Determination of plasma volume in anaesthetized piglets using the carbon monoxide (CO) method

Based on measurements of the circulating red blood cell volume (V(RBC)) in seven anaesthetized piglets using carbon monoxide (CO) as a label, plasma volume (PV) was calculated for each animal. The increase in carboxyhaemoglobin (COHb) concentration following administration of a known amount of CO into a closed circuit re-breathing system was determined by diode-array spectrophotometry. Simultaneously measured haematocrit (HCT) and haemoglobin (Hb) values were used for PV calculation. The PV values were compared with simultaneously measured PVs determined using the Evans blue technique. Mean values (SD) for PV were 1708.6 (287.3)ml and 1738.7 (412.4)ml with the CO method and the Evans blue technique, respectively. Comparison of PVs determined with the two techniques demonstrated good correlation (r = 0.995). The mean difference between PV measurements was -29.9 ml and the limits of agreement (mean difference +/-2SD) were -289.1 ml and 229.3 ml. In conclusion, the CO method can be applied easily under general anaesthesia and controlled ventilation with a simple administration system. The agreement between the compared methods was satisfactory. Plasma volume determined with the CO method is safe, accurate and has no signs of major side effects.     

Postnatal essential fatty acid deficiency in mice affects lipoproteins, hepatic lipids, fatty acids and mRNA expression

We have previously reported that essential fatty acid deficiency (EFAD) during suckling in mice resulted in an adult lean phenotype and a resistance to diet-induced obesity. We now hypothesized that postnatal EFAD would cause long-term effects on lipid metabolism. C57BL/6 mice were fed an EFAD or a control diet from the 16th day of gestation and throughout lactation. The pups were weaned to standard diet (STD) and at 15 weeks of age given either high fat diet (HFD) or STD. Lipoprotein profiles, hepatic lipids, fatty acids and mRNA expression were analyzed in 3-week-old and 25-week-old offspring. At weaning, the EFAD pups had higher cholesterol levels in both plasma and liver and 6-fold higher concentrations of hepatic cholesterol esters than control pups. Adult EFAD offspring had higher levels of hepatic cholesterol and linoleic acid, but lower levels of dihomo-γ-linolenic acid and Pparg mRNA expression in the liver. In addition, HFD fed EFAD offspring had lower plasma total cholesterol, lower hepatic triglycerides and lower liver weight compared to controls fed HFD. In conclusion, early postnatal EFAD resulted in short-term alterations with increased hepatic cholesterol accumulation and long-term protection against diet-induced liver steatosis and hypercholesterolemia.     

Left ventricular strain, rotation, and torsion as markers of acute myocardial ischemia

This study investigates how tissue Doppler imaging (TDI) and speckle tracking echocardiography (STE) describe regional myocardial deformation during controlled reductions of left anterior descending (LAD) coronary artery perfusion pressure. In eight anesthetized pigs, a shunt with constrictor was installed from the brachiocephalic artery to the LAD. Data were obtained with open shunt, followed by four degrees of stenosis (S1-S4) of increasing severity: S1, ～15%; S2, ～35%; S3, ～50%; and S4, ～60% reductions of LAD perfusion pressure. At each situation, microspheres for perfusion measurements were injected and left ventricular (LV) short- and long-axis cineloops were recorded. In the anterior wall, radial, circumferential, and longitudinal one-layer STE strain, one-layer radial TDI strain, and three-layer radial TDI and STE strain were measured. LV peak mean rotation was measured at six equidistant levels from apex to base (in 7 pigs). LV torsion was calculated from end-systolic mean rotation. With open shunt, three-layer TDI analysis showed a transmural strain gradient with no perfusion gradient. Perfusion, one-layer TDI strain, and strain in the mid- and subendocardium from three-layer TDI were reduced at S2 (P < 0.05). STE strain was not affected until S3 (P < 0.05). Peak mean rotation, increasing toward the apex, decreased at the three apical levels at S4 (P < 0.05). LV torsion did not decrease (P = 0.26). In conclusion, TDI strain detected dysfunction already with minor changes in global hemodynamics, whereas STE strain was first reduced with moderate changes. LV peak mean rotation was not reduced until severe reduction of LAD perfusion pressure, but remained increasingly counterclockwise toward the apex. LV torsion remained unaffected by ischemia.     

Protein phosphatase 2A B55 and A regulatory subunits interact with nitrate reductase and are essential for nitrate reductase activation

Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains. The activation of NR in green leaves takes place after dark/light shifts, and is dependent on photosynthesis. Previous studies using various inhibitors pointed to protein phosphatases sensitive to okadaic acid, including protein phosphatase 2A (PP2A), as candidates for activation of NR. PP2As are heterotrimeric enzymes consisting of a catalytic (C), structural (A), and regulatory (B) subunit. In Arabidopsis there are five, three, and 18 of these subunits, respectively. By using inducible artificial microRNA to simultaneously knock down the three structural subunits we show that PP2A is necessary for NR activation. The structural subunits revealed overlapping functions in the activation process of NR. Bimolecular fluorescence complementation was used to identify PP2A regulatory subunits interacting with NR, and the two B55 subunits were positive. Interactions of NR and B55 were further confirmed by the yeast two-hybrid assay. In Arabidopsis the B55 group consists of the close homologs B55α and B55β. Interestingly, the homozygous double mutant (b55α × b55β) appeared to be lethal, which shows that the B55 group has essential functions that cannot be replaced by other regulatory subunits. Mutants homozygous for mutation in Bβ and heterozygous for mutation in Bα revealed a slower activation rate for NR than wild-type plants, pointing to these subunits as part of a PP2A complex responsible for NR dephosphorylation.     

ITIH-5 expression in human adipose tissue is increased in obesity

Adipocytes secrete many proteins that regulate metabolic functions. The gene inter-α (globulin) inhibitor H5 (ITIH-5) encodes a secreted protein and is known to be expressed abundantly in the placenta. However, using gene expression profiles data we observed high expression of ITIH-5 in adipose tissue. The aim of this study was to test the hypothesis that ITIH-5 is strongly expressed in human adipocytes and adipose tissue, and is related to obesity and clinical metabolic variables. ITIH-5 adipose tissue mRNA expression was analyzed with DNA microarray and real-time PCR, and its association with clinical variables was examined. ITIH-5 protein expression was analyzed using western blot. ITIH-5 mRNA expression was abundant in human adipose tissue, adipocytes, and placenta, and higher in subcutaneous (sc) compared to omental adipose tissue (P < 0.0001). ITIH-5 mRNA and protein expression in sc adipose tissue were higher in obese compared to lean subjects (P < 0.0001 and P < 0.001, respectively). ITIH-5 mRNA expression was reduced after diet-induced weight loss (P < 0.0001). ITIH-5 mRNA expression was associated with anthropometry and clinical metabolic variables. In conclusion, ITIH-5 is highly expressed in sc adipose tissue, increased in obesity, down regulated after weight loss, and associated with measures of body size and metabolism. Together, this indicates that ITIH-5 merits further investigation as a regulator of human metabolism.     

Characterisation of the complement-regulatory proteins decay-accelerating factor (DAF,CD55) and membrane cofactor protein (MCP,CD46) on a human colonic adenocarcinoma cell line

 To avoid destruction by complement, normal and malignant cells express membrane glycoproteins that restrict complement activity. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and protectin (CD59), which are all expressed on colonic adenocarcinoma cells in situ. In this study we have characterised the C3/C5 convertase regulators DAF and MCP on the human colonic adenocarcinoma cell line HT29. DAF was found to be a glycosyl-phosphatidylinositol-anchored 70-kDa glycoprotein. Blocking experiments with F(ab′)₂ fragments of the anti-DAF monoclonal antibody BRIC 216 showed that DAF modulates the degree of C3 deposition and mediates resistance to complement-mediated killing of the cells. The expression and function of DAF were enhanced by tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β). Cells incubated with interferon γ (IFNγ) did not alter their DAF expression. Two MCP forms were expressed, with molecular masses of approximately 58 kDa and 68 kDa, the lower form predominating. MCP expression was up-regulated by IL-1β, but not by TNFα or IFNγ. Expression of DAF and MCP promotes resistance of colonic adenocarcinoma cells to complement-mediated damage, and represents a possible mechanism of tumour escape. Received: 18 July 1995 / Accepted: 4 January 1996     

Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator

Background  

Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1.     

Neonatal tetanus despite protective serum antitoxin concentration

Using the ELISA technique to estimate serum antibodies against tetanus toxin, seven neonates with clinical tetanus were found to have antibody levels 4-13 times higher than the presumed minimum protective level of 0.01 IU/ml. All but one of their mothers had been vaccinated with tetanus toxoid in pregnancy. In two other neonates, whose mothers had received multiple booster doses of toxoid during pregnancy, the anti-toxin concentrations were 100- and 400-times the presumed protective level. Therefore the toxin dose may overwhelm the pre-existing anti-toxin level and produce disease. Furthermore, multiple booster injections of tetanus toxoid may not only enhance serum anti-toxin titres, but could also lead to an ineffective immune response.