Matrix induced re-differentiation of cultured rat hepatocytes and changes of CCAAT/enhancer binding proteins

Rat hepatocytes de-differentiate and proliferate when cultured on collagen-coated dishes in a chemically defined Hepatocyte Growth Medium in the presence of hepatocyte growth factor and epidermal growth factor. The addition of biomatrix derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma stops this process and leads to re-differentiation of the cells. We monitored DNA binding activity and protein levels of CCAAT/Enhancer Binding Proteins (C/EBPs) during these events by electrophoretic mobility shift assays and western blot analysis. We used plasma protein gene expression as a marker for the proliferation and differentiation phases. During the initial proliferation phase the DNA binding activity of C/EBPs decreased about 5-10 fold, mainly due to reduction of C/EBP alpha protein to nearly undetectable levels. Addition of EHS-gel prevented the further loss of C/EBP alpha protein and established a new steady state level. Since C/EBP beta proteins were affected to a much lesser extent, the C/EBP alpha:C/EBP beta ratio was greater in the presence of EHS-gel. Transferrin, alpha 1-antitrypsin, and albumin mRNA expression increased substantially. Thus stabilized C/EBP alpha expression, an increased C/EBP alpha:C/EBP beta ratio, and increased expression of liver specific mRNAs all correlated with the transition of proliferative to differentiated cells.     

The effect of asphyxia on the pharmacokinetics of ceftazidime in the term newborn

The multiple-dose pharmacokinetics of ceftazidime (CAZ) (administered twice daily in a 50 mg/kg of body weight i.v. dose) were studied in 10 severely asphyxiated term infants with suspected septicemia on d 3 of life. Nine term infants with suspected septicemia but without asphyxia served as controls. Blood samples were collected from an arterial catheter at 0, 0.5, 1, 2, 4, 8, and 12 h after an i.v. bolus injection. A high performance liquid chromatography method was used to determine CAZ concentrations from serum. CAZ pharmacokinetics followed a one-compartment open model. The GFRs of all infants were simultaneously studied by means of the 24-h continuous inulin infusion technique. Elimination serum half-life (5.86 +/- 1.13 h versus 3.85 +/- 0.40 h) and serum trough concentrations (46 +/- 14 mg/L versus 23 +/- 7 mg/L) of CAZ were significantly (p < 0.001) increased in the asphyxiated newborn, whereas total body clearance of CAZ (128.4 +/- 25.1 mL/h versus 205.7 +/- 55.4 mL/h), CAZ clearance per kg (40.9 +/- 6.1 mL/h/kg versus 60.8 +/- 8.3 mL/h/kg), and the GFR expressed in mL/min (3.14 +/- 0.43 versus 4.73 +/- 0.89) were significantly (p < 0.001) decreased in the asphyxiated newborn. We conclude that twice daily administration of 50 mg/kg of body weight CAZ given to asphyxiated term newborns in the first days of life results in significantly higher serum trough levels in comparison with control infants. The impaired CAZ clearance is a result of a significantly decreased GFR.     

Immediate sexual rehabilitation by simultaneous placement of penile prosthesis in patients undergoing radical prostatectomy: initial results in 50 patients

Hypotonicity-induced anion permeability changes were investigated but not detected in immortalised (RBE4) rat brain endothelial cells using iodide efflux measurements. Large, rapid increases were however observed in primary cultured cells. Both cell types were reinvestigated following culture in a common growth factor-depleted medium. Responses were still undetectable in the immortalised RBE4 cells. Reduced responses were observed in the primary cultured cells that also showed altered morphology and decreased activity of another transporter, P-glycoprotein. Thus both immortalisation and different culture conditions may alter functional expression in these cells of transporters involved in hypotonicity-induced anion permeability changes.     

Interphase cytogenetics of prostatic tumor progression: specific chromosomal abnormalities are involved in metastasis to the bone

Only limited data are available on chromosomes specifically involved in the multistep tumorigenesis of prostate cancer. To investigate the cytogenetic status at different stages of prostatic tumor development, we have applied interphase in situ hybridization (ISH) with a set of (peri) centromeric DNA probes--specific for chromosomes 1, 7, 8, and Y--to routinely processed tissue sections of prostatic specimens from 75 different individuals. Our panel consisted of: 16 normal/benign prostatic hyperplasia specimens; 23 primary, localized, prostatic tumors (N0M0 stage); 20 regional lymph node metastases (M0 stage); and 16 distant metastases. Numerical aberrations of at least one chromosome were not observed in normal/benign prostatic hyperplasia cases, but were present in localized tumors (39%), regional lymph node metastases (40%), and distant metastases (69%). Within the different pTNM groups, we observed the following aberrations (listed, within each series, in decreasing order of frequency): -Y, +8, -8, +7 in primary tumors; +8, +7, -Y, +Y, -8 in regional lymph node metastases; and +8, +7, +1, -Y, -8 in distant metastases. In primary tumors, the number of aberrant cases increased significantly with local tumor stage (p < 0.05). A significant increase in gain of chromosome 8 was also observed (p < 0.02). Gain of chromosome 7 and/or 8 showed a significant increase with progression of local tumor stage (p < 0.02). Specific involvement of chromosome 8 was seen in bone metastases, but not in hematogenous metastases to other sites (p = 0.02). Comparative genomic hybridization analysis of these bone metastases disclosed centromere 8 gains as amplifications of the (whole) 8q arm, whereas centromeric loss appeared to be due to loss of 8p sequences. With progression toward metastatic disease, an accumulation of genetic changes was seen as exemplified by gain of chromosome 1, which was solely observed in distant metastases. With tumor progression, gain of chromosomes 7 and/or 8 significantly increased (p = 0.03), whereas the number of cases with aberrations of the Y chromosome did not change. Furthermore, ploidy status determined by ISH revealed a significant increase in the number of aneuploid cases along with advancement of pTNM stage (p = 0.04). Collectively, the data strongly suggest that: (a) gain of chromosome 7 and/or 8 sequences is implicated in prostatic tumor progression; (b) gain of chromosome 8 sequences is related to local tumor growth; (c) overrepresentation of 8q sequences, most likely by isochromosome 8q formation, is involved in metastatic spread to the bone; and (d) changes in the centromeric copy number, as detected by interphase ISH, might in some cases represent structural alterations, such as an isochromosome.     

Pyridoxalated hemoglobin polyoxyethylene conjugate does not restore hypoxic pulmonary vasoconstriction in ovine sepsis

OBJECTIVES: Hypoxic pulmonary vasoconstriction, a protective mechanism, minimizes perfusion of underventilated lung areas to reduce ventilation-perfusion mismatching. We studied the effects of sepsis on hypoxic pulmonary vasoconstriction and attempted to determine whether hypoxic pulmonary vasoconstriction is influenced by pyridoxalated hemoglobin polyoxyethylene conjugate, a nitric oxide scavenger. DESIGN: Prospective, randomized, controlled experimental study with repeated measures. SETTING: Investigational intensive care unit at a university medical center. SUBJECTS: Nineteen female merino sheep, divided into three groups: group 1, controls (n = 5); group 2, sheep with sepsis (n = 6); and group 3, septic sheep treated with pyridoxalated hemoglobin polyoxyethylene conjugate (n = 8). INTERVENTIONS: All sheep were instrumented for chronic study. An ultrasonic flow probe was placed around the left pulmonary artery. After a 5-day recovery, a tracheostomy was performed and a double-lumen endotracheal tube was placed. Animals in groups 2 and 3 received a 48-hr infusion of live Pseudomonas aeruginosa (6 x 10(4) colony-forming units/kg/hr). After 24 hrs, sheep in group 3 received pyridoxalated hemoglobin polyoxyethylene conjugate (20 mg/kg/hr) for 16 hrs; sheep in groups 1 and 2 received only the vehicle. Hypoxic pulmonary vasoconstriction was repeatedly tested by unilateral hypoxia of the left lung with 100% nitrogen. Hypoxic pulmonary vasoconstriction was assessed as the change in left pulmonary blood flow. MEASUREMENTS AND MAIN RESULTS: In the animals in group 1, left pulmonary blood flow decreased by 62 +/- 8 (SEM)% during left lung hypoxia and remained stable during repeated hypoxic challenges throughout the study period. After 24 hrs of sepsis, left pulmonary blood flow decreased from 56 +/- 10% to 26 +/- 2% (group 2) and from 50 +/- 8% to 23 +/- 6% (group 3). In the sheep in group 2, there was no adaptation over time. Pulmonary shunt fraction increased. Pyridoxalated hemoglobin polyoxyethylene conjugate had no effect on hypoxic pulmonary vasoconstriction or pulmonary shunt. The animals receiving the bacterial infusion developed a hyperdynamic circulatory state with hypotension, decreased systemic vascular resistance, and increased cardiac output. Pyridoxalated hemoglobin polyoxyethylene conjugate increased mean arterial pressure and systemic vascular resistance but did not influence cardiac index. Pulmonary arterial pressure was increased during sepsis and increased even further after pyridoxalated hemoglobin polyoxyethylene conjugate administration. Oxygenation and oxygen delivery and uptake were not affected by pyridoxalated hemoglobin polyoxyethylene conjugate. CONCLUSIONS: Hypoxic pulmonary vasoconstriction is blunted during sepsis and there is no adaptation over time. It is not influenced by pyridoxalated hemoglobin polyoxyethylene conjugate. Pyridoxalated hemoglobin polyoxyethylene conjugate reversed hypotension and, with the exception of an increase in pulmonary arterial pressure, had no adverse effects on hemodynamics or oxygenation.     

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.     

Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.     

Identification and characterization of heparin binding regions of the Fim2 subunit of Bordetella pertussis

Bordetella pertussis fimbriae bind to sulfated sugars such as heparin through the major subunit Fim2. The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins. Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin. The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine. These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required. Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity. Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides. A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars. A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved. The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant. Since B. pertussis fimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed.     

Novel antipsychotics and new onset diabetes

Mutations of p53 tumor suppressor gene occur in a subset of aggressive prostatic carcinomas and are detectable by immunohistochemistry. However, it is uncertain whether p53 overexpression really reflects p53 gene mutation or loss of p53 function. p21WAF1, an inhibitor of cyclin-dependent kinases, is activated by wild-type p53 protein, not by mutant type. Therefore, it is possible that combined analysis of p21WAF1 and p53 proteins aids in determining the functional status of p53 immunostaining. Routinely processed prostatic tissues from 60 patients with prostatic adenocarcinomas were examined by immunohistochemistry for p21WAF1 and p53 expression. As for tissue distribution, p21WAF1 protein was expressed mostly in the luminal layers, in contrast, p53 protein was restricted to the basal layers of benign prostatic glands. In prostatic adenocarcinomas, p21WAF1 protein was more likely to be expressed in well-differentiated areas; in contrast, p53 protein was more likely in poorly differentiated areas in the tumors. The percentage of positive nuclear areas for p21WAF1 and p53 proteins in prostatic adenocarcinomas, assessed by CAS200 computerized image analyzer, were 8.6+/-10% and 16+/-14% (mean+/-SE), respectively. The survival study showed that the p53+/ p21- phenotype showed poorer prognosis than p53+/p21+. Multivariate analysis showed that p21WAF1 expression, clinical stage, and Gleason score were independent prognosticators. In conclusion, p21WAF1 immunohistochemistry is a useful method for interpretation of p53 immunohistochemical results. Combined analysis by p21WAF1 and p53 immunostaining would predict the patient survival more accurately than p53 immunostaining alone.     

Early isotonic saline resuscitation from uncontrolled hemorrhage in rats

BACKGROUND: Attempts to modify traditional fluid resuscitation have been based on animal models that evaluate several variables including anesthesia. This study presents the effects of early saline resuscitation from severe uncontrolled hemorrhage unanesthetized rats. METHODS: Sixty-three female Sprague-Dawley rats were equally divided into three groups: group A, nonresuscitated; and groups B and C, resuscitated ;with isotonic saline (40 and 80 mL/kg, respectively). Hemodynamics, blood loss, survival time, and mortality were recorded for 360 minutes after the hemorrhage, which was initiated by 75% resection of the tail. RESULTS: In group C, 80 mL/kg of saline significantly lowered mortality (24% vs 76% and 71% for groups A and B, respectively) with concomitant increases in mean survival time (241 +/- 103 min vs 146 +/- 108 and 175 +/- 92 min for groups A and B, respectively). There were no statistically significant differences in blood loss, hematocrit, or hemodynamic parameters among the groups. CONCLUSIONS: Early and adequate isotonic saline resuscitation of unanesthetized rats improved outcome despite continuing hemorrhage. The significantly lower mortality rate and increased survival time were not a result of transiently improved arterial pressure and did not correlate with blood loss. No significant bleeding increases were noted in the resuscitated groups.