Mammalian Prion protein expression in yeast; a model for transmembrane insertion.

The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian microsomes, 50% being found as transmembrane (TM) proteins with the central TM1 segment spanning the membrane. TM1 hydrophobicity is marginal for lateral membrane insertion, which is primarily driven by hydrophobic interaction between the ER translocon and substrates in transit. Most inserted TM1 has its N-terminus in the ER lumen (Ntm orientation), as expected for arrest of normal secretion. However, 20% is found in inverted Ctm orientation. These are minor species in vivo, presumably a consequence of efficient quality control. PrP mutations that increase TM1 hydrophobicity result in increased Ctm insertion, both in vitro and in mouse brain, and a strong correlation is found between CtmPrP insertion and neuropathology in transgenic mice; a copper-dependent pathogenicity mechanism is suggested. PrP fusions with a C-terminal epitope tag, when expressed in yeast cells at moderate levels, appear to interact efficiently with the translocon, providing a useful model for testing the effects of PrP mutations on TM insertion and orientation. However, secretion of PrP by the mammalian translocon requires the TRAP complex, absent in yeast, where essentially all PrP ends up as TM species, 85-90% Ntm and 10-15% Ctm. Although yeast is, therefore, an incomplete mimic of mammalian PrP trafficking, effects on Ctm insertion of mutations increasing TM1 hydrophobicity closely reflect those seen in vitro. Electrostatic substrate-translocon interactions are a major determinant of TM protein insertion orientation and the yeast model was used to investigate the role of the large negative charge difference across TM1, a likely cause of translocation delay that would favor TM insertion and Ctm orientation. An increase in ΔCh from -5 to -7 caused a marked increase in Ctm insertion, while a decrease to -3 or -1 allowed 35 and about 65% secretion, respectively. Utility of the yeast model and the role of this charge difference in driving PrP membrane insertion are confirmed.

Telemetry capsule for measuring contractile motion in the small intestine.

The aim of this study was to develop a capsule which can measure contractions in the small intestine. Currently available methods cause discomfort to the patient while taking measurements; with the development of a telemetry capsule that can measure contractions, patients can avoid pain and continue with ordinary activities while the information is automatically collected from the external receiver. In order to develop the contraction force measuring capsule, various types of silicone transducers were designed and implemented to measure the contraction pressure in the small intestine. The minimum resolution of the implemented transducer was 0.05 mbar, which was small enough to measure contractions. The transducer was assembled with telemetry modules and packaged as a capsule (Φ13 × L25 mm) that had a similar size to a commercial capsule endoscope. In order to verify the function of the capsule, in vitro experiments were conducted and contractile motion was measured as 16.6 cycles per minute (CPM).

Colorectal polyps: a frequently-missed cause of rectal bleeding in Egyptian children.

OBJECTIVES: Colorectal polyps are important causes of rectal bleeding but they have been infrequently reported in Egyptian children. The prevalence and characteristics of colorectal polyps in a consecutive cohort of Egyptian children with rectal bleeding are presented. METHODS: A total of 174 children aged 2-12 years [mean (SD) 6.4 (3.7)] with fresh rectal bleeding were enrolled prospectively. Rectal examination, laboratory investigations and fibre-optic colonoscopy were performed in all patients. RESULTS: The source of bleeding was diagnosed as colorectal polyps in 100 patients (57.4%) and was owing to other causes in 74. The interval between onset of symptoms and presentation ranged from 2 to 48 months [mean (SD) 18.3 (16)]. In patients with other causes, rectal bleeding was attributed to intestinal amoebiasis (42), diarrhoea/dysentery (18), severe constipation (2) and intestinal schistosomiasis (2). Polyps were solitary in 56 children (56%) and ranged from 2 to 5 in 34 (34%) and >5 in 10 (10%). Polyps were confined to the rectum in 68 children, were rectosigmoid in 20, in the descending colon in 8, and splenic flexure in 4. Polyps were juvenile in 84 children (84%), inflammatory in 10 (10%) and hyperplastic, schistosomal or adenomatous in 2 each (6%). Colonoscopic polypectomy was successful and arrested the bleeding in all cases. CONCLUSION: In Egyptian children, colorectal polyps are relatively common and an easily treatable cause of fresh rectal bleeding. They should be high on the list of differential diagnoses.

Preoperative computed tomography-guided hookwire needle localization of a peritoneal multilocular inclusion cyst.

Preoperative hookwire localization of breast lesions is a well established technique to aid surgeons in localizing breast tumors. We describe the innovative use of a standard hookwire with CT guidance to localize an intraperitoneal inclusion cyst.

Is stored expressed breast milk an alternative for working Egyptian mothers?

Expression and storage of breast milk is way to maintain breastfeeding when mother and infant are separated, if the nutritional value can be conserved. Three expressed breast milk samples were collected from 61 healthy lactating mothers in Cairo, Egypt, for determination of total protein, fat, lactose and zinc content, as well as vitamins C, A and E concentrations. One sample was analysed immediately without storage, 1 after storage for 24 hours in a refrigerator (4 degrees C) and 1 after storage for 1 week in a home freezer (-4 degrees C to -8 degrees C). Refrigeration and freezing of breast milk caused a statistically significant decline in levels of vitamins C, A and E. Nevertheless, the values of all nutrients were still within the international reference ranges for mature breast milk.

Is stored expressed breast milk an alternative for working Egyptian mothers?

Expression and storage of breast milk is way to maintain breastfeeding when mother and infant are separated, if the nutritional value can be conserved. Three expressed breast milk samples were collected from 61 healthy lactating mothers in Cairo, Egypt, for determination of total protein, fat, lactose and zinc content, as well as vitamins C, A and E concentrations. One sample was analysed immediately without storage, 1 after storage for 24 hours in a refrigerator [4 degrees C] and 1 after storage for 1 week in a home freezer [-4 degrees C to -8 degrees C]. Refrigeration and freezing of breast milk caused a statistically significant decline in levels of vitamins C, A and E. Nevertheless, the values of all nutrients were still within the international reference ranges for mature breast milk

Gilbert's syndrome is a contributory factor in prolonged unconjugated hyperbilirubinemia of the newborn.

OBJECTIVE: Prolonged neonatal jaundice, beyond day 14 of life, is very common and of concern to the clinician. The aim of this study was to investigate whether a genetic mutation in the bilirubin UGT1A1 gene, which has been associated with Gilbert's syndrome in adults, is a contributory factor in prolonged neonatal jaundice. STUDY DESIGN: Blood was collected from 85 term newborns with unexplained hyperbilirubinemia, and DNA was prepared. The neonates were divided into 6 groups depending on whether they were breast-fed or bottle-fed and whether they had acute, prolonged, or very prolonged jaundice. UGT1A1 TATA promoter genotyping (DNA test for Gilbert's syndrome) was performed on all samples, and analysis of the entire UGT1A1 coding sequence was performed in a representative sample (11 of 26) of very prolonged cases. RESULTS: In addition to the known common UGT1A1 TATA alleles (TA6 and TA7), a novel TATA allele (TA5) in a neonate with very prolonged jaundice was identified. Statistical analysis of the TATA genotype distributions within the group of breast-fed neonates revealed significant differences among the acute, prolonged, and very prolonged subgroups (.05 > P >.01): the incidence of familial hyperbilirubinemia genotypes (7/7 and 5/7) is 5 times greater in very prolonged cases (31%) relative to acute cases (6%). Neonates with prolonged jaundice from family pedigrees were observed to demonstrate the Gilbert's phenotype as children or young adults. CONCLUSIONS: A genetic predisposition to develop prolonged neonatal hyperbilirubinemia in breast-fed infants is associated with TATA box polymorphism of the UGT1A1 gene and will be recognized as Gilbert's syndrome in adulthood.

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase.

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.

Effect of NADH-X on cytosolic glycerol-3-phosphate dehydrogenase.

At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, with respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cytosolic fraction. It was a much less potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidation catalyzed by this enzyme from all three sources. Glycerol-3-phosphate dehydrogenase purified from muscle cytosol contains tightly bound NADH-X, NAD, and ADP-ribose, each in amounts of about 0.1 mol per mole of enzyme polypeptide chain. A deproteinized supernatant of this enzyme contained these three ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with a Ki, in the reaction with NADPH at pH 7.05, in the range of 0.2 microM with respect to the total concentration of ligands ([ADP-ribose] + [NAD] + [NADH-X] = 0. 2 microM). However, only the NADH-X component could account for the potent inhibition because NAD, ADP-ribose, and the primary acid product (which can be produced from NADH-X) each had a Ki considerably higher than 0.2 microM. Although at pH 7.05 NADH-X inhibited NADPH oxidation considerably more than NADH oxidation, the reverse was the case at pH 7.38. Since the enzyme purified from muscle contains tightly bound NADH-X, NADH-X might become attached to the enzyme in vivo where it could play a role in regulating the ratio of NADH to NADPH oxidation of the enzyme.

The kinetics of bioaccumulation of zinc, copper, lead and cadmium by oysters (Crassostrea iredalei and C. belcheri) under tropical field conditions.

A study on the kinetics of accumulation and depuration of Zn, Cu, Pb and Cd by the oysters (Crassostrea iredalei and Crassostrea belcheri) cultured at two locations in the Merbok Estuary, Malaysia was conducted. A first-order kinetic model was employed to fit the experimental data in order to estimate the rate constants for uptake and elimination processes and to predict the bioconcentration factors (BCF). Among the four metals studied, only the Zn accumulation process could not be modelled using first-order kinetics. The elimination rate constants estimated from depuration data for C. iredalei are found to be much greater than those from accumulation data. The results suggest that the values of kinetic parameters and BCFs derived under conditions of both aqueous and dietary exposure are probably more site- than species-specific.

Hemoglobin and hematocrit values of Saudi newborns in the high altitude of Abha, Saudi Arabia.

A study was designed to determine the red cell values (hemoglobin and hematocrit) of neonates born in the high altitude of Abha and to compare these values with known values of other lowland areas of Saudi Arabia. From the cord blood of 587 normal, appropriate for gestational age and term infants born in 1993 in Abha Maternity Hospital, the ranges of Hb and Hct were 130 to 240 g/L and 0.24 to 0.79 L/L respectively. The mean Hb was 187 g/L. There was no significant difference between the male and female values. Also, 17% of the infants in this study were polycythemic, while no polycythemia was recorded in these lowland areas, and only 2% to 4% in the general global newborn population. It was therefore revealed that Abha newborns had higher red cell values at birth when compared to other newborns in the low altitude areas of Riyadh and Jeddah (P <0.001). We postulate that the high altitude (2700 meters above sea level) of Abha, and therefore its relative hypoxia, has induced high red cell values in infants born in the city. This phenomenon therefore warrants the adoption of higher red cell reference values and not necessarily those already documented in other Saudi newborn populations.

Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages.

Using the equilibrium dialysis apparatus, an aqueous suspension of predominantly aggregated Re lipopolysaccharide (ReLPS) from Escherichia coli D31 m4 (99.9% at 82.5 microM) can be processed to yield a solution of monomeric ReLPS at a saturation concentration of 77 ng/ml (3.4 x 10(-8) M). We compared the in vitro biological activities of these two physically distinct types of ReLPS preparations in two select assays, reaction in the Limulus amebocyte lysate (LAL) assay and induction of Egr-1 mRNA in macrophages. These assays were chosen for their rapid response times and relatively short incubation periods. The monomeric ReLPS was 179- and 1000-fold more active than the aggregated ReLPS preparation in the LAL assay and induction of Egr-1 mRNA by thioglycollate-elicited murine peritoneal macrophages, respectively. These results clearly showed that the monomeric ReLPS is the more active form. The lower biological activities of the aggregated ReLPS preparation might be due to the presence of a small amount of monomeric ReLPS (0.01-0.6%) produced during its preparation and the incubation periods in the biological assays. Thus, aggregated ReLPS may be relatively inactive.

Effect of pH on solubility and ionic state of lipopolysaccharide obtained from the deep rough mutant of Escherichia coli.

The dissociation of the highly aggregated form of lipopolysaccharide (LPS) from Gram-negative bacteria to the monomeric (or soluble) form is though to be the initial step in the activation of responding cells (macrophages, B-cells, neutrophils, monocytes, and endothelial cells) by LPS. This process is presently not adequately understood. Using the equilibrium dialysis apparatus and a highly purified and well-characterized radiolabeled deep rough chemotype LPS ([14C]ReLPS) from Escherichia coli D31m4, we have examined the effect of pH on its solubility (CT) and ionic states in aqueous media. The solubility range of [14C]ReLPS suspended in 50 mM Tris-HCl-100 mM KCl buffer (or 50 mM MES-100 mM KCl buffer at pH 6.5) was determined to be from (2.91 +/- 0.01) x 10(-8) to (4.55 +/- 0.07) x 10(-8) M over a pH range of 6.50-8.20, respectively. These experimental data satisfactorily fitted the curve generated by the solubility equation CT = S0(1 + K5/[H+])/([H+]/K4' + 1), where S0 is the concentration of the tetraanionic ReLPS, K5 is the dissociation constant of the tetraanionic ReLPS in solution, and K4' is the dissociation constant of the trianionic ReLPS at the surface of the solid particles in suspension. The increase in solubility of ReLPS with increase in pH from 7.00 to 8.20 is primarily caused by the formation of the pentaanionic form from the tetraanions. The pK5 (primarily the second dissociation of the 1-phosphate) of ReLPS was determined to be 8.58 from experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)