Studies on the heterogeneity of human serum Lp lipoproteins and on the occurrence of double Lp lipoprotein variants

Lp lipoproteins have been prepared by a mild method from the serum of a large number of individuals. Approximately 25% of the individuals tested showed the presence of a double Lp peak in analytical ultracentrifuge diagrams. These double peaks were designated Lp(a)-1 and Lp(a)-2 to distinguish them from the single Lp(a) peak. The mean viscosity-corrected sedimentation coefficient, S _{1.004, 20 C} and density of the single Lp(a) peak were 15.8±1.8s (n=32) and 1.076±0.01 g/ml, of the Lp(a)-1 peak were 13.5±1.1s (n=14) and 1.064±0.007 g/ml, and of the Lp(a)-2 peak were 16.8±1.7s (n=14) and 1.074±0.009 g/ml. Absorption tests using a double and single Lp preparation showed that both Lp peaks in the double variants possess Lp(a) specificity. Evidence is lacking as yet for individual specificities for either Lp(a)-1 or Lp(a)-2. Interand intra-individual heterogeneity among Lp lipoproteins is discussed.This investigation was supported by National Institutes of Health grant HI-09739, U.S. Atomic Energy Commission contract (11-1)-1552, and National Institutes of Health research program project 1P01-GM-15419.     

Induction of antibodies to a tumor-associated antigen by immunization with a whole melanoma cell vaccine

Summary Urinary-tumor-associated antigen (U-TAA) is a glycoprotein present in the urine of melanoma patients. Previous studies have addressed the role of U-TAA in immunoprognosis. The present investigation was undertaken to determine whether the administration of whole melanoma cell vaccine (MCV) could induce the formation of anti-(U-TAA) antibodies in melanoma patients. The subjects of this study were stage II and III melanoma patients receiving MCV alone or in conjunction with cyclophosphamide. Anti-(U-TAA) IgM and IgG antibody levels were determined by enzyme immunoassay in sequential serum samples from 15 stage II and III melanoma patients receiving MCV. U-TAA purified from the urine of a melanoma patient was used as a target in this assay. The mean anti-(U-TAA) IgM titer prior to vaccination was similar to that of a non-vaccinated melanoma control group (1:1138±214, n=15 vs 1:1334±254, n=7; P=0.375) but prevaccination IgG levels were generally higher than in the control group (1:3984±602 vs 1:2595±423; 0.1>P>0.05). While only 6 of the 15 patients demonstrated a rise in levels of IgG antibodies (mean 1:2964±1047 pre-MCV to 1:9958±2677 post MCV, P<0.01), 11 of the 15 patients demonstrated a greater than twofold rise in their anti-(U-TAA) IgM titers following vaccination (1:1051±259 pre-MCV to 1:2518±576 post-MCV; P<0.005). In addition, patients with visceral metastases consistently elicited anti-(U-TAA) responses equivalent to those with more limited disease. Concomitant administration of cyclophosphamide did not affect the response rates or peak antibody levels. The possibility that these antibody responses were actually against histo-compatibility locus antigens (HLA) (contaminating our U-TAA preparation) was ruled out because the target antigen (U-TAA) was devoid of HLA, and because the induction of anti-(U-TAA) antibodies did not correlate with the induction of anti-HLA antibodies. These results demonstrate augmentation of anti-(U-TAA) IgM and IgG antibodies by immunization with the MCV.     

Mechanism of exocrine pancreatic insufficiency in streptozotocin-induced diabetes mellitus in rat: Effect of cholecystokinin-octapeptide

This study investigated the effects of cholecystokinin-octapeptide (CCK-8) on pancreatic juice flow and its contents, and on cytosolic calcium (Ca²⁺) and magnesium (Mg²⁺) levels in streptozotocin (STZ)-induced diabetic rats compared to healthy age-matched controls. Animals were rendered diabetic by a single injection of STZ (60 mg kg^–1, I.P.). Age-matched control rats obtained an equivalent volume of citrate buffer. Seven weeks later, animals were either anaesthetised (1 g kg^–1 urethane; IP) for the measurement of pancreatic juice flow or humanely killed and the pancreas isolated for the measurements of cytosolic Ca²⁺ and Mg²⁺ levels. Non-fasting blood glucose levels in control and diabetic rats were 92.40 ± 2.42 mg dl^–1 (n= 44) and >500 mg dl^–1 (n= 27), respectively. Resting (basal) pancreatic juice flow in control and diabetic anaesthetised rats was 0.56 ± 0.05 ul min^–1 (n= 10) and 1.28 ± 0.16 ul min^–1 (n= 8). CCK-8 infusion resulted in a significant (p < 0.05) increase in pancreatic juice flow in control animals compared to a much larger increase in diabetic rats. In contrast, CCK-8 evoked significant (p < 0.05) increases in protein output and amylase secretion in control rats compared to much reduced responses in diabetic animals. Basal [Ca²⁺]_i in control and diabetic fura-2-loaded acinar cells was 109.40 ± 15.41 nM (n= 15) and 130.62 ± 17.66 nM (n= 8), respectively. CCK-8 (10^–8M) induced a peak response of 436.55 ± 36.54 nM (n= 15) and 409.31 ± 34.64 nM (n= 8) in control and diabetic cells, respectively. Basal [Mg²⁺]_i in control and diabetic magfura-2-loaded acinar cells was 0.96 ± 0.06 nM (n= 18) and 0.86 ± 0.04 nM (n= 10). In the presence of CCK-8 (10^–8) [Mg²⁺]_i in control and diabetic cells was 0.80 ± 0.05 nM (n= 18) and 0.60 ± 0.02 nM (n= 10), respectively. The results indicate that diabetes-induced pancreatic insufficiency may be associated with derangements in cellular Ca²⁺ and Mg²⁺ homeostasis. (Mol Cell Biochem 261: 83–89, 2004)     

Evidence for permanent water channels in the basolateral membrane of an ADH-sensitive epithelium

Summary The transepithelial water permeability in frog urinary bladder is believed to be essentially dependent on the ADH-regulated apical water permeability. To get a better understanding of the transmural water movement, the diffusional water permeability (P _d) of the basolateral membrane of urinary bladder was studied. Access to this post-luminal barrier was made possible by perforating the apical membrane with amphotericin B. The addition of this antibiotic increasedP _d from 1.12±0.10×10^–4 cm/sec (n=7) to 4.08±0.33×10^–4 cm/sec (n=7). The effect of mercuric sulfhydryl reagents, which are commonly used to characterize water channels, was tested on amphotericin B-treated bladders. HgCl₂ (10^–3 m) decreasedP _d by 52% andpara-chloromercuribenzoic acid (pCMB) (1.4×10^–4 m) by 34%. The activation energy for the diffusional water transport was found to increase from 4.52±0.23 kcal/mol (n=3), in the control situation, to 9.99±0.91 kcal/mol (n=4) in the presence of 1.4×10^–4 m pCMB. Our second approach was to measure the kinetics of water efflux, by stop-flow light scattering, on isolated epithelial cells from urinary bladders.pCMB (0.5 or 1.4×10^–4 m) was found to inhibit water exit by 91±2%. These data strongly support the existence of proteins responsible for water transport across the basolateral membrane, which are permanently present.     

Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling

Nitrate-selective microelectrodes were used to measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old barley (Hordeum vulgare L.) seedlings grown in nutrient solution containing 10 mol · m^–3 nitrate. Measurements in each cell type grouped into two populations with mean (±SE) values of 5.4 ± 0.5 mol · m^–3 (n=19) and 41.8 ± 2.6 mol · m^–3 (n = 35) in epidermal cells, and 3.2 ± 1.2 mol · m^–3 (n = 4) and 72.8 ± 8.4 mol · m^–3 (n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell sampling procedure was used to withdraw a vacuolar sap sample from individual epidermal and cortical cells. Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 ± 5.3 mol · m^–3 (n = 10) in epidermal cells and 101.2 ± 4.8 mol · m^–3 (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements in each cell type, supporting the hypothesis that the higher of the two populations of electrode measurements in each cell type are vacuolar in origin. Differences in the absolute values obtained by these methods are probably related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay against concentration. Furthermore, a 28-h time course for the accumulation of nitrate measured with electrodes in epidermal cells showed the apparent cytoplasmic measurements remained constant at 5.0 ± 0.7 mol · m^–3, while the vacuole accumulated nitrate to 30–50 mol · m^–3. The implications of the data for mechanisms of nitrate transport at the plasma membrane and tonoplast are discussed.Symbol _n ² Chi-squared with n degrees of freedom R.-G.Z. was awarded a Sino-British Friendship Scholarship sponsored by the British Council and H.-W.K. was supported by an AFRC Linked Research Grant to A.D.T for collaboration with R.A.L. We wish to thank Dr. K. Goulding for advice on ion chromatography, Dr. K. Moore for assistance with statistical analysis and Dr. J.H. Williams for advice on the microsample analysis.     

Rate of activation and deactivation of K∶Cl cotransport by changes in cell volume in hemoglobin SS,CC and AA red cells

Red blood cells (RBC) of subjects homozygous for hemoglobin A (AA), C (CC) and S (SS) exhibit different cell volumes which might be related to differences in cell volume regulation. We have investigated how rapidly K:Cl cotransport is activated and deactivated to regulate the cell volume in these cells. We measured the time course of net K⁺ efflux after step changes in cell volume and determined two delay times: one for activation by cell swelling and a second for deactivation by cell shrinkage. Cell swelling induced by 220 mOsm media activated K⁺ efflux to high values (10–20 mmol/ liter cell x hr) in CC and SS; normal AA had a threefold lower activity. The delay time for activation was very short in blood with a high percentage of reticulocytes (retics): (SS, 10% retics, 1.7±0.3 min delay, n=8; AA, 10% retics, 4±1.5 min, n=3; CC, 11.6% retics, 4±0.3, n=3) and long in cells with a smaller percentage of reticulocytes: (AA, 1.5% retics, 10±1.4 min, n=8; CC whole blood 6% retics, 10±2.0 min, n=10, P<0.02 vs. SS). The delay times for deactivation by cell shrinking were very short in SS (3.6±0.4 min, n=8, P<0.02) and AA cells with high retics (2.7±1 min, n=3) and normal retics (2.8±1 min, n=3), but 8–15-fold longer in CC cells (29±2.8 min, n=9).Density fractionation of CC cells (n=3) resulted in coenrichment of the top fraction in reticulocytes and in swelling-activated cotransport (fourfold) with short delay time for activation (4±0.3 min) and long delay for deactivation (14±4 min). The delay time for activation, but not for deactivation, increased markedly with increasing cell density. These findings indicate that all CC cells do not promptly shut off cotransport with cell shrinkage and high rates of cellular K⁺ loss persist after return to isotonic conditions.In summary, (i) K:Cl cotransport is not only very active in young cells but it is also very rapidly activated and deactivated in young AA and SS cells by changes in cell volume. (ii) Delay times for cotransport activation markedly increased with RBC age and in mature cells with low cotransport rates, long delay times for activation were observed. (iii) The long delay time for deactivation exhibited even by young CC cells induces a persistent loss of K⁺ after cell shrinkage which may contribute in vivo to the uniformly low cell volume, low K⁺ and water content of CC cells.This research was supported by National Institutes of Health grants Shannon Award HL-35664, HL-42120, Sickle Cell Center grant HL-38655, and a Grant-in-Aid of the New York Branch of the American Heart Association. The technical help of Sandra M. Suzuka, M.S. is gratefully acknowledged.     

Water and salt permeability of gastric vesicles

Summary Volume-dependent changes in light scatter have been shown to be a linear function of the osmotic gradient imposed upon gastric vesicles purified from hog gastric mucosa. Observation of the light scattered 90° to incident, using the Durrum stop flow system D-110, indicates that the vesicles exposed to hypertonic medium undergo rapid shrinkage due to water loss from the vesicle interior. The rate constant for this water movement is 1.1±0.09 sec^–1 (n=10) and is linearly dependent on temperature between 16 and 36°C. The activation energy of 13.93±0.60 kcal mole^–1 (n=3), calculated from an Arrhenius plot, is inconsistent with water movement facilitated by a large-pore aqueous channel. A slower reswell phase, dependent on solute entry into the intravesicular space, follows the water-dependent shrink phase. KCl entry, studied because of the intravesicular requirement for active K⁺/H⁺ transport, exhibits two entry stages. The faster, described by a single exponential imposed upon a constantly sloping background, has a rate constant of 7.75±0.48×10^–3 sec^–1 (n=15). The slower phase, which typically accounts for 90% of the reswell process, demonstrates a rate constant of 1.94±0.23×10^–4 sec^–1 (n=15). In the presence of valinomycin or nigericin, two fast rate constants and one slow rate constant of swelling are observed. The rate constant of the faster reswell phase is increased from 7.75±0.48×10^–3 sec^–1 (n=15) to 15.74±3.7×10^–3 sec^–1 (n=5) and 17.23±3.4×10^–3 (n=3) by the addition of nigericin (1 g ml^–1) and valinomycin (4.5 m), respectively. The second part of the faster reswell phase is approximately that seen in the control population. Transport-dependent volume changes of significant magnitude can be demonstrated following the addition of ATP to vesicles equilibrated with 150mm KCl. The volume change is a function of HCl leak rate and is abolished by ionophores which eliminate the transport-dependent pH gradient. So ₄ ^–- substitution, which eliminates the overshoot phenomena observed in KCl medium, also eliminates the shrinkage resulting from ATP addition.     

Sulfhydryl oxidase immunoreactivity in seminiferous tubules of infertile men

Summary Sulfhydryl oxidase (SOx) immunoreactivity was investigated in the seminiferous epithelium of human biopsy material from the testes of 33 adult men with disturbed fertility. SOx immunoreactivity was expressed in normal seminiferous epithelium in type-A spermatogonia (27±4% of all spermatogonia) (n=4), in spermatocytes and round spermatids. Mature spermatozoa as well as Sertoli cells were unlabelled. within the interstitium, Leydig cells were immunopositive. In biopsies of oligozoospermic men showing hypospermatogenesis (n=24), an increase in labelled spermatogonia up to more than 90% was observed in biopsies, where seminiferous epithelia revealed only spermatogonia and Sertoli cells. Within the group of oligozoospermic patients there was a significant increase of labelled spermatogonia from 43±13% (>20 mill/ejaculate) (n=7) to 55±16% ( 20 and >20 mill/ejaculate) (n=6) to 68±8% (<5 mill/ejaculate) (n=11) and a significant (P=0.01) decrease of score count from 7.0±2.7 to 2.0±1.8. In this group the increase of labelled spermatogonia was correlated with sperm concentrations in the ajaculate (correlation coefficient: r=-0.6). In biopsies of azoospermic patients showing maturation arrest at the level of spermatocytes or spermatids (n=5) the percentage of labelled spermatogonia was within the range of 24% to 59%. Immunoreactivity in Sertoli cells was only found in single degenerating cells and in tubules showing Sertoli Cell Only Syndrome (SCO) without lumen formation. Sertoli cells within immature seminiferous cords were immunonegative, indicating that Sertoli cell SOx immunoreactivity is rather a sign of physiological alterations in degenerating cells than dependent on the stage of differentiation. Leydig cells did not show changes of immunoreactivity in any biopsy. It is concluded that SOx expression in spermatogonia may serve as a marker for spermatogenic efficiency.     

A functional polymorphism in the PTHR1 promoter region is associated with adult height and BMD measured at the femoral neck in a large cohort of young caucasian women

The parathyroid hormone type 1 receptor (PTHR1) mediates the actions of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHRP). Interacting with this receptor, PTHRP contributes to skeletal development through the regulation of chondrocyte proliferation and differentiation. Recently, a tetranucleotide repeat—(AAAG) _n —in the P3 promoter of the PTHR1 gene has been shown to have functional activity in vitro, and homozygosity for (AAAG)₆, or the 6/6 genotype, has been associated with greater adult height compared to the 5/5 genotype. In this study, we evaluated the association of (AAAG) _n with height and bone mineral density (BMD) measured at lumbar spine (LS) and femoral neck (FN) in a cohort of 677 young caucasian women 18–35 years of age. Genomic DNA was amplified and genotyped by comparison with sequenced controls following electrophoretic separation through high-resolution polyacrylamide gels. Allele frequencies for (AAAG) _n were: 76.8% (n=5); 20.9% (n=6); 1.8% (n=7); 0.18% (n=8); 0.27% (n=9); 0.08% (n=2), and there was no evidence for Hardy–Weinberg disequilibrium. Analysis of variance showed that subjects bearing one or two (AAAG)₆ alleles (6/X & 6/6) were significantly taller (165.7±0.5 cm) than the others (X/X, 164.5±0.3 cm; P=0.034). This association was significant after adjusting for multiple covariates—current age, age at menarche, physical activity, smoking status, and intakes of caffeine and calcium. Comparison of genotype groups for BMD was not significant at LS, but BMD was significantly higher at FN in the group with at least one (AAAG)₆ allele (adjusted means: 1.021±0.008 vs. 0.999±0.006 g/cm², P=0.032). In conclusion, our data show that subjects bearing one or two (AAAG)₆ alleles are taller than subjects without, reinforcing the notion that in vivo variation in promoter activity of the PTHR1 gene may be a relevant genetic influence on final adult height and BMD.     

Effects of angiotensin-II receptor blocker candesartan cilexetil in rats with dilated cardiomyopathy

We examined effects of an angiotensin-II receptor blockers, candesartan cilexetil, in rats with dilated cardiomyopathy after autoimmune myocarditis. Candesartan cilexetil showed angiotensin-II blocking action in a dose-dependent manner in rats with dilated cardiomyopathy. Twenty-eight days after immunization, surviving Lewis rats were divided into four groups and given candesartan cilexetil at 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg per day (Group-C0.05, n = 15, Group-C0.5, n = 15 and Group-C5, n = 15, respectively) or vehicle alone (Group-V, n = 15). After oral administration for 1 month, the left ventricular end-diastolic pressure and heart weight/body weight ratio were lower in Group-C0.05 (13.3± 1.1 mmHg and 3.7± 0.2 g/kg, respectively), in Group-C0.5 (8.0± 0.9 mmHg and 3.3± 0.1 g/kg, respectively) and in Group-C5 (5.5± 1 mmHg and 3.1± 0.1 g/kg, respectively) than in Group-V (13.5± 1.0 mmHg and 3.8± 0.2 g/kg, respectively). The area of myocardial fibrosis was also lower in Group-C0.05 (25± 3%), in Group-C0.5 (20± 3%), and in Group-C5 (12± 1%) than in Group-V (32± 4%). Furthermore, expressions of transforming growth factor-1 and collagen-III mRNA were suppressed in Group-C0.05 (349± 23% and 395± 22%, respectively), Group-C0.5 (292± 81% and 364± 42%, respectively) and in Group-C5 (204± 63% and 259± 33%, respectively) compared with those in Group-V (367± 26% and 437± 18%, respectively). These results suggest that candesartan cilexetil can improve the function of inefficient heart. (Mol Cell Biochem 269: 137–142, 2005)     

Copper and Zinc in the Serum,Urine, and Hair of Patients with Wilson’s Disease Treated with Penicillamine and Zinc

The purpose of this study was to determine the different levels of copper and zinc in the serum, urine, and scalp hair of patients with Wilson’s disease receiving different, currently accepted methods of treatment to reduce the copper load (penicillamine—group 1, n = 8; zinc—group 2, n = 8; penicillamine+zinc—group 3, n = 8). Blood, urine, and hair samples were collected from the patients. All three treatments resulted in a significant decrease of the serum copper levels. Significantly increased levels of zinc in the serum were detected in the patients in groups 2 and 3 (19.1 and 18.8 μmol/l, respectively; p < 0.05). Copper excretion in the urine significantly increased during its administration to groups 1 and 3 (11.5 and 7.94 μmol/24 h respectively; p < 0.001) due to the effect of penicillamine. The administration of zinc as monotherapy (group 2) or in combination with penicillamine (group 3) led to an increase of its excretion (25.3 and 22.4 μmol/24 h, respectively; p < 0.01). Only an insignificant rise of the copper content in the hair was found in all three groups of patients. The content of zinc in the hair did not differ significantly in any of the groups in comparison with the control group.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Determination of 5-aminolevulinic acid in blood plasma, tissues and cell cultures by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method for determining 5-aminolevulinic acid (ALA) in biological samples is described. ALA is derivatized with o-phthaldehyde to give a compound with favorable properties for high-performance liquid chromatography with electrochemical detection. The method does not require extensive pretreatment of the samples and its detection limit is in the range of 1 pmol/20 μl injection. This method was applied to the determination of plasma ALA from normal and lead-exposed subjects, where 0.26±0.08 μM (n=30) and 2.6±0.75 μM (n=30), respectively were found. We also determined ALA in rat tissues, namely liver and brain, and the uptake of ALA by cultured fibroblasts and hepatocytes to illustrate the diversified applicability of the method.     

Effects of halothane,isoflurane, sevoflurane and desflurane on contraction of ventricular myocytes from streptozotocin-induced diabetic rats

Various clinically used volatile general anaesthetics (e.g. sevoflurane, halothane, isoflurane and desflurane) have been shown to have significant negative inotropic effects on normal ventricular muscle. However, little is known about their effects in ventricular tissue from diabetic animals. Streptozotocin (STZ)-induced diabetes is known to induce changes in the amplitude and time course of shortening and one report suggests that the inotropic effects of anaesthetics are ameliorated in papillary muscles from diabetic animals. The aim of these studies was to investigate this further in electrically stimulated (1 Hz) ventricular myocytes. Cells were superfused with either normal Tyrode (NT) solution or NT containing anaesthetic (1 mM) for a period of 2 min (at 30–32°C). Myocytes from STZ rats were shown to have a significantly longer time to peak shortening (p > 0.001, n= 50) and the amplitude of shortening tended to be greater but this was not significant (p= 0.13, n= 50). Halothane, isoflurane, desflurane and sevoflurane significantly (p < 0.05) reduced the magnitude of shortening of control cells by 72.5 ± 3.2%, 46.5 ± 9.7%, 28.9 ± 4.3% and 22.8 ± 5.6%, respectively (n > 11 per group) but their steady-state negative inotropic effect was found to be no different in cells from STZ-treated rats (73.0 ± 4.8%, 40.7 ± 4.7%, 25.0 ± 5.2% and 19.8 ± 5.2%, respectively, n > 10 per group). Therefore, we conclude that the inotropic effects of volatile anaesthetics were not altered by STZ treatment. (Mol Cell Biochem 261: 209–215, 2004)     

Diversity of K+ channels in the basolateral membrane of restingnecturus oxyntic cells

Summary Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa ofNecturus maculosa. In cell-attached patches with pipette solution containing 100mm KCl, four major classes of K⁺ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec fora, and 8 msec forb). Some channels showed subconductance levels. The maximal inward conductanceg _max was 31±5 pS (n=13) and the reversal potentialE _rev was atV _p=–34±6 mV (n=9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels withg _max=40 ±18 pS (n=3) andE _rev was atV _p=–31±5 mV (n=3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1mm) to the bath solution in cell-attached patches increased the open probabilityP _o of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels withg _max=21±3 pS (n=4) andE _rev was atV _p=–24±9 mV (n=4). These channels exhibited very high open probability (P _o=0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26±2 pS (n=3) andE _rev atV _p=–33±3 mV (n=3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K⁺ in the pipette solution replaced by Na⁺, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K⁺ over Na⁺ and Cl^–. A variety of K⁺ channels contributes to the basolateral K⁺ conductance of resting oxyntic cells.     

Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively

We have separated and quantified two new minor hemoglobins named HbA_1d3a and HbA_1d3b. The level of HbA_1d3a was significantly higher in uremic than in non-uremic patients (3.00 ± 0.50% vs. 1.28 ± 0.26% of total hemoglobin). It correlated well with carbamylated hemoglobin (r=0.80, n=81, p<0.002) and with plasma urea concentration (r=0.78, n=81, p<0.002). These data and the electrospray ionization mass spectrometric analysis provide strong evidence that HbA_1d3a is an α-chain modified by carbamylation. The HbA_1d3b level in the diabetic patients was found to be 1.6-fold that in non-diabetic subjects (3.00 ± 0.49 vs. 1.90 ± 0.33). This was attributed to HbA_1d3 modified by glycation. Indeed HbA_1d3b correlated significantly with HbA_1c (r=0.71, p<0.002) and with serum glucose level (r=0.62, p<0.002). These two new minor hemoglobins may serve as complements for the objective assessment of averagd long-term uremia and glycemia in uremic and diabetic patients.     

Hydraulic Conductivity (Lp) and Its Activation Energy (Ea), Cryoprotectant Agent Permeability (Ps) and ItsEa, and Reflection Coefficients (?) for Golden Hamster Individual Pancreatic Islet Cell Membranes

Long-term cryopreservation of islets of Langerhans would be advantageous to a clinical islet transplantation program. Fundamental cryobiology utilizes knowledge of basic biophysical characteristics to increase the understanding of the preservation process and possibly increase survival rate. In this study several of these previously unreported characteristics have been determined for individual islet cells isolated from Golden hamster islets. Using an electronic particle counting device and a temperature control apparatus, dynamic volumetric response of individual islet cells to anisosmotic challenges of 1.5 M dimethyl sulfoxide (DMSO) and 1.5 M ethylene glycol (EG) were recorded at four temperatures (8, 22, 28, and 37°C). The resulting curves were fitted using Kedem and Katchalsky equations which describe water flux and cryoprotectant agent (CPA) flux based on hydraulic conductivity (L_p), CPA permeability (P_s), and reflection coefficient (?) for the membrane. For Golden hamster islet cells,L_p,P_s, and ? for DMSO at 22°C were found to be 0.23 ± 0.06 μm/min/atm, 0.79 ± 0.32 × 10⁻³cm/min, and 0.55 ± 0.37 (n= 11) (mean ± SD), respectively. For EG at 22°C,L_pequaled 0.23 ± 0.06 μm/min/atm,P_sequaled 0.63 ± 0.20 × 10⁻³cm/min, and ? was 0.75 ± 0.17 (n= 9). Arrhenius plots (lnL_por lnP_sversus 1/temperature (K)) were created by adding the data from the other three temperatures and the resulting linear regression yielded correlation coefficients (r) of 0.99 for all four plots (L_pandP_sfor both CPAs). Activation energies (E_a) ofL_pandP_swere calculated from the slopes of the regressions. The values for DMSO were found to be 12.43 and 18.34 kcal/mol forL_pandP_s(four temperatures, totaln= 52), respectively. For EG,E_aofL_pwas 11.69 kcal/mol andE_aofP_swas 20.35 kcal/mol (four temperatures, totaln= 58).     

Pinocytosis and locomotion in amoebae

Summary Different methods were used to demonstrate the existence of Ca⁺⁺-binding sites (Ca⁺⁺-bs) at the plasma membrane ofAmoeba proteus. In pinocytoting animals the number (indicated by the average distanced in nm) and size (average longitudinal axiss in nm) of Ca⁺⁺-bs at the cytoplasmic surface of the cell membrane were significantly increased (d=162±15;n=41 ands=93±5;n=47) in comparison to controls (d=208 ±21;n=37 ands=59±8;n=45). The ratio of P: Ca obtained by X-ray microanalysis was in the range of 1.5. The differences observed in the two experimental groups of amoebae are explained by conformational changes in the molecular structure and an increased Ca⁺⁺-permeability of the plasma membrane during induced pinocytosis.Microplasmodia of the acellular slime moldPhysarum polycephalum investigated for comparison were found to have no Ca⁺⁺-bs at the interior cell surface.     

Genetic diversity of Listeria monocytogenes serotype 1/2a strains collected in Brazil by Multi-Virulence-Locus Sequence Typing

Listeria monocytogenes is an opportunistic pathogen with the ability to adapt to different environmental conditions, resulting in safety issues for food producers. Foods contaminated by L. monocytogenes can represent a risk if consumed by susceptible individuals such as elderly, pregnant women and the immunocompromised. The aim of this study was to evaluate the genetic diversity of a collection of L. monocytogenes isolated from different matrices in Brazil during the period of 1979–2015. A total of 51 L. monocytogenes serotype 1/2a strains isolated from clinical samples (n = 3) and food samples (n = 48) were characterized by Multi-Virulence-Locus Sequence Typing (MVLST). The strains were assigned to nine virulence types (VT): VT-11 (n = 3, 5·9%), VT-45 (n = 27, 52·9%), VT-59 (n = 11, 21·6%), VT-68 (n = 3, 5·9%), VT-94 (n = 2, 3·9%), VT-107 (n = 2, 3·9%), VT-184 (n = 1, 1·9%), VT-185 (n = 1, 1·9%) and VT-186 (n = 1, 1·9%); and four of them (VT-11, VT-45, VT-59 and VT-68) have already been associated with cases of listeriosis worldwide. The VT-11, VT-59 (Epidemic Clone V) and VT-186 were identified in blood culture samples, as well as in different classes of foods. It is recommended that the epidemiological surveillance agencies evaluate the risk that foods contaminated with L. monocytogenes VTs pose to susceptible populations.     

Aflatoxin concentrations in chilies vary depending on variety

Aflatoxin (AF) contamination was determined in 40 chili samples, representing 3 varieties from Punjab, Pakistan, by HPLC with fluorescence detection. Total AF were determined in varieties Longi (n = 15), Wonder hot (n = 12), and Skyline 1 (n = 13), and the concentrations were high in some samples. AF were detected in 9 (60%), 8 (67%), and 7 (54%) of Longi, Wonder hot, and Skyline 1 samples, respectively; concentration ranged from 4.7 to 34.6, 1.0 to 14.3, and 7.8 to 15.6 μg/kg, respectively. The percentage of samples greater than the European Union statutory limit for AFB₁ and total AF in spices were 53% and 53%, 53% and 8%, and 53% and 38% for Longi, Wonder hot, and Skyline 1, respectively. Significant differences at α < 0.05 between mean concentration of AF in Longi (15.9 ± 0.6 μg/kg) and Wonder hot (5.8 ± 0.3 μg/kg) were determined. In conclusion, choosing the variety demonstrating the lowest AF concentration from various chili samples will assist in improving product quality. This preferred variety is Wonder Hot as described herein.