Dual Gas Treatment With Hydrogen and Carbon Monoxide Attenuates Oxidative Stress and Protects From Renal Ischemia-Reperfusion Injury.

BACKGROUND: Hydrogen (H2) and carbon monoxide (CO) gas are both reported to reduce reactive oxygen species and alleviate tissue ischemia-reperfusion (I-R) injury. The present study was conducted to evaluate the effects of a mixture of H2 gas and CO gas (dual gas) in comparison with hydrogen gas (H2: 2%) alone on I-R renal injury (composition of dual gas; N2: 77.8%; O2: 20.9%; H2: 1.30%; CO: 250 parts per million). METHODS: Adult male Sprague-Dawley rats (body weight 250-280 g) were divided into 5 groups: (1) sham operation control, (2) dual gas inhalation (dual treatment) without I-R treatment, (3) I-R renal injury, (4) H2 gas alone inhalation (H2 treatment) with I-R renal injury, and (5) dual treatment with I-R renal injury. I-R renal injury was induced by clamping the left renal artery and vein for 45 minutes followed by reperfusion, and then contralateral nephrectomy was performed 2 weeks later. Renal function was markedly decreased at 24 hours after reperfusion, and thereafter the effects of dual gas were assessed by histologic examination and determination of the superoxide radical, together with functional and molecular analyses. RESULTS: Pathologic examination of the kidney of I-R rats revealed severe renal damage. Importantly, cytoprotective effects of the dual treatment in comparison with H2 treatment and I-R renal injury were observed in terms of superoxide radical scavenging activity and histochemical features. Rats given dual treatment and I-R renal injury showed significant decreases in blood urea nitrogen. Increased expression of several inflammatory cytokines (tumor necrosis factor-α, interleukin-6, intracellular adhesion molecule-1, nuclear factor-κB, hypoxia inducible factor-1α, and heme oxygenase-1) was attenuated by the dual treatment. CONCLUSIONS: Dual gas inhalation decreases oxidative stress and markedly improves I-R-induced renal injury.

The influence of growth hormone/insulin-like growth factor deficiency on prostatic dysplasia in pbARR2-Cre, PTEN knockout mice.

BACKGROUND: Elevated insulin-like growth factor-I (IGF-I) serum levels and phosphatase and tensin homolog (PTEN) loss are prostate cancer (PCa) risk factors that enhance androgen-responsive and castration-resistant PCa xenografts growth. METHODS: The impact of suppressed growth hormone (GH)/IGF-I levels on neoplastic initiation of PTEN-deficient prostate epithelia was assessed histologically and by epithelial-to-mesenchymal marker expression in Ghrhr D60G homozygous (lit/lit) and heterozygous (lit/+) pbARR2-Cre, PTEN(fl/fl) (PTEN-/-) mice. How suppressed GH/IGF-I levels impacted growth of PTEN-/- mouse-derived prostate cells (MPPK) was examined by growth and survival signaling of cells cultured in lit/+ or lit/lit serum. RESULTS: Body weight, prostate weight and serum GH and IGF-I levels were reduced in lit/lit relative to lit/+ PTEN-/- littermates. While the anterior lobes of lit/+ PTEN-/- prostates consistently presented swollen, indicative of ductal blockage, the degree of prostatic dysplasia in 15- and 20-week-old lit/lit and lit/+ PTEN-/- mice was indistinguishable as measured by normalized prostatic weight, tissue histology, or probasin, PSP94, E-cadherin, N-cadherin and vimentin expression. However, growth and AKT activation of MPPK cells was decreased when cultured in lit/lit serum as compared with lit/+ serum and restored in lit/lit serum supplemented with IGF-I and, to a lesser extent, GH. CONCLUSIONS: These results suggest that initiation of prostate carcinogenesis by loss of PTEN is not influenced by germline variation of genes encoding signaling molecules in the GH/IGF-I axis, but suggests that these factors may affect the progression of dysplastic phenotype and supports previous studies, indicating that the GH/IGF milieu does impact the growth of PTEN-deficient dysplastic prostatic cells once transformed.

Esophageal hernia in dementia: surgeon's role for mixed-type esophageal hernia in an elderly woman with dementia.

The mixed-type esophageal hernia is an indication for operation to prevent stomach volvulus and perforation. However, preventive operation is meaningful depending on the physical status. We encountered an 84-year-old, demented, bed-ridden woman of mixed-type esophageal hernia complicated with severe reflux esophagitis. First, the patient was conservatively treated by intravenous hyperalimentation and H2 blocker but, with onset of delirium, she removed the venous route twice. Subsequently, she was tightly restrained to the bed to avoid removing the line. Ethical deliberation for the patient tightly fixed to the bed and intravenous alimentation for her life prompted us to reconsider hernia operation after discussion with surrogate decision makers. The patient recovered uneventfully after operation, and movement without intravenous route or without any restraints was maintained by oral feeding assisted by gastrostomy feeding. In the coming decade, when senior patients are expected to increase, such operations can be forwarded to respect the patients' quality of life.

Tie-2 and angiopoietin-1 expression in human thyroid tumors.

Tie-2 is an endothelial cell-specific receptor tyrosine kinase involved in vascular maturation and remodeling. Although its expression is considered to be restricted to vascular endothelial cells and hematopoietic progenitors, our immunohistochemical and in situ hybridization studies showed that Tie-2 and its ligand, angiopoietin (Ang)-l were expressed not only in benign and malignant human thyroid tumor cells but also in hyperplastic regions of adenomatous goiter. To confirm the expression in these tissues further, we used a laser capture microdissection system to isolate epithelial tumor cells from tissue specimens selectively, and demonstrated the expression of Tie-2 and Ang-1 mRNAs in tumor cells by RT-PCR analysis. Furthermore, Tie-2 and Ang-1 mRNAs and proteins were also detected in rat thyroid cell lines, FRTL-5 and PCCL-3. Our results suggest that Ang-1/Tie-2 signaling may be involved in the proliferation of thyroid epithelial cells.

Mutational analysis of the BMP-1 gene in patients with gastroschisis.

BACKGROUND: Gastroschisis is a rare abdominal wall defect. Although the pathogenesis of gastroschisis is unknown, there is some evidence of the genetic etiology of gastroschisis. Recently, a functionally null deletion of the mouse bone morphogenic protein-1 (BMP-1) gene resulted in a phenotype that resembled a human neonate with gastroschisis. BMP-1 thus became the first potential candidate gene for gastroschisis. METHODS: To explore this possibility the authors collected blood samples from 11 patients who had gastroschisis. Mutational analysis of exons 2 to 15 of the human BMP-1 gene was performed using genomic polymerase chain reaction, single-strand conformation polymorphism analysis and direct sequencing methods. RESULTS: No mutation of the human BMP-1 gene was observed in any of these patients. CONCLUSION: Although heterogeneous etiologies might be proposed for gastroschisis, our results provide further evidence of a nongenetic etiology for gastroschisis. J Pediatr Surg 36:885-887.

Metastases to the penis from carcinoma of the prostate.

A 58-year-old man presented with dysuria at the Osaka Medical College Hospital in November 1996. Laboratory examination revealed elevated serum prostate-specific antigen (PSA) to > 100 ng/mL. Adenocarcinoma of the prostate with metastasis to the bone was diagnosed after a biopsy of the prostate and bone scintigraphy; hormonal therapy was administered. Although bone metastasis was well controlled and the serum PSA level declined to within normal levels (2.0 ng/mL), several painless nodules were found on the penile glans. Biopsy of the nodules showed that the penile tumor was a metastasis from the prostate cancer. The patient underwent partial penectomy for relief from penile pain. The serum PSA level showed no elevation 3 months after the partial penectomy, suggesting that careful observation of prostate cancer patients is necessary, even when oseous metastasis is well controlled and serum PSA levels are kept within normal ranges by hormonal therapy. The case also indicates that urologists should consider the possibility of metastasis to the penis from prostate cancer.

Post-translational proteolytic processing of procollagen C-terminal proteinase enhancer releases a metalloproteinase inhibitor.

Activity of matrix metalloproteinases (MMP) is regulated by a family of proteins called tissue inhibitors of metalloproteinases (TIMP). Four TIMPs have been cloned, and their molecular weights range from 29,000 to 20,000. By reverse zymography, we have observed a metalloproteinase inhibitor with an apparent molecular weight of 16, 500 from medium conditioned by human brain tumor cells. Antibodies directed against TIMPs failed to react with the 16,500 molecular weight inhibitor, indicating that it was not a truncated form of a known TIMP. The inhibitor was isolated from conditioned medium using affinity and ion exchange chromatography. N-terminal sequences of the inhibitor matched amino acid sequences within the C-terminal domain of a protein known as procollagen C-terminal proteinase enhancer (PCPE). Thus, the inhibitor was named CT-PCPE. Comparison of the N-terminal domain of TIMP with CT-PCPE revealed that both contained six cysteine residues. As in the case of TIMP, reduction and alkylation abolished the inhibitory activity of CT-PCPE. Purified CT-PCPE inhibited MMP-2 with an IC(50) value much greater than that of TIMP-2. This implies that MMPs may not be the physiologic targets for CT-PCPE inhibition. However, these results suggest that CT-PCPE may constitute a new class of metalloproteinase inhibitor.

Structural organization and expression patterns of the human and mouse genes for the type I procollagen COOH-terminal proteinase enhancer protein.

The procollagen C-proteinase enhancer (PCPE) is a glycoprotein that potentiates enzymatic cleavage of the type I procollagen C-propeptide by bone morphogenetic protein-1 (BMP-1). The human PCPE gene (PCOLCE) was previously mapped to 7q22, an area frequently disrupted in uterine leiomyomata, while disruption of the rat PCPE gene leads to anchorage-independent growth and loss of contact inhibition in rat fibroblasts. Here we describe the entire intron/exon organizations of PCOLCE and the mouse PCPE gene (Pcolce) and analyze expression of PCOLCE RNA in various human adult and fetal tissues and of Pcolce RNA at various stages of mouse development. PCOLCE and Pcolce are shown to be small genes 6.0 and 6.5 kb, respectively, with a conserved intron/exon structure comprising 9 exons. A notable difference between the two genes derives from insertion of multiple Alu sequences immediately upstream and downstream and within PCOLCE. Temporal expression of PCPE mRNA is shown to differ from that of BMP-1 and type I procollagen during mouse development, consistent with possible additional functions for PCPE beyond enhancement of C-proteinase activity. Consistent with a possible role in leiomyomata, PCOLCE is shown to be expressed at relatively high levels in uterus.

Changes in cyclin B during oocyte maturation and early embryonic cell cycle in the newt, Cynops pyrrhogaster: requirement of germinal vesicle for MPF activation.

When full-grown oocytes of the newt Cynops pyrrhogaster were treated with progesterone in O-R2 solution containing antibiotics, approximately 85% of the oocytes completed meiosis synchronously. Maturation-promoting factor (MPF) activity appeared just before germinal vesicle breakdown (GVBD) and the oocytes maintained high MPF activity throughout metaphase I and metaphase II of meiosis. A slight decrease of MPF activity was observed at the first polar body emission. The distribution of cyclin B1 was investigated with anti-cyclin B1 antibody. No cyclin B1 was found in the oocytes before progesterone treatment. Cyclin B1 appeared in the cortex of animal hemispheres, especially around and inside germinal vesicle just before GVBD. A large amount of cyclin B1 accumulated at metaphase I, approximately half disappeared at the first polar body emission, and then cyclin B1 accumulated again at metaphase II. An inactive form of cdc2 kinase was observed in both the germinal vesicles and the oocyte cytoplasm, while an active form appeared at the M phase. No MPF was observed in the oocytes from which the germinal vesicle had been removed. A cdk7-like molecule was localized in the germinal vesicle, but not in oocyte cytoplasm, indicating that inactive cdc2 kinase associated with cyclin B1 derived from cytoplasm is activated by phosphorylation in the germinal vesicle. The changes in the amount of cyclin B1 were synchronous with the first cell cycle after fertilization. Cyclin B1 was primarily localized in the cortex of the animal hemisphere. A shift in band mobility upon electrophoresis of cyclin B1 was observed from samples taken during the cell cycle; this shift was probably due to the protein's phosphorylation state.

Transforming growth factor-beta regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes.

Transforming growth factor-beta1 (TGF-beta1) induces increased extracellular matrix deposition. Bone morphogenetic protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix deposition; it is the procollagen C-proteinase (PCP) that processes procollagen types I-III, and it may also mediate biosynthetic processing of lysyl oxidase and laminin 5. Here we show that BMP-1 is itself up-regulated by TGF-beta1 and that secreted BMP-1, induced by TGF-beta1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-beta1 elevated levels of BMP-1 mRNA approximately 7-fold and elevated levels of mRNA for mammalian tolloid (mTld), an alternatively spliced product of the BMP1 gene, to a lesser extent. Induction of RNA was dose- and time-dependent and cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-beta1 in MG-63 and other fibrogenic cell cultures, were predominantly in forms in which proregions had been removed to yield activated enzyme. TGF-beta1 treatment also induced procollagen N-proteinase activity in fibrogenic cultures, while expression of the procollagen C-proteinase enhancer (PCPE), a glycoprotein that stimulates PCP activity, was unaffected. In contrast to fibrogenic cells, keratinocytes lacked detectable PCPE under any culture conditions and were induced by TGF-beta1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes.

Effect of LDL-apheresis on the pharmacokinetics of the lipophilic antilipidemic agent probucol.

The effect of LDL-apheresis on the pharmacokinetics of antilipidemic agents has not been evaluated thoroughly. In this study, we investigated the effect of LDL-apheresis on the pharmacokinetics of probucol, a lipophilic antilipidemic agent, by studying its distribution and changes in the blood concentration of probucol after LDL-apheresis. The concentrations of lipoproteins were measured before and after LDL-apheresis in eight patients with familial hypercholesterolemia taking probucol. Concentrations of probucol in the various lipoprotein fractions and plasma were measured by HPLC. The serum concentrations of probucol before and after LDL-apheresis were 39.8 +/- 3.3 and 16.5 +/- 1.6 micrograms/ml, and the correlation coefficient between the changes in the serum probucol concentration and those in the serum cholesterol concentration before and after LDL-apheresis was significant (r = 0.73, P < 0.01). Changes in the probucol and cholesterol concentrations after LDL-apheresis were mainly found in the LDL fraction. The calculated reductions in the serum contents of probucol and cholesterol were similar to the contents of probucol and cholesterol in the irrigation fluid of the dextran sulfate column. These data suggest that changes of probucol concentration in plasma by LDL-apheresis are mainly due to reductions in the LDL fraction.

A translocation interrupts the COL5A1 gene in a patient with Ehlers-Danlos syndrome and hypomelanosis of Ito.

Ehlers-Danlos syndrome (EDS) is a genetically and pathogenetically heterogeneous group of disorders of which at least 11 types have been described. All are connective tissue disorders characterized by defects of the skin, ligaments and blood vessels with the clinical spectrum ranging from innocuous findings to lethality. Mutations in the genes encoding the major fibrillar collagen types I and III have been demonstrated in EDS types VII and IV, respectively, while mutations in the lysyl hydroxylase and ATP7A genes, with roles in collagen cross-linking, are responsible for EDS types VI and IX. The biochemical and molecular bases for the most common forms of EDS (types I, II and III) are unknown. Here, we describe a balanced translocation between chromosome 9 and an X chromosome that disrupts the minor fibrillar collagen type V gene COL5A1 in a patient with both EDS type I and hypomelanosis of Ito. The breakpoint occurs at 9q34 within COL5A1 intron 24 and interestingly, within a LINE-1 (L1) element at Xp21.1. A fusion mRNA between COL5A1 and an Alu sequence is produced, but no aberrant protein is detectable. Rather, the amount of type V collagen is reduced in the patient's fibroblasts, suggesting haploinsufficiency as a cuase of the phenotype. This demonstrates that a mutation in a type V collagen gene, COL5A1, results in EDS type I, and shows the involvement of L1 sequences in a constitutional chromosomal translocation. Because collagen type V is a heteromorphic protein in which molecules may be composed of polypeptides encoded by three COL5A genes, this suggests all three genes as candidates for mutations in EDS.

Effects of fibronectin-type V collagen recombinant fusion protein on cell adhesion and cell proliferation.

An expression vector pTF7520-Col-V-In, which encodes a fusion protein of the cell-binding domain of fibronectin (C277) and the insulin- and heparin-binding domain of the alpha 1 chain of human type V collagen, was constructed. E. coli transfected with this plasmid synthesized a 50-kDa fusion protein. This fusion protein, C277-V, was purified from the crude extract by a single step heparin HPLC. Similar amounts of insulin bound to purified C277-V and to the alpha 1 chain of type V collagen as judged by the binding of peroxidase-conjugated insulin. Cell-adhesive activity of C277-V was lower than that of the original fibronectin fragment C274, but similar numbers of cells adhered to both protein substrates when the culture dishes were coated with 1 mM of each protein. Insulin bound to the C277-V substratum stimulated the growth of mouse mammary tumor MTD cells in serum-free culture medium.

Complete primary structure of human collagen alpha 1 (V) chain.

Several cDNA clones, encoding prepropeptide of human collagen alpha 1(V) chain, have been isolated. The prepropeptide (1838 amino acids length) of the alpha 1(V) chain was composed of a putative signal peptide, a large NH2-terminal noncollagenous region, a main collagenous region, and a COOH-terminal noncollagenous region. The signal peptide contained many leucine residues. The NH2-terminal noncollagenous region was much larger than those of the other collagens and had a region homologous to the COOH-terminal domain of laminin A chain, but it did not contain a cysteine-rich region that was maintained in the region of the other collagens. This region also contained probable tyrosine sulfation sites, and short collagenous sequences that were interrupted by three noncollagenous segments. The main collagenous region of the alpha 1(V) chain consisted of 338 repeats of Gly-X-Y-triplet. This region had a high degree (82%) of homology with the amino acids of the collagen alpha 1(XI) chain. The COOH-terminal noncollagenous region resembled that of the alpha 1(XI) chain, too, and 8 residues of cysteine that were important for the formation of the triple helix structure of collagens were observed. These results suggest that the alpha 1(V) chain belongs to the fibrillar collagen relative to the alpha 1(XI) chain, but codon usage of the alpha 1(V) cDNA was clearly different from those of the other fibrillar collagens including the alpha 1(XI), while it was similar to type IV collagen. This result supposes a different evolution of the alpha 1(V) gene from those of the other fibrillar collagens.

Insulin binds to type V collagen with retention of mitogenic activity.

The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity.

Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human x rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched. Chromosome-specific Southern profiles can be used to monitor chromosomes in hybrid cells or to characterize chromosome aberrations, such as deletions.

Unusual findings of M-mode echocardiogram in a case with left ventricular thrombus.

The unusual echo findings of the left ventricular thrombus by M-mode echocardiogram is reported. The patient was a 41-year-old man with hypokinesis of the inferior and inferolateral wall due to acute myocardial infarction. The findings of M-mode echocardiogram revealed dense thick linear echoes just below the anterior wall of the apex, which had a higher density than the myocardium and looked "band like". These abnormal echoes obtained by M-mode echocardiogram were also detected through examinations by 2-dimensional echocardiogram, computed tomogram and left ventriculogram, and were assumed to arise from the index finger-sized thrombus in the left ventricle at the time of surgical procedure. Although this abnormal echo, revealed to have a "band like" feature by M-mode echocardiogram, is usually seen in the left ventricular myxoma, we assume that this "band like" echo by M-mode echocardiogram arose from the left ventricular thrombus under conditions such as myocardial infarction, ventricular aneurysm, dilated cardiomyopathy and long-term congestive heart failure.

Effects of nifedipine on platelet function.

Effects of nifedipine on platelet aggregation were studied both in vitro and in vivo. From in vitro experiments, nifedipine inhibited platelet aggregation in a dose-dependent manner. The inhibition by nifedipine (final concentration 10 micrograms/ml) on epinephrine-induced and collagen-induced platelet aggregation was more than 90%, greater than that on adenosine diphosphate (ADP)-induced aggregation. The consumption ratio of small platelets (less than or equal to 6.4 fL) was higher than that of large platelets (greater than 6.4 fL), suggesting that nifedipine inhibits the aggregation of large platelets more effectively. Changes in the effects of nifedipine on platelet aggregation associated with exercise were also studied in six healthy volunteers. While platelet aggregability increased after exercise without administration of nifedipine, it was inhibited 90 minutes after the drug's administration (10 mg). The inhibition of collagen-induced and ADP-induced (2 microM) aggregation by nifedipine was particularly significant.

Platelet aggregation in variant angina: its relation to plasma catecholamines.

We concurrently measured plasma catecholamine levels and platelet aggregation in patients with variant angina, effort angina and old myocardial infarction. Platelet aggregability was lower in variant angina than in effort angina and old myocardial infarction. Plasma concentrations of epinephrine and norepinephrine in the variant angina group showed higher values than those in the effort angina group and the old myocardial infarction group. Moreover, there was a statistically significant negative correlation between plasma catecholamine concentration and ADP-induced platelet aggregation. These findings suggest that the lower platelet aggregation is related to the higher concentration of plasma catecholamine in variant angina.