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Objective: To investigate the expressions of 78-kDa glucose-regulated protein (GRP78) and Caspase-12 and their relationship with apoptosis in renal cortex of diabetic rats., Methods: Uninephrectomized Wistar rats were used to induce diabetes by intraperitoneal injection of Streptozotocin (STZ 65 mg/kg). After 8 weeks, the expression and distribution of GRP78, Caspase-12, proliferating cell nuclear antigen (PCNA) were examined by immunohistochemistry. Flow cytometry was used to detect the levels of protein of GRP78 and Caspase-12. Apoptosis was evaluated by means of terminal deoxynucleotidyl transferase-mediated d-UDP nick-end labeling (TUNEL) and Flow cytometry. Serum creatinine, blood urea nitrogen and 24-hour urine protein excretion were checked., Results: Compared with those in normal control group, the numbers of apoptosis and the expression of GRP78, Caspase-12 in glomerular and tubular cells were much higher in the diabetic kidneys at 8 weeks. There was no significant difference between group A and group B., Conclusion: Activation of endoplasmic reticulum stress may play an important role in the development of diabetic nephropathy.

Objectives: To track bone marrow stem cells (BMSCs) labeled by enhanced green fluorescent protein (EGFP) and superparamagnetic iron oxide (SPIO)-poly-L-lysine (PLL) compound by MRI in vitro for autotransplantation into pancreas of type 1 diabetes miniature pigs., Methods: The BMSCs were isolated by density gradient centrifugation and attachment culture from type 1 diabetes minipigs' bone marrow. Expressional intensity of EGFP in BMSCs transfected lentivirus-EGFP with a multiplicity of infection (MOI) of 30:1 reached the highest level after 96 h from transfection, while the positive rate was 43.2%. Different magnetic resonance scanning protocols were carried out on various density BMSCs labeled by different concentration of SPIO in various time-point in vitro., Results: When SPIO concentration was 25 mg/L (count in Fe(3+)), the positive Fe(3+)-labeling rate of BMSCs was 93.1%. Most of SPIO particles in BMSCs' cytoplasm were observed in secondary lysosomes, but they were not detected in important organelle as cell nucleus. Comparing with gelatin the MRI of BMSCs labeled with SPIO in the condition with 1 × 10(4)/ml cells density and 25 mg/L Fe(3+) concentration in vitro, the signal intensity changes (ΔSI) after BMSCs labeled with SPIO 3 weeks and 6 weeks in TSE T(1)WI, TSE T(2)WI and FLASH T(2) WI sequences were 12%, 41%, 63% and 7%, 28%, 46% respectively (P < 0.01 and P < 0.05, respectively)., Conclusions: The data showed that the porcine BMSCs labeled with SPIO and EGFP could be traced successfully in vitro by MRI in the suitable sequences.

Objective: To observe the morphologic changes and the expression of suppressor of cytokine signaling-1/3 (SOCS-1/3) in renal tubular epithelial cells induced by high glucose (HG) and to investigate their significance., Methods: The renal tubular epithelial cell line (HKCs) cultured in vitro were divided into blank control group, HG group, and Janus kinase 2 inhibitor AG490 group. HKC of blank control group was cultured for 8 hours in 5.5 mmol/L glucose, and the other two groups were cultured in 300.0 mmol/L glucose or 300.0 mmol/L glucose+10 μmol/L AG490 for 2, 4, 6, 12, 24, 48 hours (n=6). The morphology and ultrastructure were observed with inversion microscope and electron microscope at different time points. Protein expression of SOCS-1/3 was assayed by immunocytochemistry and Western blotting; SOCS-1/3 mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR)., Results: Under inversion microscope it was showed that 12 hours after being cultured with HG, the cells assumed a spindle-shape, with irregular protrusions, and cellular membrane became indistinguishable with prolongation of time, with increase of intracellular granules. Under the electron microscope, it was seen that there was distinct decrease in microvilli on the cell membrane and mitochondria, with an increase in rough endoplasmic reticulum. The cellular changes were not obvious in AG490 group. Furthermore, immunocytochemistry and Western blotting showed that the immunoreactivity was localized in the cytoplasm as well as in the nuclei, and there was basic expression of SOCS-1/3 protein in normal HKC (0.218±0.023, 0.337±0.009). HG was shown to induce up-regulation of the expression of SOCS-1/3 protein at 4, 6, 12, 24 hours compared with blank control group. The expression of SOCS-1 was highest at 4 hours (1.022±0.072), and that of SOCS-3 was highest at 6 hours (1.256±0.105, both P<0.01), while the expression of SOCS-1/3 protein in AG490 group was lower than that in HG group (4 hours SOCS-1: 0.589±0.167, 6 hours SOCS-3 : 0.656±0.075, both P<0.05). However, HG induced a higher expression of SOCS-1/3 mRNA at 2, 4, 6, 12 hours compared with blank control group. The expression of SOCS-1 was highest at 4 hours (1.716±0.098 vs. 0.475±0.045, P<0.05), and that of SOCS-3 was highest at 6 hours (2.848±0.116 vs. 0.749±0.086, P<0.01), while the expression of SOCS-1/3 mRNA in AG490 group was lower (4 hours SOCS-1: 0.865±0.075, 6 hours SOCS-3: 0.923±0.116, both P<0.05)., Conclusion: HG could produce morphology and ultrastructure changes in renal tubular epithelial cell, and it induces up-regulation of SOCS-1/3 expression. These changes might be related with negative regulation of Janus kinase (JAK)/signal transducers and activators of transcription (STAT)/SOCS pathway.

Objective: To investigate the effect of AG490, a Janus kinase 2 inhibitor, on epithelial-myofibroblast transdifferentiation induced by interleukin-1β (IL-1β)., Methods: Cultured human renal tubular epithelial cell line (HKCs) were divided into three groups: blank control group, IL-1β (5 ng/ml) group and AG490 group (IL-1β 5 ng/ml+AG490 10 μmol/L). The cells in all groups were collected at 24, 48, 72 hours after intervention. Immunocytochemistry and Western blotting analysis were used to determine the expressions of cytokeratin-18 (CK-18) and α-smooth muscle actin (α-SMA)., Results: The higher expression of CK-18 (1.25±0.08) and mild expression of α-SMA (0.17±0.01) were found in blank control group. In IL-1β group, the protein level of CK-18 was gradually decreased with prolongation of stimulus (24 hours : 0.69±0.04, 48 hours: 0.52±0.03, 72 hours: 0.30±0.01), while the expression level of α-SMA was gradually increased (24 hours: 0.56±0.04, 48 hours: 1.05±0.07, 72 hours: 1.43±0.07), and the difference between blank control group and IL-1β group was statistically significant (all P<0.05). The administration of AG490 could restore the expression of CK-18 (24 hours: 1.07±0.07, 48 hours: 0.93±0.06, 72 hours: 0.83±0.06), and inhibit the expression of α-SMA induced by IL-1β (24 hours: 0.33± 0.01, 48 hours: 0.52±0.01, 72 hours: 0.61±0.04). There was significant difference between AG490 group and IL-1β group (all P<0.05). The results of immunocytochemistry and that of Western blotting were identical., Conclusion: IL-1β can induce the transdifferentiation of renal tubular epithelial cells, up-regulate the expression of α-SMA, induce the renal tubular epithelial cells to transform to myofibroblast, while AG490 can inhibit the effect of IL-1β.

Store-operated Ca(2+) entry (SOCE) has recently been proposed to contribute to Ca(2+) influx in vascular smooth muscle cells (VSMCs). Imperatorin is known for its potent vasodilatory effects as a dietary furanocoumarin. The study was designed to examine the hypothesis that SOCE have a functional role in imperatorin-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery rings were measured by a sensitive myograph system. Laser scanning confocal microscopy was used to determine the intracellular Ca(2+) concentration of fluo-3-loaded VSMCs. Imperatorin (1-100 μM) relaxed artery rings precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca(2+) influx due to SOCE. Imperatorin didn't inhibit SOCE-mediated increases in cytosolic Ca(2+) levels evoked by the emptying of the stores. In isolated artery rings, imperatorin didn't inhibit SOCE-induced contractions due to store depletion. Our results exclude SOCE mechanism of vasodilatation by imperatorin. But imperatorin is partly similar with nifedipine in vasorelaxation effect., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: To investigate the effects of Bai-Chuan capsule (BC) on the left ventricular hypertrophy in spontaneously hypertensive rats (SHR)., Methods: Taking SHR and Wistar Kyoto rats (WKY) as the model group and the control group respectively, Captopril as positive drug, administered BC 0.3 g/kg, Captopril 3.75 mg/kg or 0.5% CMC-Na to each group by ig for 3 months, and measured the change of blood pressure. The left ventricular mass index was calculated after rats were sacrificed. Left ventricle was used to pathological observations, plasma angiotensin II and aldosterone were measured by radioimmunoassay., Conclusion: BC can inhibit left ventricular hypertrophy, plasma level of angiotensin II and aldosterone to some extent in SHR.

Objective: To explore the effect of high fat diet on the expression of sterol regulatory element binding protein-1 (SREBP-1), transforming growth factor-beta1 (TGF-beta1) and alpha-smooth muscle actin (alpha-SMA) in renal tubular cells and extracellular matrix accumulation in Wistar rats., Methods: The Wistar rats were treated with high fat diet for 12 weeks and renal lipid deposit was detected by the method of Oil Red O staining. The immunohistochemistry and Western blot were used to investigate the expression of SREBP-1, TGF-beta1, alpha-SMA and fibronectin (FN) protein. The expression of SREBP-1 mRNA was determined with in situ hybridization. Masson staining was for the detection of extracellular matrix (ECM) accumulation., Results: The weight of rats raised by high fat diet increased, in company with the high serum glucose, serum triglyceride and serum insulin. The Oil Red O staining revealed that the renal proximal tubular epithelial cells showed significant lipid droplet in high fat diet rats. SREBP-1 protein and mRNA were located in the renal tubular cells and the expressions of high fat diet rats were higher than those of normal control rats. They were respectively 1.88 times and 1.85 times than those of normal control group. TGF-beta1 and alpha-SMA protein were also located in renal tubular cells and high fat diet up-regulated the expression of them. ECM accumulation was detected with Masson staining and the result showed that high fat diet treatment increased interstitial ECM product and FN protein was found high expression., Conclusion: High fat diet may induce lipid droplet deposit in renal tubular cells by up-regulation of the expression of SREBP-1, which causes ECM accumulation by increasing the expression of TGF-beta1 and alpha-SMA.

Objective: To investigate changes in proliferative activity of myoblasts in skeletal muscle and potential role of phosphorylated Akt on it, so that a better understanding in mechanisms of skeletal muscle atrophy after burn injury will be got., Methods: One hundred and twenty Wistar rats were randomly divided into 2 groups: control and severe thermal injury group. Rats in severe thermal injury group were subjected to a 40% total body surface area full-thickness scald injury, and Tibialis Anterior (TA) muscles were collected on 0, 1, 4, 7, 10, 14 days post-injury. After muscle mass determined, immunohistochemical double staining was used for detection of Proliferative Cell Nuclear Antigen (PCNA) of myoblasts. Protein expression of total Akt and phosphorylated Akt was determined by Western Blot., Results: Burn injury induced significant reduction of TA muscle mass and maximal reduction of it appeared by 4 days after injury (P < 0.01). Proliferative activity of myoblasts decreased significantly from the first day post-injury (P < 0.01) and increased slowly to basal level of controls after 7 days post-injury. The phosphorylated Akt was undetectable in both of controls and injured samples before 4 days but increased significantly after 7 days post-injury (P < 0.01), though total Akt expression had no significant alteration at any time points (P > 0.05)., Conclusions: Decrease in proliferative activity of myoblasts may be one of the contributors of significant atrophy of skeletal muscle after burn injury. Effect of phosphorylated Akt on proliferation attenuated in early stage and increased significantly in later stage after burn injury may partly explain the changes in proliferative activity of myoblasts.

Objective: To investigate changes in skeletal muscle apoptosis after a severe thermal injury in rats., Methods: One hundred Wistar rats were randomly divided into two groups: sham thermal injury group and severe thermal injury group. They were subdivided into 1, 4, 7, 10, 14 days post-injury with 10 rats in each subgroup. Rats in severe thermal injury group were subjected to a 40% total body surface area full-thickness scald injury. Both weight and tibialis anterior (TA) mass of rats were weighed on 1, 4, 7, 10, 14 days post-injury. Electron microscope was used for observing ultrastructural changes in skeletal muscle, including apoptosis. Tissues of tibialis anterior from burn and sham burn animals were then examined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemistry., Results: Compared with sham thermal injury group, body weight and TA mass of rats were decreased from first day on, and it dropped to the lowest level at 4 days (P<0.05 and P<0.01), and started to regain from 7 days on (all P<0.01). Electron micrographs showed condensation of chromatin around the periphery of the nucleus, blebbing of the sarcolemma, and free of myofibrils near myonuclei in a large area in skeletal muscle of thermally injured rats. Sporadic TUNEL positive myonuclei were also seen under light microscope in skeletal muscle in thermal injury group. There were no characteristic signs of apoptosis in skeletal muscle in rats of sham group., Conclusion: There are skeletal muscle apoptosis after severe thermal injury. It may contribute to atrophy of skeletal muscle after burn injury in rats.

Objective: To investigate changes in apoptosis-related ligands in serum in rats with severe scald and the effect of intensive insulin therapy on the changes., Methods: One hundred and fifty Wistar rats were randomly divided into 3 groups: sham burn (SB), scald (S) and treatment (T) groups. Rats in S and T groups were inflicted with 40% TBSA full-thickness burn, followed by intraperitoneal injection with 40 mL/kg of isotonic saline for resuscitation. Rats in T group were subcutaneously injected insulin in a dose of 0.25 U/100 g 24 hours after burn injury, and every 12 hours for 5 days (0.25, 0.50, 0.75, 1.00, 1.25 U/100 g each day, respectively) to control the level of blood glucose between 3 and 6 mmol/L. Rats in SB group were sham scalded at 37 degrees C without resuscitation. Blood was drawn from abdominal aorta on 1, 4, 7, 10, 14 post burn day (PBD) for determination of serum levels of TNF-alpha, soluble Fas ligand (sFasL) and soluble Fas receptor (sFas) by enzyme-linked immunosorbent assay (ELISA), and insulin by radioimmunity assay (RIA)., Results: The serum level of TNF-alpha in S group peaked on 1 PBD (30.9 +/- 8.7) ng/L, which showed statistically significant difference when compared with that of SB and T groups (12.7 +/- 2.8) ng/L, (16.8 +/- 4.7) ng/L, respectively, P < 0.01), then lowered gradually to become similar to that of SB group on 7 PBD. The level of TNF-alpha in T group increased gradually, but was obviously lower than that of S group on 1, 4, 7 PBD (P < 0.01). The level of sFasL in S (on 7-14 PBD) and T (4-10 PBD) groups was significantly higher than that in SB group (P < 0.05), then lowered to normal level. The levels of sFas on 4-10 PBD in T group were obviously higher than that in S and SB group (P < 0.05). Ratio of sFasL to sFas in serum of S group was higher than that in SB group on 7, 10 PBD, which was higher than that in T group on 7 PBD (P < 0.05). There was significant decrease in serum level of insulin in S group compared with that of SB group on 4-10 PBD (P < 0.05). The level of insulin in T group increased on 1 PBD, peaked on 4 PBD (327 +/- 15 microU/mL), which was significantly higher than that in SB and S groups (42 +/- 15, 28 +/- 10 microU/mL, respectively, P < 0.01), then decreased gradually to normal level., Conclusions: Insulin may inhibit apoptosis after burn by down-regulating secretion of apoptotic ligands.

Objective: To investigate the effects of fluvastatin on activation of Janus kinase 2 (JAK2) and signal transducers and activators of transcription 1, 3 (STAT1, 3) in glomerular mesangial cells(GMCs) under high concentration of glucose., Methods: Rat GMCs were cultured in vitro, and they were treated with glucose and fluvastatin respectively. Tyrosine phosphorylation of JAK2 (p-JAK2) expression was detected by immunoprecipitation and Western blotting analysis. The protein expressions of JAK2, STAT1, p-STAT1, STAT3 and p-STAT3 were assessed by Western blotting. The protein synthesis of transforming growth factor-beta1 (TGF-beta1) and fibronectin (FN) in the supernatants of the GMCs were determined by enzyme-linked immunoadsorbent assay (ELISA). TGF-beta1 mRNA was assessed by reverse transcription and polymerase chain reaction (RT-PCR)., Results: Compared with low glucose control group, the expressions of p-JAK2 (802+/-124 vs.204+/-31), p-STAT1 (2,856.6+/-337.8 vs. 617.7+/-76.2), p-STAT3 (3,049.8+/-421.3 vs. 946.7+/-141.2) and TGF-beta1 mRNA were significantly up-regulated in GMCs under high glucose medium, and the concentration of TGF-beta1 in the supernatants [(2.87+/-0.34) microg/L vs. (1.20+/-0.11) microg/L] and FN [(6.34+/-0.61) mg/L vs. (3.24+/-0.26) mg/L, both P<0.01] were higher in the supernatants. The expression levels of p-JAK2 (412+/-67), p-STAT1 (1,178.4+/-137.1), p-STAT3 (1,572.6+/-181.2) and TGF-beta1 mRNA were significantly lower in fluvastatin group than those in high glucose group. The concentration of TGF-beta1 [(1.94+/-0.27) microg/L] and FN [(4.27+/-0.33)mg/L] in the supernatants in fluvastatin group were lower than those in high glucose control group (all P<0.05)., Conclusion: Fluvastatin can inhibit overproduction of TGF-beta1 and FN in GMCs under high concentration of glucose, the underlying mechanism may partly be attributable to its influence on phosphorylation of JAK/STAT.

We identified and characterized a novel swine gene Bcl10 with GenBank (Accession No. EU088132) which was homologous to human BCL10 (B-cell CLL/lymphoma 10) gene. The full-length cDNA of 925 bp for swine BCL10 was in silico cloned, and then its ORF of 702 bp coding 233 amino acid residues was verified by RT-PCR and DNA sequencing. NCBI BLAST assay indicated that this cDNA contained three extrons with a length of 57, 289 and 356 bp respectively, and it was located on the chromosome 4 of pig genome. Using semi-quantitative PCR, preliminary expression profiles of swine BCL10 were verified in different swine tissues. The expression of swine BCL10 was verified by GFP marker and RT-PCR assay. We found that, BCL10 expressed in high level in swine spleen, and with a modest level in thymus, brain and lymph node; low level mRNA was expressed in liver and not detectable level in kidney. The swine BCL10 gene was cloned to the GFP-containing eukaryotic expression vector pEGFP-C1 and transfected to PK-15 cell line by lipofectin. BCL10 was ex-pressed effectively in PK-15 cells. In summary, we cloned a novel swine BCL10 gene, and investigated its expression char-acters. This will be the fundament of the future research on its function.

Objective: To investigate the expression and implication of osteopontin (OPN) in renal cortex of diabetic rats., Methods: Experimental diabetes was reproduced in uninephrectomized rats by giving streptozotocin (STZ). The animals were divided into two groups: control group and diabetic group (each n=24). Immunohistochemistry and Western blotting were used to detect the protein expression of OPN, CD68, and proliferating cell nuclear antigen (PCNA). At the same time the mRNA expression of OPN was detected by in situ hybridization., Results: Compared with control group, blood glucose, blood urea nitrogen (BUN), 24-hour urine protein quantum and quantification of OPN protein significantly increased while creatinine clearance rate (CCr) was significantly decreased in diabetic rat group 1, 2, 4 and 8 weeks after STZ injection. The protein of PCNA were also higher than that of control group 1 and 2 weeks after STZ injection (all P<0.05), and the increase in CD68 was observed at week 4 and week 8., Conclusion: Overexpression of OPN may be responsible for the proliferation of tubular cells at early stage of diabetic nephropathy and for the accumulation of macrophage at later stage.

Objective: To investigate the clinical implication of platelet-derived growth factor (PDGF)-D and PDGF-beta in IgA nephropathy in childhood., Methods: Forty-seven children with IgA nephropathy and 26 controls were enrolled for study, and their serum, urine and renal biopsy specimens were examined. The patients were divided into control group [including serum, urine specimens of 13 healthy children and 13 renal biopsy samples of non-IgA nephropathy in children], mild proliferation (MP) group (13 patients), focal proliferation (FP) group (19 patients), and proliferation sclerosis (PS) group (15 patients). Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to determine contents of PDGF-D, PDGF-beta and PDGF-B in blood, urine and renal tissues. The levels of 24-hour urinary protein excretion, serum albumin (Alb), serum blood urea nitrogen (BUN) and creatinine (Cr) were also determined., Results: Compared with control group, levels of PDGF-D and PDGF-B were progressively elevated in blood and urine of IgA nephropathy children with increase in severity of glomerular damage (all P<0.01). Serum as well as urinary PDGF-D and PDGF-B levels were positively correlated with 24-hour urinary protein excretion (PDGF-D blood: r=0.546, urine: r=0.760; PDGF-B blood: r=0.634, urine: r=0.577, respectively, P<0.01), while negatively correlated with serum Alb levels in IgA nephropathy patients (PDGF-D blood: r=-0.649, urine: r=-0.528; PDGF-B blood: r=-0.613, urine: r=-0.531, respectively, P<0.01). Contents of PDGF-D and PDGF-beta in renal tissue were much higher than those of control group (P<0.01). Along with the increase in severity of glomerular pathology, their contents increased gradually. PDGF-B was only significantly expressed in renal tissue in FP group and PS group., Conclusion: PDGF-D might significantly enhance the development of mesangial proliferation and tubulointerstitial fibrosis. In comparison with PDGF-B, PDGF-D appears to reflect more sensitive to the severity and prognosis of IgA nephropathy.

Aim: The aim of the present study was to further elucidate the mechanism of the protective role of fluvastatin on diabetic nephropathy., Methods: Streptozotocin-induced diabetic rats were treated daily with fluvastatin (4 mg/kg body weight) by gavage. The animals were killed 4 weeks later and urine and blood samples were collected. The kidney tissues were removed and subjected to the following experiments. Rat glomerular mesangial cells (GMC) were cultured under normal glucose (5.5 mmol/L), high glucose (HG, 30 mmol/L), HG+AG490 (10 micromol/L), or HG with fluvastatin (1 micromol/L). Glomeruli or the GMC lysate was immunoprecipitated and/or immunoblotted with antibodies against Janus kinase 2 (JAK2), SH2-domain containing tyrosine phosphatase-1 (SHP-1), phosphospecific SHP-2, and signal transducer and activators of transcription (STAT), respectively. Transforming growth factor-beta (TGF-beta1) mRNA was measured by RT-PCR. The protein synthesis of TGF-beta1 and fibronectin in the culture medium of GMC was detected by ELISA., Results: The phosphorylation levels of JAK2, STAT1, STAT3, and SHP-2 increased significantly, and SHP-1 phosphorylation was reduced in glomeruli of diabetic rats. Treatment with fluvastatin reduced phosphorylation levels of JAK2, STAT1, STAT3, and SHP-2 in glomeruli of diabetic rats, but it had no effect on the dephosphorylation of SHP-1. The exposure of GMC to 30 mmol/L glucose caused the activation of JAK2, STAT1, STAT3, and SHP-2. It upregulated TGF-beta1 expression and increased protein synthesis of fibronectin. These high glucose-induced changes were suppressed by fluvastatin, as well as AG490, a JAK2 inhibitor., Conclusion: The regulation of the phosphorylation of JAK/STAT by fluvastatin may be responsible for its renal protective effects on diabetic nephropathy.

Objective: To evaluate the effect of JAK/STAT signaling pathway activation on the transdifferentiation and secretion of transforming growth factor-beta1 (TGF-beta1) induced by high glucose in renal proximal tubular epithelial cells., Methods: Human kidney cells (HKC) were cultured and then divided into four groups: low glucose (LG) group, high glucose (HG) group, high mannitol (LG + M) group, and HG + AG490 group. Immunoprecipitation and Western blot analysis were used to determine the expression of tryosine phosphorylated Janus kinase 2 ( p-JAK2). The protein expressions of STAT1, STAT3, p-STAT1, and p-STAT3 and the expressions of alpha-SMA and E-Cadherin were observed by Western blot. The contents of TGF-B1, fibronectin and type I collagen in the supernatants of the cultured HKC were detected by enzyme-linked immunosorbent assay (ELISA). The expression of TGF-beta1 mRNA was measured by reverse transcription and polymerase chain reaction (RT-PCR)., Results: Compared with LG group, the expressions of JAK2, p-STAT1, p-STAT3, and TGF-beta1, mRNA were significantly increased in HG group from 6 to 72 hours. Meanwhile, the contents of TGF-beta1 and collagen I in the supernatants and the expression of alpha-SMA increased and the expression of E-Cadherin decreased. The expressions of JAK2, p-STAT1, p-STAT3, and TGF-beta mRNA as well as the levels of TGF-beta1 and collagen I in the supernatant s in HG + AG490 group were significantly lower than in the HG group. The expressions of alpha-SMA and E-Cadherin were also decreased in HG + AG490 group., Conclusion: Activation of JAK/STAT signaling pathway may be involved in the high glucose-induced transdifferentiation and overproduction of TGF-beta1, and ECM proteins in HKCs.

A rapid, simple and sensitive method was demonstrated for the determination of phenolic compounds in water samples by alternating-current oscillopolarographic titration. With the presence of sulfuric acid, phenol could be transferred into a nitroso-compound by reacting with NaNO2. The titration end-point was obtained by the formation of a sharp cut in the oscillopolarographic with infinitesimal NaNO2 on double platinum electrodes. The results showed that phenol concentration had an excellent linear relationship over the range of 4.82 x 10(-6)-9.65 x 10(-3) mol/L, the RSD of the proposed method was lower than 1.5%, and the spiked recoveries of three real water samples were in the range of 95.6%-106.9%.

Background: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive., Materials and Methods: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot., Results: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells., Conclusions: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.

Objective: To study the metastasis feature of the primary and metastatic lymph node lesions in supraglottic or hypopharyngeal cancer., Methods: The expression of CD44 and nm23-H1 in specimens from the primary and metastatic lymph node lesions of the 41 cases with supraglottic or hypopharyngeal cancer were studied with immunohistochemistry method and flow cytometry., Results: No correlation was found between the expression of CD44, nm23-H1 and the tumor differentiation of the supraglottic or hypopharyngeal cancer, but their expression related with the clinical staging. The CD44 and nm23-H1 positive expression rates in the primary and metastatic lymph node lesions were 75.6% (31/41), 85.4% (35/41) and 34.1% (14/41), 26.8% (11/41) respectively (P >0.05). The average fluorescence index of CD44 and nm23-H1 in the primary and metastatic lymph node lesions were 1.27 +/- 0.18, 1.33 +/- 0.16 and 1.11 +/- 0.19, 1.08 +/- 0.15 (x +/- s) respectively (P >0.05)., Conclusions: The expressions of CD44 and nm23-H1 in the metastatic lymph node tumor had no difference compared with that in primary tumor of the supraglottic or hypopharyngeal cancer. The difference of metastasis potentials between the primary and metastatic lymph node lesions in the same patient was not proved in this study and should be further investigated from multiple oncogens markers.

Objective: To investigate the characteristics of sensory gating P50 of patients with first-episode schizophrenia., Methods: The auditory evoked potentials P50 were recorded in 66 patients (Group Sch) with first-episode schizophrenia and 92 normal controls (Group NC) by using conditioning/testing paradigm presented with auditory double click stimuli., Results: The value of S1-P50 of Group Sch was 3 microV +/- 2 microV, significantly lower than that of Group NC (6 microV +/- 3 microV, P <0.01). The value of S2-P50 of Group Sch was 4 microV +/- 2 microV, significantly higher than that of Group NC (2 microV +/- 1 microV, P < 0.01). The S2/S1 ratio of Group Sc was 81% +/- 40%, significantly higher than that of Group NC (42% +/- 21%, P < 0.01). The value of S2 - S1 of Group Sch was 2 microV +/- 1 mciroV, significantly lower than that of Group Sch (3 mciroV +/- 2 microV). The value of 100 (1-S2/S1) of Group Sch was 19% +/- 17%, significantly lower than that of Group NC (58% +/- 21% , P < 0.01)., Conclusion: Sensory gating deficit exists in the patients with first-episode schizophrenia, manifested in deficiency of inhibition that can be quantitatively observed by measuring auditory evoked potential P50.

Objective: To investigate the relationship of tubular epithelial-myofibroblast transdifferentiation and the expressions of hepatocyte growth factor (HGF) and Smad7, and to elucidate the role of HGF and Smad7 in diabetic nephropathy., Methods: Diabetes was induced in male Wistar rats with right nephrectomy and streptozotocin (STZ) administration. The expressions of cytokeratin 18 (CK18), alpha-smooth muscle actin (alpha-SMA), HGF and Smad7 were assayed with immunohistochemistry. The expressions of alpha-SMA, HGF were assayed with flow cytometry. The expression of Smad7 was assessed by Western blot., Results: Compared with those in kidneys of the control group, diabetic kidneys showed down-regulated expression of CK18 and up-regulated expression of alpha-SMA. The expressions of HGF and Smad7 were increased significantly in the kidneys of diabetic rats at first, then they decreased gradually, but were still higher than those of control., Conclusion: The tubular epithelial cells may undergo phenotypic alterations and change to myofibroblasts. The absence of up-regulation of HGF and Smad7 may be related with the transdifferentiation of tubular cells in this animal model.

Objective: To investigate the effects of losartan on Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 in glomeruli of diabetic rats., Methods: Sixty Wistar male rats were randomly divided into control group (n=20), diabetes group (n=20), and losartan treatment group (n=20). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg). Losartan (10 mg/kg) was administered daily by gavage from the day following the induction of diabetes. The animals were sacrificed in the weeks 2 and 4 after STZ injection. The renal cortical tissues were obtained and glomeruli were isolated. The protein expressions of JAK2, STAT3 and tyrosine phosphorylated STAT3 (p-STAT3) were assessed respectively by Western blot. Immunoprecipitation and Western blot analysis were used to determine tyrosine phosphorylated JAK2 (p-JAK2). JAK2 and STAT3 mRNA were assayed by reverse transcription-polymerase chain reaction (RT-PCR)., Results: Compared with the control group rats, the respective expression of JAK2, p-JAK2, STAT3, p-STAT3, JAK2 mRNA and STAT3 mRNA was significantly increased in the diabetic glomeruli without losartan treatment (all P<0.01). After treatment with losartan, the expression of p-JAK2 and p-STAT3 in the diabetic glomeruli was down-regulated (all P<0.05). However losartan had no effect on the expression of JAK2, STAT3, JAK2 mRNA and STAT3 mRNA in the diabetic glomeruli., Conclusion: JAK2 and STAT3 signal proteins may be involved in the kidney damage associated with diabetes. Regulation of phosphorylation of JAK2 and STAT3 may be responsible for the renal protective effects of losartan in diabetic rats.

Objective: To investigate the protective effects of Lovastatin on renal function in experimental diabetic nephropathy in rats. and the function of cAMP-responsive element binding protein (CREB1) in this duration., Methods: Diabetes was induced in male Wistar rats by intrapetitoneal injection of STZ (65 mg/kg). Three groups were divided as: Sham group, diabetic control group and Lovastatin treatment group. Lovastatin (20 mg/kg) was administered daily by gavage from the next day of the induction diabetes for 4 weeks. Upro, ucr in urine and Glu, Scr, BUN in serum were determined. Immunohistochemistry and computer image-pattern analysis system were used to analyze expression of PCNA and p-CREB1. Western blot were performed to valuate the expression of p-CREB1 with their specific corresponding antibodies. CREB1 mRNA was measured by RT-PCR., Results: After Lovastatin treatment, renal function were ameliorative in diabetic rats. Decrements were also found in the expression of and PCNA, p-CREB1 and its mRNA in the treatment group compared with the diabetic group., Conclusion: Inhibition of glomerular cAMP-responsive element binding protein 1 may be responsible for the Lovastatin protective function that allevate the renal proliferation and hypertrophy in diabetic rats.

Objective: To investigate the effects of lovastatin on renal function, activity and expression of the p38 mitogen-activated protein kinase (MAPK) and cAMP responsive element-binding protein (CREB) in experimental diabetic nephropathy in rats., Methods: Eighteen uninephrectomized male Wistar rats were randomly divided into three groups: control (n=6), diabetic (n=6) and lovastatin treatment group (n=6). Diabetes was induced by intraperitoneal injection of STZ (65 mg/kg). Lovastatin (20 mg/kg) was administered daily by gavage from the next day of the induction diabetes for 4 weeks. Four weeks later, animals were sacrificed and samples were collected to determine various parameters, including protein and creatinine in urine (Upro and UCr), and glucose (Glu), creatinine (SCr), blood urea nitrogen (BUN) in serum. Immunohistochemistry and computer image-pattern analysis system were used to analyze activation of P-p38 MAPK and P-CREB, the expression of transferase growth factor-beta(1) (TGF-beta(1)), fibronectin (FN), laminin (LN) in renal glomeruli were also measured., Results: Expression of P-p38 MAPK and P-CREB in diabetic glomeruli in diabetic rats were higher than the controls, the same as the expression of TNF-beta(1), FN, LN(all P<0.01). After lovastatin treatment, expression of P-p38 MAPK, P-CREB and TGF-beta(1), FN, LN were markedly decreased compared with diabetic group(all P<0.01)., Conclusion: Inhibition of glomerular p38 MAPK signal transduction pathway may be responsible for the decrement of extracellular matrix accumulation and renal protective effects of lovastatin in uninephrectomized rats.

Aim: To investigate the effect of puerarin on expressions of MMP-2 and TIMP-2 in the kidney of diabetic rats., Methods: Uninephrectomized male Wistar rats were used to induce diabetes by intraperitoneal injection of streptozocin (65 mg x kg(-1)). Puerarin was given daily by intraperitoneal injection from the third day of induction of diabetes for 16 weeks. Using in situ hybridization and immunohistochemistry to detect MMP-2, TIMP-2 mRNA expressions and MMP-2, TIMP-2, collagen IV and Laminin expressions in diabetic kidneys with image analysis system, Flow cytometry was used to detect the expressions of TGFbeta1, MMP-2 and TIMP-2., Results: Compared with those in kidneys of control group, expressions of MMP-2 mRNA and proteins were lower, while the expressions of both TGFbeta1 and TIMP-2 were higher in the diabetic kidney (P < 0.05). The level of MMP-2 expression was advanced, while expression of TIMP-2 was reduced by puerarin treatment (P < 0.05)., Conclusion: Puerarin showed some renal protective effect on diabetic nephropathy, partly through inhibition of excessive deposition of glomeruli extracellular matrix by up-regulating MMP-2 and down-regulating TIMP-2 expressions besides reducing the blood glucose.

Objective: To observe the injury on micro-skin induced by a self designed micro-skin machine., Methods: Micro-skin was produced either with the machine or by hand. Cells at the edge of micro-skin were observed by transmission electron microscope. succinic dehydrogenase activity in supernatant of cultivated cells was analyzed, and the cell proliferation of micro-skin was assessed by (3)H-TdR. Twenty patients were enrolled in the study for the observation of the wound healing time between the two groups of micro-skin after being grafted., Results: Transmission electron microscope examination revealed that the cellular injury at the edge of the micro-skin in machine-made group was mild compared with that in man-made group. (3)H-TdR rate was elevated but the activity of succinic dehydrogenase in the supernatant of cultured cells decreased in supernatant of cultured cells of machine produced micro-skin. Wound healing time was shortened in machine made group. (P < 0.05)., Conclusion: The cellular injury at the edge of micro-skin in the machine made group was mild when compared with that in the man-made group with cell proliferation accelerated and wound healing time shortened.

Objective: To detect gene mutations of p53 gene (exon 4-6) in fibroblasts., Methods: Samples of keloids were taken from 15 patients. The mutations of p53 gene were detected using polymerase chain reaction, the single-strand conformational polymorphism(SSCP) analysis and DNA sequencing., Results: Gene mutations in p53 gene exon 4, 5, and 6 were identified in all the patients with keloids., Conclusion: Gene mutations resulted in keloid p53 protein losing its functions of suppressing cell processes and conducting apoptosis.

Objective: To investigate whether there are abnormal fibroblasts in the surrounding skin of keloids for better understanding of the etiological feature of keloids., Methods: Fresh samples were used for cell culture. Flow cytometry was used for analyzing cell cycles of fibroblasts derived from keloids and the surrounding skin. Proliferative cell proportions were compared between every two groups. P53 exon 4, 5 and 6 were amplified by PCR. DNA was sequenced to examine the structure of the destination gene., Results: The proliferative cell proportion of the fibroblasts derived from the surrounding skin is not so high as that from the verge of keloids, but is higher than the normal skin fibroblasts derived from other part of the keloid patients or persons without keloids (P < 0.05). Mutations (point and frameshift mutations) of P53 exon 4 (6/6) and exon 5(2/6) in fibroblasts derived from the surrounding skin of keloids were identified. Fibroblasts derived from keloids have the same mutations as its surrounding skin., Conclusion: There are abnormal fibroblasts in the surrounding skin of keloids. It may be the consequence of keloid infiltrative growth and a reason of easy recurrence of keloid after therapy.

Aim: To investigate the interaction between hepatitis C virus core protein and translin protein and its role in the pathogenensis of hepatocellular carcinoma and lymphoma., Methods: With the components of the yeast two hybrid system 3, "bait" plasmids of HCV core the gene was constructed. After proving that hepatitis C virus core protein could be firmly expressed in AH109 yeast strains, yeast two- hybrid screening was performed by mating AH109 with Y187 that transformed with liver cDNA library plasmids-pACT2 and then plated on quadruple dropout (QDO) medium and then assayed for alpha-gal activity. Sequencing analysis of the genes of library plasmids in yeast colonies that could grow on QDO with alpha-gal activity was performed. The interaction between HCV core protein and the protein we obtained from positive colony was further confirmed by repeating yeast two - hybrid analysis and coimmunoprecipitation in vitro., Results: A gene from a positive colony was the gene of translin, a recombination hotspot binding protein. The interaction between HCV core protein and translin protein could be proved not only in yeast, but also in vitro., Conclusion: The core protein of HCV can interact with translin protein. This can partly explain the molecular mechanism for hepatocellular carcinoma and lymphoma caused by HCV.

Objective: To investigate the biological function of augmenter of liver regeneration (ALR), we used yeast-two hybrid technique to detect proteins in hepatocytes interacting with ALR., Methods: ALR bait plasmid was constructed by using yeast-two hybrid system 3, then transformed into yeast AH109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in a 2XYPDA medium. Diploid yeast was plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selection and screening. After extracting and sequencing of the plasmid from blue colonies. Analysis was performed by bioinformatics., Results: Of 36 colonies sequenced, 14 are metallothionein, 12 albumin, and 3 selenoprotein P. One colony is a new gene with unknown function., Conclusion: The successful cloning of gene of ALR interacting protein has paved the way for studying the physiological function of ALR and associated proteins.

Aim: To investigate the effects of angiotensin converting enzyme inhibitor (ACEI) on apoptosis and the expression of Fas and Fas-L in the kidney of diabetic rats., Methods: Uninephrectomized Spraque-Dawley rats were used to induce diabetes by intraperitoneal injection of streptozotocin (65 mg.kg-1). Benazepril (10 mg.kg-1) was given daily by gavage from the next day of the induction to diabetes for 12 weeks. Apoptosis was evaluated by means of terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL). Flow cytometry and immunohistochemistry were used to detect the expression of Fas and Fas-L., Results: Compared with those in the kidneys of control group, apoptotic cells were more in number and the expression of Fas and Fas-L was higher in the diabetic kidneys (P < 0.05). The number of apoptotic cells and the level of expression of Fas and Fas-L were reduced by benazepril treatment (P < 0.05)., Conclusion: Angiotensin converting enzyme inhibitor benazepril showed some renal protective effect on diabetic nephropathy, partly through inhibiting apoptosis by down-regulating Fas and Fas-L expression.

Objective: To study the function of augmenter of liver regeneration (ALR) as a regulatory factor that specifically stimulates hepatic cell regeneration, we constructed yeast expressive vector of ALR and expressed it in yeast cells., Methods: Total RNA was extracted from HepG2 cells, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the coding region of ALR. The products were cloned into PGEM-T vector and sequenced, then cloned into PGBK T7 vector. The recombinant plasmid PGBK T7-ALR was transformed into yeast AH109. The yeast protein was extracted and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization technique., Results: DNA sequencing results confirmed that the coding region of ALR was correctly inserted into the yeast expression vector, and Western blotting assay showed that recombinant ALR was successfully expressed in yeast. Its molecular weight was identical to the theoretical value of 15 000 Da; the protein was found inside the yeast cells., Conclusion: The successful expression of ALR in yeast cells makes it possible to study further on its biological function.

Objective: To detect Fas DNA mutations (exon 1-6) in the fibroblasts of patients with keloids, thereby to understand the clinicopathological implications of altered structure of the keloids., Methods: PCR followed by single-strand conformational polymorphism analysis and direct DNA sequencing were used to detect Fas gene mutations in 15 patients with keloids., Results: Insertion and point mutations were identified on the boundary between intron 5 and exon 6 in two patients, while no Fas mutations were found in the fibroblasts derived from normal skin samples of any of the patients., Conclusion: Nonfunction of Fas protein may be related to aberrant gene structure that codes for the transmembrane domain.

The aim of this study was to investigate whether the water particles produced by a nebulizer can reach the alveoli of the lung when they are inhaled with the air. For the purpose of demonstrating water particle incorporation into the lung, light and electron microscopic radioautography with tritiated thymidine was employed. Tritiated thymidine was dissolved in distilled water and nebulized to fine water particles with a new type nebulizer named Shinki, then inhaled by mice. The lungs were taken out and processed by either rapid-freezing and freeze-substitution for dry-mounting radioautography or conventional chemical fixation for wet-mounting radioautography. By wet-mounting radioautography, silver grains were observed in the nuclei of a few alveolar type 2 cells and interstitial cells, demonstrating DNA synthesis. By dry-mounting radioautography numerous silver grains were located diffusely in all the epithelial and interstitial cells, demonstrating diffuse localization of soluble radioisotope-labeled compounds. These results show that water particles produced by the Shinki nebulizer can directly reach the alveoli and thus, better therapeutic effect can be acquired with the Shinki nebulizer than with the usual nebulizer. Further, the nebulizer can be used for the radioautographic studies of the lung in demonstrating incorporation of some drugs into alveolar tissues.

Serum sickness nephritis was induced in male Fisher 344/JCL rats by injecting egg albumin into the foot pads and peritoneal cavity. The alteration of anionic sites in the glomerular basement membrane (GBM) of the rats with significant proteinuria was studied with a quick-freezing and deep-etching method using polyethyleneimine as a cationic probe. In control rats, anionic sites were located around the fibrils of the lamina rara externa, which radiated perpendicularly from the lamina densa to podocyte cell membranes. In the glomeruli of proteinuric rats, many electron-dense deposits were observed in the subepithelial side of the GBM, where the fibrils of the lamina rara externa were usually obscured and anionic sites around them could not be recognized. However, in some areas, a clear boundary could be observed between deposits and the lamina densa. Electron micrographs of freeze-fractured deposits showed that the fibrils radiated perpendicularly from the lamina densa and that anionic sites around them had been preserved. These results suggest that some of the deposits simply passed through the GBM and masked transiently the fibril structures of the GBM, but others probably destroyed these fibril structures, including anionic sites.

A morphometric study was undertaken to examine age-related changes in glomerular ultrastructure and anionic sites in ddY male mice at various ages. A progressive increase in glomerular extracellular matrices, including thickening of the glomerular basement membrane (GBM), formation of GBM nodules, and mesangial matrix increase, was found to be the primary age-related ultrastructural change in aging mice; there were also electron-dense deposits in mesangial and subepithelial regions. The extent of GBM thickening in mice was less than was reported in rats. Rather, the GBM nodules, which had the same electron density as the lamina densa (LD) and protruded on the subepithelial side of the GBM, were more striking. Quantitative evaluation showed that GBM thickness, number and size of GBM nodules, and the area of the mesangial matrix were significantly correlated with the age of the mice. The distribution of anionic sites in the glomeruli of aging animals was described for the first time. No statistically significant differences were noted between the number of glomerular anionic sites in the different age groups. These results indicate that the increase in glomerular extracellular matrices reported in aged rats was also present in aged mice, although the extent of various changes was different. The results also indicate that this increase in glomerular extracellular matrices with age was not accompanied by significant alteration in glomerular anionic sites.

A 73-year-old Japanese man with a history of partial gastrectomy due to gastric cancer 4 years previously was admitted because of intermittent fever. The patient developed abdominal pain, erythema, and myalgia in addition to the fever during the final clinical course, and died of acute heart failure. Autopsy disclosed atrophy of the left lobe of the liver and acute myocardial infarction. Neither metastasis nor recurrence of the cancer was observed. Small- and medium-sized arteries of the visceral organs showed various stages of necrotizing vasculitis with narrowing of the lumina. The vasculitis was most prominent in the left lobe of the liver and in the heart. Narrowing of the portal vein due to portal tract inflammation in addition to vasculitis of the hepatic arteries may have induced ischemia and infarction, which had resulted in atrophy of the left hepatic lobe.

Chronic serum sickness nephritis was induced in rats by sensitization with egg albumin. The glomeruli were then examined by the quick-freezing and deep-etching method with immunohistochemical identification of immune complex deposits on replica membranes. In control rats, the glomerular basement membrane showed a three-layered structure. The middle layer was composed of a compact meshwork of fine fibrils that was connected to adjacent endothelial and epithelial cells by perpendicular fibrils in the inner and outer layers. The mesangial matrix contained a similar but looser meshwork structure. In the nephritic rats, small granular deposits were observed within the meshwork, distorting its fine structure. Larger nodular aggregates extended continuously from the middle layer to the epithelial side. Disruption of the connecting fibrils overlying these larger aggregates was noted. The deposits were stained by antiegg albumin or antirat IgG antibodies. Gold particles immunostaining for the sensitizing antigen were also localized within the deposits. These findings suggest that the deposition of immune complexes occurs in the fibrillary meshworks of the mesangium and lamina densa in this experimental model, with the resultant distortion of their meshwork structures and the formation of nodular aggregates.

Histopathological analysis was performed in the first renal biopsy specimens of patients over and under 10 yrs of IgA nephritis. They were divided clinically into two groups, the one with remission and the other with prolonged disease state respectively. Increased mesangial sclerosis, frequent occurrence of segmental glomerular lesions and tubulointerstitial change were significantly evident in the group with prolonged disease state. It is suggested that similar glomerular events are progressing in IgA nephritis which is carried over to adult age.

Serum sickness nephritis was induced in male Fisher rats by immunization with egg albumin (EA). Correlations of subepithelial deposits (SD) with size and charge barriers of the glomerular filter were investigated using native (NF) and cationized (CF) ferritin as tracer probes. In proteinuric animals large numbers of NF molecules perfused from the abdominal aorta were observed to cross the glomerular basement membrane (GBM) and enter SD. The concentration of NF molecules was higher in GBM segments with SD than in GBM segments without SD, and the concentration of these molecules was higher within SD than in the intervening GBM. In contrast, CF clusters were fewer in number in the lamina rara externa (LRE) of GBM segments with SD than in the GBM segments without SD. CF particles could not be observed within SD, even in the areas of podocyte detachment. It is suggested that permeability in GBM segments with SD increases and that the development of proteinuria in this model can be attributed to alterations in both charge- and size-selective barriers to glomerular filtration.

A rare case of small cell carcinoma (SCC) of the gallbladder combined with adenocarcinoma is reported. The patient was a 70-year-old Japanese man, who died of the disease shortly after the onset of symptoms. Autopsy disclosed a small tumor (1.0 cm in longest diameter) in the fundus of the gallbladder, with widespread metastasis. Histochemically, the tumor cells showed negative reactions for argyrophilic and argentaffin stainings, a weak immunohistochemical reaction only for neuron-specific enolase, and negative reactions for all of the other neurosecretory markers used, including neurofilament, chromogranin, somatostatin, gastrin and leu-7. However, electron microscopic examination revealed a few typical neurosecretory granules (NSG) in the cytoplasm of some tumor cells. We suggest that: 1. The presence of NSG in the cytoplasm of tumor cells is the most reliable diagnostic criterion for SCC. 2. SCC, at least the combined type, arises from a multipotential stem cell.

An autopsy case of diffuse malignant peritoneal mesothelioma in a young woman who showed a high serum level of CA125 is reported. Autopsy revealed extensive tumor involvement of the visceral and parietal peritoneum. The liver, spleen and other abdominal viscera were encased by tumor nodules. Histologically, the polygonal tumor cells were arranged mostly in a sheet-like fashion with a few tubular or papillary forms. No PAS reaction-positive mucin was recognized, but there was a strongly positive colloidal iron reaction. The colloidal iron positivity was effaced after combined treatment with hyaluronidase and sialidase. Immunohistochemically the tumor cells showed strongly positive reactions for CA125, epithelial membrane antigen (EMA) and cytokeratin, weak positivity for carcinoembryonic antigen (CEA) and focal positivity for vimentin. Ultrastructurally, the most characteristic feature was the expression of numerous long microvilli projecting from the tumor cell surfaces and abundant long desmosomes between the tumor cells. We consider that pretreatment using a combination of hyaluronidase and sialidase might be useful for the diagnosis of malignant mesothelioma. CA125 staining should be performed routinely in cases where this tumor is suspected.

The three-dimensional ultrastructure of glomerular sclerosis in the chronic phase of Masugi nephritis was investigated using a quick-freezing and deep-etching method. Newly formed mesangial matrix, which was increased in the axial portions, was composed of fine fibrillar networks similar to those in the lamina densa of the basement membrane. These fibrils were 10-20 nm in diameter and directly attached to the cell membranes of mesangial cells, endothelial cells and podocytes by connecting fibrils. Moreover, thicker fibrils with diameters of 20-30 nm were present in the networks and were connected with cross-bridges. A newly formed matrix of fine fibrillar networks was also seen in the areas of mesangial interposition in the glomerular capillary wall. The border between the matrix and lamina densa was unclear. The fibrils organizing the networks of lamina densa of the glomerular loop were thickened, with some decoration. Connecting fibrils were disrupted in the areas of endothelial detachment. It is suggested that prolonged tissue injury with endothelial detachment might induce mesangial sclerosis composed of fine fibrillar networks. The increase in density of the networks seemingly interfere with the contractile function of mesangial cells, which is followed by alteration of mesangial flow.

The method for the analysis of anileridine, levorphanol, nalbuphine and ethamivan in urine by means of GC/NPD and GC/MSD is described. TFAA and MSTFA-MBTFA have been used in this procedure for TFA and TMS derivatization. The parent forms and the metabolites of the four drugs can be found by GC/NPD screening and GC/MSD confirmation. The method is reliable, fast and sensitive.

The analytical method for doping control analysis has been studied. Twenty-nine drugs including eighteen narcotic analgesics, nine beta-blockers, one stimulant and one internal standard were analysed simultaneously by gas chromatograph equipped with a nitrogen-phosphorous detector and gas chromatograph combined with a mass selective detector after derivatization of the drugs.

Alterations in glomerular basement membrane (GBM) anionic sites associated with immune deposits (ID) were observed using polyethyleneimine (PEI) as a cationic probe in serum sickness nephritis induced by egg albumin (EA). The anionic sites were fewer in number than in other GBM segments and were irregular in distribution in most, but not all, of the segments of the GBM with ID on the epithelial side of the lamina densa (LD). The disappearance of anionic sites was obvious in areas where the internal aspects of the lamina rara externa (LRE) of the GBM were occupied by ID, even if the ID were very small. In contrast, the disappearance of anionic sites was not evident, even though no change in anionic sites was found in some areas, where the ID had departed from the internal aspects of the LRE and a pale band was seen between the ID and the LD. Further, PEI aggregates, showing localization of anionic sites, were seen within the low density ID, but no PEI aggregates were seen within the high density ID. The results suggest that: 1) whether or not ID induce the disappearance of anionic sites is independent of the size of the ID, but is dependent on the density of and the place occupied by the ID, and 2) the ID seem to induce the disappearance of anionic sites by masking rather than destroying them.

A method for the separation and identification of 13 beta-blockers using GC/MS and GC/NPD are described. The TFA and TMS derivatives were prepared by adding TFAA, MSTFA and MBTFA to the standard drugs. The mixture was heated at 65 degrees C for 20 min and chromatographed on FS capillary columns (HP-5). Preliminary results show that the derivatization with MSTFA is better than that with TFAA or MBTFA. There is not much difference between TFAA and MBTFA derivatization. The method is reliable, rapid and simple and may be used to screen these drugs and further applied to the analysis of biological fluids such as plasma and urine.

Serum sickness nephritis was induced in Fisher rats by immunization with egg albumin (EA) and correlations between immune complex deposition, alterations of podocytes and development of proteinuria were analysed. Immunoelectron microscopy showed that EA, rat IgG and C3 were confined to the electron-dense deposits (Ds). From 3 weeks, when significant proteinuria had developed, the subepithelial region was filled with large numbers of Ds on the peripheral capillary wall as well as in the paramesangium. The loss of slit diaphragms and detachment of foot processes overlying Ds were observed and the escape of Ds into urinary space was frequently detected. Morphometric evaluation showed that the volume of subepithelial Ds and the number of the sites of podocytic detachment correlate significantly with the amount of proteinuria. In addition, the native ferritin injected via the abdominal aorta was seen in large amounts in the urinary space near the areas devoid of epithelial covering. The development of podocytic detachment was clearly coincident with the appearance of proteins with a larger molecular weight in urine. From these results, it is suggested that the loss of slit diaphragms and the detachment of podocytes resulting from the progressive accumulation of Ds will allow the leakage of proteins of larger molecular weight across the capillary wall. These podocytic lesions may be one of the main aetiologies for the development of heavy proteinuria in this model.