Influence of oxygen partial pressures on protein synthesis in feeding crabs

Many water-breathing animals have a strategy that consists of maintaining low blood PO2 values in a large range of water oxygenation level (4-40 kPa). This study examines the postprandial changes in O2 consumption, arterial blood PO2, and tissue protein synthesis in the shore crab Carcinus maenas in normoxic, O2-depleted, and O2-enriched waters to study the effects of this strategy on the O2 consumption and peptide bond formation after feeding. In normoxic water (21 kPa), the arterial PO2 was 1.1 kPa before feeding and 1.2 kPa 24 h later. In water with a PO2 of 3 kPa (arterial PO2 0.6 kPa), postprandial stimulation of protein synthesis and O2 consumption were blocked. The blockade was partial at a water PO2 of 4 kPa (arterial PO2 0.8 kPa). An increase in environmental PO2 (60 kPa, arterial PO2 10 kPa) resulted in an increase in protein synthesis compared with normoxic rates. It is concluded that the arterial PO2 spontaneously set in normoxic Carcinus limits the rates of protein synthesis. The rationale for such a strategy is discussed.     

Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids

Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function.     

Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria

There are about 2.5 million glomeruli in the kidneys each consisting of a barrel of glomerular basement membrane surrounded by glomerular endothelial cells on the inside and glomerular epithelial cells with established foot processes (podocytes) on the outside. Defects in this filtration apparatus lead to glomerular vascular leak or proteinuria. The role of vascular endothelial growth factor (VEGF) in the regulation of glomerular vascular permeability is still unclear. Recent studies indicate that patients receiving anti-VEGF antibody therapy may have an increased incidence of proteinuria. In a different setting, pregnancies complicated by preeclampsia are associated with elevated soluble VEGF receptor 1 protein (sFlt-1), endothelial cell dysfunction and proteinuria. These studies suggest that neutralization of physiologic levels of VEGF, a key endothelial survival factor, may lead to proteinuria. In the present study, we evaluated the potential of anti-VEGF neutralizing antibodies and sFlt-1 in the induction of proteinuria. Our studies demonstrate that anti-VEGF antibodies and sFlt-1 cause rapid glomerular endothelial cell detachment and hypertrophy, in association with down-regulation of nephrin, a key epithelial protein in the glomerular filtration apparatus. These studies suggest that down-regulation or neutralization of circulating VEGF may play an important role in the induction of proteinuria in various kidney diseases, some forms of cancer therapy and also in women with preeclampsia.     

In vitro selection of second site revertants analysis of the hairpin ribozyme active site

We have used in vitro genetics to evaluate the function and interactions of the conserved base G8 in the hairpin ribozyme catalytic RNA. Second site revertant selection for a G8X mutant, where X is any of the other three natural nucleobases, yielded a family of second site suppressors of the G8U mutant, but not of G8C or G8A, indicating that only G and U can be tolerated at position 8 of the ribozyme. This result is consistent with recent observations that point to the functional importance of G8 N-1 in the chemistry of catalysis by this ribozyme reaction. Suppression of the G8U mutation was observed when changes were made directly across loop A from the mutated base at substrate position +2 or positions +2 and +3 in combination. The same changes made in the context of the natural G8 sequence resulted in a very large drop in activity. Thus, the G8U mutation results in a change in specificity of the ribozyme from 5'-N / GUC-3' to 5'-N / GCU-3'. The results presented imply that G8 interacts directly with U+2 during catalysis. We propose that this interaction favors the correct positioning of the catalytic determinants of G8. The implications for the folding of the ribozyme and the catalytic mechanism are discussed.     

Human apurinic/apyrimidinic endonuclease (Ape1) and its N-terminal truncated form (AN34) are involved in DNA fragmentation during apoptosis

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.     

Ghrelin can bind to a species of high density lipoprotein associated with paraoxonase

Ghrelin is a 28-residue peptide hormone that is principally released from the stomach during fasting and prior to eating. Two forms are present in human plasma: the unmodified peptide and a less abundant acylated version, in which octanoic acid is attached to the third residue, a serine, via an ester linkage. The acylated form of ghrelin acts as a ligand for the growth hormone secretagogue receptor and can stimulate the release of growth hormone from the pituitary gland. It also initiates behavioral and metabolic adaptations to fasting. Here we show that an immobilized form of ghrelin specifically binds a species of high density lipoprotein associated with the plasma esterase, paraoxonase, and clusterin. Both free ghrelin and paraoxon, a substrate for paraoxonase, can inhibit this interaction. An endogenous species of ghrelin is found to co-purify with high density lipoprotein during density gradient centrifugation and subsequent gel filtration. This interaction links the orexigenic peptide hormone ghrelin to lipid transport and metabolism. Furthermore, the interaction of the esterified hormone ghrelin with a species of HDL containing an esterase suggests a possible mechanism for the conversion of ghrelin to des-acyl ghrelin.     

Causes of local recurrence after curative surgery for rectal cancer

The rate of local recurrence (LR) has been 20-40% after resective surgery for rectal cancer by the traditional - Miles or Dixon - operative technics. The authors performed curative resection in 358 patients with rectal cancer in a 10 year period (01.01.1990 - 31.12.2000) in the Surgical Department of Szeged University. Since 01.01.1996 the authors changed this type of surgery for the Heald technics (total mesorectal excision - TME - with sharp dissection, using the UltraCision device) for the surgical treatment of middle or lower third rectal cancer. To compare the results of the two procedures, the authors analysed their material in two periods: Period I: 01.01.1991 - 31.12.1992: 62 patients operated on with the traditional operative technics; LR 15% within 2 years after surgery. Period II: 01.01.1997 - 31.12.1998: 78 patients operated on with the Heald technics (TME with sharp dissection); LR 6.4% within 2 years after surgery. Based on their results, the authors found that the modern operative technics by Heald, used in the second period of the study, was a relevant factor decreasing LR from 15% to 6.4%, while the gender, age of the patients, ratio of the abdominoperineal extirpation versus anterior resection (APRE/AR) and the free margin of more than 3 cm proved to be irrelevant.     

Description and mission evaluation of the Hungarian case-control surveillance of congenital abnormalities, 1980-1996

BACKGROUND: The Hungarian Case-Control Surveillance of Congenital Abnormalities was established in 1980. This article describes how the Hungarian Case-Control Surveillance of Congenital Abnormalities was first organized and is currently maintained. The baseline statistics are provided and potential venues of postmarketing surveillance of drug teratogenicity and other public health tasks and research are proposed. METHODS: Cases with congenital abnormalities and patient controls with Down syndrome were selected from the Hungarian Congenital Abnormality Registry. Population controls without congenital abnormalities were selected from the National Birth Registry on the basis of three matching criteria: sex, week of birth, and district of parent's residence. Three sources of information concerning drug exposures, maternal disorders, and pregnancy complications, among others, were used: (1) prospective and medically recorded data from antenatal care logbooks and discharge summaries; (2) retrospective maternal self-reported data obtained with a structured questionnaire in all the three study groups; and (3) data collected by regional nurse in house visits to nonrespondent cases and patient controls. Twenty-five congenital abnormality groups were evaluated. During the 17-year period of data collection, 22,843 cases, 38,151 population controls, and 834 patient controls were incorporated into the data set, constituting the largest population-based case-control data set of congenital abnormalities to date. RESULTS: Demographic features of pregnant women and informative offspring are presented along with the distribution of 25 main groups of congenital abnormalities. CONCLUSIONS: This system is appropriate for postmarketing the surveillance of drug teratogenicity, for the improvement of congenital abnormality diagnosis, to get informed consent, to have a communication with parents and to provide material for research.