JK132, the novel alpha(IV)-related chain differs from other known alpha(IV) chains.

Keratin 12-deficient mice have fragile corneal epithelia.

PURPOSE: Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. METHODS: One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. RESULTS: The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice. CONCLUSIONS: The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.