Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin.

AIMS/OBJECTIVE: Nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease. Recent studies have demonstrated that podocyte injury is involved in the onset of and progression to renal insufficiency. Here, we describe a novel, highly sensitive ELISA for detecting urinary podocalyxin, a glycoconjugate on the podocyte apical surface that indicates podocyte injury, particularly in the early phase of diabetic nephropathy. METHODS: Urine samples from patients with glomerular diseases (n = 142) and type 2 diabetes (n = 71) were used to quantify urinary podocalyxin by ELISA. Urine samples were obtained from 69 healthy controls for whom laboratory data were within normal values. Podocalyxin was detected in urine by immunofluorescence, immunoelectron microscopy and western blotting. RESULTS: Morphologically, urinary podocalyxin was present as a vesicular structure; western blotting showed it as a positive band at 165-170 kDa. Levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetes. In patients with diabetes, urinary podocalyxin was higher than the cut-off value in 53.8% patients at the normoalbuminuric stage, 64.7% at the microalbuminuric stage and 66.7% at the macroalbuminuric stage. Positive correlations were observed between urinary podocalyxin levels and HbA(1c), urinary ß(2) microglobulin, α(1) microglobulin and urinary N-acetyl-ß-D-glucosaminidase, although urinary podocalyxin levels were not correlated with other laboratory markers such as blood pressure, lipid level, serum creatinine, estimated GFR or proteinuria. CONCLUSIONS/INTERPRETATION: Urinary podocalyxin may be a useful biomarker for detecting early podocyte injury in patients with diabetes.

Development of a novel high power sub-THz second harmonic gyrotron.

Record-breaking high power coherent radiation at a subterahertz frequency region from a gyrotron utilizing second harmonic resonance modes was attained with a simple cavity. In order to aim at high power and high frequency simultaneously, the oscillation mode was selected carefully enough to realize stable radiation free from mode competition. The cavity radius was determined from the viewpoints of the oscillation frequency, the coupling coefficient between the electron beam, and the rf-electric field. The cavity length was also optimized for the highest perpendicular efficiency. In addition, a new electron gun which is capable of generating a thin laminar beam for a large current was introduced. Consequently, single mode second harmonic radiation with powers of 52 and 37 kW at frequencies of about 349 and 390 GHz, respectively, was achieved.

Aberrant maspin expression in gallbladder epithelium is associated with intestinal metaplasia in patients with cholelithiasis.

OBJECTIVE: Aberrant expression of maspin protein related to DNA hypomethylation in the promoter region is frequently observed in gallbladder carcinomas, whereas the non-tumorous gallbladder epithelium is maspin negative. We investigated maspin expression in non-tumorous gallbladder epithelium in patients with cholelithiasis. METHODS: An immunohistochemical study of maspin expression was performed in 69 patients with cholelithiasis and 30 patients with gastric cancer without cholelithiasis. RESULTS: Immunoreactivity for maspin was observed in focal and patchy regions of the gallbladder epithelium. Positive immunoreactivity for maspin was significantly associated with the presence of intestinal metaplasia in patients with cholelithiasis (p<0.05). CONCLUSION: The high incidence of aberrant maspin expression in both intestinal metaplasia and carcinoma of the gallbladder supports the assumption that intestinal metaplasia of the gallbladder may predispose to gallbladder carcinoma.

Frequent microsatellite alterations on chromosome 3p in esophageal squamous cell carcinoma.

By the microsatellite assay, two types of genetic alterations, loss of heterozygosity (LOH) and replication error (RER), were examined using 7 dinucleotide repeat markers [D3S1317 (3p26); CI3-1169 (3p25); CI3-946 (3p25); D3S1255 (3p24.2-25); CI3-771 (3p21.3); CI3-1413 (3p14.1-14.3); and CI3-373 (3p13)] on the short arm of chromosome 3p as well as 3 markers [D2S123 (2p15-16), IFNA (9p22), and D16S408 (16q12.1-13)] on other chromosomes in 35 patients with esophageal squamous cell carcinoma. On 3p, LOH was detected in 34% (12 of 35) and RER was detected in 60% (21 of 35) at single or multiple loci. RER occurred at a similar frequency in all stages and did not correlate with clinicopathological characteristics. On the other hand, LOH at the 3p25 locus was more frequently detected in carcinomas with lymph node metastasis than in those without it (P < 0.05). The incidences of microsatellite alterations were low on the chromosomes other than 3p, except at D2S123, where the incidence of RER was 20%. These findings suggest that RER on 3p is an early event and that a tumor suppressor gene which is involved in the progression of esophageal squamous cell carcinoma may exist near the 3p25 locus.

Mutations of the APC gene occur during early stages of gastric adenoma development.

Mutations of the adenomatous polyposis coli (APC) gene have recently been shown to play an important role in colorectal tumorigenesis. We investigated mutations of the APC gene in 30 gastric adenomas obtained endoscopically. Mutations of the APC gene were examined by polymerase chain reaction-single-strand conformation polymorphism analysis followed by sequencing of the polymerase chain reaction products. Mutations were detected in 20% (6 of 30) of gastric adenomas. In addition, deletion of the remaining allele that subsequently led to complete inactivation of the APC gene was confirmed in one-half (3 of 6) of the tumors with APC gene mutations. Sequencing analysis confirmed that the mutations resulted in truncation of the gene products or in an amino acid change. The incidences of mutations of the APC gene remained constant regardless of the size or degree of histological atypia. Our observations suggest that mutations of the APC gene, similarly to those in colorectal tumorigenesis, occur during the early stages of gastric adenoma development.

The herbal medicine sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1 phase.

Water-soluble ingredients of the herbal medicine sho-saiko-to dose-dependently inhibited the proliferation of a human hepatocellular carcinoma cell line (KIM-1) and a cholangiocarcinoma cell line (KMC-1). Fifty % effective doses on day 3 of exposure to sho-saiko-to were 353.5 +/- 32.4 micrograms/ml for KIM-1 and 236.3 +/- 26.5 micrograms/ml for KMC-1. However, almost no suppressive effects were detected in normal human peripheral blood lymphocytes or normal rat hepatocytes. Sho-saiko-to suppressed the proliferation of the carcinoma cell lines significantly more strongly than did each of its major ingredients, i.e., saikosaponin a, c, and d, ginsenoside Rb1 and Rg1, glycyrrhizin, baicalin, baicalein, and wogonin, or another herbal medicine, juzen-taiho-to (P < 0.05 or 0.005). Because such ingredients are barely soluble in water, there could be synergistic or additive effects of the ingredients in sho-saiko-to. Morphological, DNA, and cell cycle analyses revealed two possible modes of action of sho-saiko-to to suppress the proliferation of carcinoma cells; (a) it induces apoptosis in the early period of exposure and (b) it induces arrest at the G0/G1 phase in the late period of exposure.

Inactivation of the CDKN2 gene by homozygous deletion and de novo methylation is associated with advanced stage esophageal squamous cell carcinoma.

We examined the genomic status of the CDKN2 gene including de novo methylation of 5' CpG islands in primary and metastatic tumor samples from 31 patients with esophageal squamous cell carcinoma. One somatic frame shift mutation (1 of 31; 3.2%) was identified by PCR-single strand conformational polymorphism analysis and DNA sequencing. Homozygous deletion and de novo methylation of the gene were confirmed in 5 (16%) and 6 (19%) of 31 patients, respectively. Homozygous deletion and de novo methylation were significantly associated with silencing of gene expression (P < 0.01). Aberrations of the CDKN2 gene were detected in tumors with lymph node metastasis and muscular invasion (12 of 22; 54%) and in none of stage I tumors (0 of 9.0%; P < 0.05). These results suggest that homozygous deletion and de novo methylation are predominant mechanisms of inactivation of the CDKN2 gene and may be associated with metastatic and invasive phenotypes of esophageal squamous cell carcinoma.

Two distinct regions of deletion on the long arm of chromosome 5 in differentiated adenocarcinomas of the stomach.

Frequent loss of heterozygosity (LOH) on the long arm of chromosome 5 (5q) has been reported in many types of human malignancies, including gastric carcinoma. One of the targets of 5q-LOH in colorectal carcinoma is certainly the adenomatous polyposis coli (APC) gene on 5q21. However, other evidence has suggested the presence of another tumor suppressor gene in this region which may be inactivated in gastric carcinoma. In the present study, to determine the location of the putative tumor suppressor gene on 5q, LOH at nine microsatellite loci on 5q were investigated at 38 differentiated adenocarcinomas of the stomach that probably did not carry APC mutations. LOH at any locus on 5q occurred in 37% (14 of 38) of the tumors. Although many tumors exhibited large interstitial deletions on 5q that included the APC locus (5q21), we have identified minimum regions of deletion as the D5S428 locus and the interferon regulatory factor-1 (IRF-1) locus. Thus, at least two putative tumor suppressor genes, which play a crucial role in the genesis of differentiated adenocarcinoma of the stomach and are distinct from the APC gene, lie on 5q.

Effects of diltiazem and long-term beta 1-adrenergic blockade on hemodynamics and blood flow during exercise in patients with stable angina pectoris.

The short-term effects of oral diltiazem on hemodynamics and distribution of cardiac output at rest and during semiupright bicycle exercise were evaluated in eight patients with stable effort angina on long-term beta 1-adrenergic blockade. Cardiac output and iliofemoral blood flow were measured using thermodilution. The patients were exercised to the same work load on a bicycle before and 2 h after oral diltiazem (60 mg in two patients and 120 mg in six). At maximal exercise, diltiazem reduced heart rate from 94 +/- 5 to 88 +/- 6 beats/min (p less than 0.01), mean arterial pressure from 139 +/- 5 to 127 +/- 4 mm Hg (p less than 0.01) and systemic vascular resistance from 9.7 +/- 0.7 to 8.4 +/- 0.4 x 10(2) dynes.s.cm-5 (p less than 0.05) compared with control. During exercise, cardiac output, iliofemoral blood flow, mean pulmonary wedge pressure and mean right atrial pressure were not altered, but stroke volume increased from 119 +/- 11 to 131 +/- 10 ml (p less than 0.05). Maximal ST segment depression during exercise was decreased and angina was less. Diltiazem does not alter the distribution of the cardiac output during exercise but improves ischemia.

Loss of heterozygosity at the DCC gene locus is not crucial for the acquisition of metastatic potential in oesophageal squamous cell carcinoma.

Tumour specimens from 111 patients with oesophageal squamous cell carcinoma were screened for loss of heterozygosity (LOH) at the deleted colorectal carcinoma (DCC) gene locus. DCC-LOH occurred in 10 of 61 informative cases (16%). No statistically significant correlation was observed between DCC-LOH and lymph node metastasis, histopathological grade or tumour stage. The survival of patients exhibiting DCC-LOH was not statistically different from that of patients without LOH. These results suggest that LOH at the DCC locus is not related to the acquisition of metastatic potential or the state of tumour cell differentiation in oesophageal squamous cell carcinoma.

ST monitoring for myocardial ischemia during and after coronary angioplasty.

We performed 12-lead electrocardiographic monitoring in 97 patients during coronary angioplasty (PTCA) of a single vessel to correlate ischemic ST changes with clinical, angiographic and coronary hemodynamic variables and to determine the optimum lead or combination of leads for their detection. Ischemia (chest pain or ST change, group A) occurred in 79 patients (80%), but in only 15 of 23 patients (65%) with collaterals (p less than 0.05). Ischemia occurred more often in left anterior descending and left circumflex PTCA than right coronary PTCA, but pain was the only manifestation more often in left circumflex and right coronary PTCA. Ischemic ST change was silent in 16% and this proportion did not differ in clinical or angiographic groups except for diabetes with 3 of 5 (60%) having silent ischemia (p less than 0.05). Patients in group A (ischemia) compared to group B (no ischemia) had less severe lesions (85 +/- 9 vs 91 +/- 7%, p less than 0.01), higher transstenotic gradients (62 +/- 19 vs 53 +/- 9 mm Hg, p less than 0.05) and lower distal occluded pressures (24 +/- 11 vs 33 +/- 10 mm Hg, p less than 0.01), suggesting less collateral flow. Compared with a 12-lead electrocardiogram, the best single lead for detecting ST change during PTCA in each artery had a sensitivity of 80% and this increased to 93% using the best 2 leads. The best 3 leads (V3/III/V5 for left anterior descending and III/V2/V5 for right coronary and left circumflex) increased sensitivity to 100%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of nisoldipine on systemic and leg blood flow, oxygen transport and metabolism, and hemodynamics during exercise in effort angina pectoris.

The acute effects of 10 mg of oral nisoldipine on hemodynamics, oxygen transport and metabolism, and distribution of cardiac output, at rest and during semiupright bicycle exercise, were evaluated in 10 men with effort angina receiving long-term beta 1 blockade. Cardiac output and leg blood flow were measured using the thermodilution technique. At rest, nisoldipine decreased systemic resistance from 18.9 +/- 1.0 to 15.9 +/- 1.2 dynes.s.cm-5.10(2) (p less than 0.05) and cardiac output increased from 4.8 +/- 0.2 to 5.3 +/- 0.3 liters/min (p less than 0.05) without changing leg blood flow. During maximal exercise with nisoldipine, systemic resistance was reduced (10.6 +/- 0.9 to 8.6 +/- 0.5 dynes.s.cm-5.10(2), p less than 0.05) and cardiac output increased 18% (10.3 +/- 0.7 to 12.2 +/- 0.6 liters/min, p less than 0.05) when compared with control values. Exercise heart rate was higher with nisoldipine (113 +/- 4 vs 106 +/- 4 beats/min, p less than 0.01), but the mean arterial pressure was not significantly changed, giving a higher rate-pressure product. The increase in mean pulmonary artery wedge pressure was attenuated (26 +/- 3 vs 30 +/- 3 mm Hg during control exercise, p less than 0.05), but ST depression was unaltered. Exercise leg flow was reduced by nisoldipine from 4.3 +/- 0.4 to 3.9 +/- 0.3 liters/min (p = 0.07) and the proportion of cardiac output distributed to the legs was reduced from 42 +/- 3 to 33 +/- 3% (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and function of interleukin-8 in human hepatocellular carcinoma.

Expression and functions of interleukin (IL)-8, a pro-inflammatory cytokine with angiogenesis action, was examined in 23 surgically resected hepatocellular carcinoma (HCC) specimens and 7 HCC cell lines. In all HCC tissues, IL-8 expression was confirmed with reverse-transcription polymerase chain reaction method and enzyme-linked immunosorbent assay, and immunohistochemistry showed HCC cells were the major producer of IL-8 in the tissues. Microvessel density was measured by the double immunohistochemical staining of muscular vessels in HCC tissues, but the density was not related to the level of IL-8 in the HCC tissues. On the other hand, in the co-culture of human umbilical vein endothelial cells (HUVEC) and a HCC cell line (KIM-1), IL-8 produced by KIM-1 significantly accelerated the proliferation of HUVEC. In addition, cases with a high IL-8 level in cancerous tissue had a significantly higher frequency of portal vein invasion, venous invasion and bile duct invasion (p<0.05). In the cultures of 7 HCC cell lines IL-8 secretion into culture medium increased with the treatment of IL-1beta or tumor necrosis factor-alpha. This showed IL-8 expression is regulated by inflammatory cytokines. IL-8 produced by HCC is an angiogenesis factor of HCC, but it could have a much more important role in the invasion and metastasis of HCC.

An alpha-fetoprotein-producing human gallbladder carcinoma cell line (KMG-C) shows more aggressive biologic behavior than an alpha-fetoprotein-non-producing cell line.

We have established a new human alphafetoprotein (AFP)-producing gallbladder carcinoma (GBC) cell line, termed KMG-C, from a 67-year-old Japanese male. KMG-C and its reconstituted tumors in nude mice showed the morphological features of an adenocarcinoma. Functionally, KMG-C secreted AFP, carcinoembryonic antigen, and carbohydrate antigen 19-9, as well as 7 serum proteins, including albumin and C-reactive protein. KMG-C showed more malignant biological behavior than an AFP-nonproducing GBC cell line, KMG-A, established originally from the tumor of the same patient; KMG-C had a shorter doubling time, higher tumorigenicity, and an aneuploid DNA index. Our results suggest that AFP-producing GBC cells may have more malignant biological characteristics than AFP-non-producing GBC cells in GBCs having both of these components.

Deletion type hepatocyte growth factor has different effects on growth and c-met expression in 10 different human hepatocellular carcinoma cell lines.

We examined expression of c-met protein, and the mitogenic and morphologic effects of deletion type hepatocyte growth factor (dHGF) by using 10 human hepatocellular carcinoma (HCC) cell lines having different morphologic and biologic features. c-met protein was detected at varying levels in all cells, regardless of the histological grades. Among the 7 lines expressing c-met at high levels, mitogenic effects of dHGF were stimulative for 2 lines; suppressive for 3 lines; and not distinguishable for the other 2 lines. Furthermore, mitogenic effects of dHGF were different in two clonally related cell lines, having different morphologic and biologic features, even though expression of c-met protein was comparable. dHGF induced scattering of cells and morphologic changes in two lines with suppressing and unaffected growth. In the 3 lines expressing c-met at relatively low levels, no remarkable mitogenic or morphogenic effects were detected. These results suggest that the expression levels of c-met protein were not related to the differentiation levels of HCC cells, and dHGF may cause different biological effects on the cells with almost identical c-met protein expression.

Subsite preferences of pepstatin-insensitive carboxyl proteinases from prokaryotes: kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase.

Kumamolysin, a carboxyl proteinase from Bacillus novosp. MN-32, is characterized by its thermostability and insensitivity to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidated using two series of synthetic chromogenic substrates: P(5)-P(4)-P(3)-P(2)-Phe*Nph (p-nitrophenylalanine: *cleavage site)-P(2)'-P(3)', in which the amino acid residues at the P(5)-P(2), P(2)' and P(3)' positions were systematically substituted. Among 74 substrates, kumamolysin was shown to hydrolyze Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this peptide were K(m) = 41+/-5 microM, k(cat) = 176+/- 10 s(-1), and k(cat)/K(m) = 4.3+/-0.6 mM(-1) x s(-1). These systematic analyses revealed the following features: (i) Kumamolysin had a unique preference for the P(2) position. Kumamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P(2) position; this was also observed for the pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al. (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomonas sp. 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas sp. T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P(2) position. (ii) Kumamolysin preferred such charged amino acid residues as Glu or Arg at the P(2)' position, suggesting that the S(2)' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4. In general, the S(2)' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydrophilic nature of the S(2)' subsite was confirmed to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases from prokaryotes.

Lack of mutations of the adenomatous polyposis coli gene in oesophageal and gastric carcinomas.

The adenomatous polyposis coli (APC) gene is the target of the loss of chromosome 5q heterozygosity observed frequently in gastrointestinal tract carcinomas and is inactivated in these carcinomas. We screened 94 gastrointestinal tract carcinomas for APC mutations, by polymerase chain reaction single-strand conformation polymorphism (SSCP) analysis. Mutations were detected in 8 of 21 (38%) colorectal carcinomas in the mutation cluster region of the APC gene whereas no mutation was detected in any of 49 oesophageal and 24 gastric carcinomas, even though SSCP analysis was extended to include the 5' half of the APC gene exon 15. Direct DNA sequencing revealed that six of eight (75%) mutations in colorectal carcinomas resulted in truncated gene products. These findings confirm the significance of APC gene mutations in colorectal, but not oesophageal or gastric carcinomas. Some other tumour suppressor genes near the APC gene may be the target of the frequent allelic loss of chromosome 5q in oesophageal and gastric carcinomas.

Near-infrared estimation of O2 supply and consumption in forearm muscles working at varying intensity.

We estimated a blood flow index, O2 supply index, and O2 consumption index from near-infrared (NIR) signals during venous occlusion imposed at rest and immediately after handgrip exercise with loads equal to 5, 10, 15, 20, 25, and 30% of the maximum voluntary contraction. We also estimated forearm blood flow (BFfa) by strain-gauge plethysmography and forearm O2 consumption (VO2fa) by the invasive method. There was a significant correlation between the rate of increase in total hemoglobin during venous occlusion obtained from NIR signals and BFfa in each subject (r = 0.853 approximately 0.981, P < 0.001). There was also a significant correlation (r = 0.854 approximately 0.944, P < 0.001) between the O2 consumption index estimated from NIR signals and VO2fa. The mean values for O2 supply index in five subjects increased with exercise intensity, while the O2 consumption index showed no further increase about 25% of maximum voluntary contraction. We found significant positive correlations between the O2 supply index and BFfa (r = 0.986, P < 0.001) and the O2 consumption index and VO2fa (r = 0.976, P < 0.001) during exercise at 5-30% of maximum voluntary contraction. These results demonstrate that analysis of NIR signals during venous occlusion provides an advantageous method of estimation of O2 supply and consumption in working muscles during exercise of varying intensity.

Antagonism of the emetic action of xylazine by alpha-adrenoceptor blocking agents.

The intramuscular injection of xylazine (2 mg/kg) evoked vomiting in 81% of the dogs studied. Adrenoceptor antagonists showing alpha 2-blocking activity, yohimbine, tolazoline and phentolamine, antagonized the xylazine-induced vomiting in a dose-dependent manner. Of these antagonists, yohimbine was the most effective, since the maximal antagonistic effect was seen at 0.5 mg/kg yohimbine, a dose at which the other drugs had less or no effect. The adrenoceptor antagonists showing alpha 1-blocking activity, prazosin and phenoxybenzamine, at the doses studied did not prevent the emesis induced by xylazine. A beta-adrenoceptor antagonists, propranolol, was ineffective in reducing xylazine-induced vomiting. The dopamine receptor antagonists, metoclopramide and domperidone, did not prevent xylazine-induced vomiting nor did yohimbine antagonize apomorphine-induced vomiting. The xylazine-induced vomiting was not prevented by atropine, naloxone or hexamethonium. These results indicate that the xylazine-induced vomiting is mediated by alpha 2-adrenoceptors and does not appear to involve beta-adrenoceptors, cholinoceptors, dopamine or opiate receptors in the emetic pathway.

Central alpha-adrenoceptor subtypes involved in the emetic pathway in cats.

The intracerebroventricular (i.c.v.) injection of clonidine, xylazine, adrenaline and methoxamine elicited dose-dependent vomiting in cats in that order of potency. The vomiting induced by clonidine, xylazine and adrenaline was antagonized by i.c.v. yohimbine and phentolamine possessing alpha 2-adrenoceptor-blocking activity, but not by prazosin showing alpha 1-adrenoceptor-blocking activity. In contrast, methoxamine-induced vomiting was antagonized by prazosin, but not by yohimbine. The vomiting induced by xylazine and adrenaline was not prevented by i.c.v. 6-hydroxydopamine treatment, but was prevented by i.c.v. reserpine treatment. Ablation of the area postrema with some damage to extremely adjacent areas abolished the vomiting induced by each alpha-adrenoceptor agonist. These results indicate that both central alpha 1- and alpha 2-adrenoceptors are involved in the emetic pathway in cats, although alpha 2-adrenoceptors seem to have the main role. It is also suggested that monoamines, and in particular 5-hydroxytryptamine in the brain, are involved in the regulation of alpha-adrenoceptor-mediated vomiting.