Membrane transport of long-chain fatty acids: evidence for a facilitated process

In mammalian cells, membrane uptake of long-chain fatty acids is mediated by two separate components; a passive component that is a linear function of the concentration of free fatty acid in the extracellular medium and a saturable component that exhibits the characteristics of a protein-facilitated process. This review summarizes the body of work that has accumulated related to the mechanism of fatty acid transport. Evidence in support of a facilitated uptake process is presented with relation to the different cell types or membrane systems where it was collected. The evidence includes saturation kinetics, competition between different substrates, and sensitivity to a variety of inhibitors. Recent knowledge related to membrane proteins thought to be implicated in the uptake process is reviewed. Factors that may modulate uptake or alter the relative contribution of passive versus facilitated components are briefly discussed. These include the molar ratio of fatty acid to its physiological carrier, plasma albumin and the metabolic or hormonal milieu.     

The effect of age-dependent increase in fat mass on peripheral insulin action is saturable

Insulin resistance and increased fat mass (FM) are common in human aging. We aimed to investigate the relationship between the age-dependent increase in FM and insulin resistance (by euglycemic hyperinsulinemic clamp technique), in a homogenous rodent model. The decline in insulin responsiveness was linear until late adulthood when body weight, FM, and epididymal fat reached a critical amount (r > .750, for all). Above this critical point, there was no further decline in insulin responsiveness with aging and with increased BW (p < .00001 for all spline curve analyses). This decline in insulin-mediated glucose uptake was accounted for by a decrease in whole body glycolytic rate with no change in the rate of glycogen synthesis. Thus, in this homogenous model, an early increase in FM is associated with impairment in insulin action until a critical FM is achieved, after which there is no additional insulin resistance with aging. We suggest that decreasing insulin responsiveness, in a heterogeneous group such as humans, will only occur within a specific accretion of visceral or total FM.     

No change in NMDA receptor-mediated response rise-time during development: evidence against transmitter spillover

Glutamatergic transmission was examined in tadpole optic tectum to test the possibility that transmitter concentration reaching N-methyl-D-aspartate (NMDA) receptors increases over development. Pharmacologically isolated NMDA receptor-mediated transmission was monitored with whole-cell recordings. Synaptic responses were recorded from cells at different locations in the optic tectum, corresponding to different stages of development. Rise-times and decay-times of NMDA currents were analyzed. We found no significant correlation between rise-time and developmental stage. As NMDA rise-times can correlate with concentration for glutamate concentrations below 200 microM, these results argue that, if there is developmental variation in transmitter concentration, this occurs for values greater than 200 microM. Furthermore, we found a correlation between rise-times and decay-times, consistent with a model in which transmitter concentration is high and rise-time is controlled by channel closings. These results argue against synaptic models in which low concentrations of transmitter (as by spillover from nearby release sites) selectively activates NMDA receptors.     

No evidence for temporal decay in working memory.

What drives forgetting in working memory? Recent evidence suggests that in a complex-span task in which an irrelevant processing task alternates with presentation of the memoranda, recall declines when the time taken to complete the processing task is extended while holding the time for rehearsal in between processing steps constant (Portrat, Barrouillet, & Camos, 2008). This time-based forgetting was interpreted in support for the role of time-based decay in working memory. In this article, we argue the contrary position by (a) showing in an experiment that the processing task in Portrat et al.’s (2008) study gave rise to uncontrolled post-error processes that occupied the attentional bottleneck, thus preventing restorative rehearsal, and (b) showing that when those post-error processes are statistically controlled, there is no evidence for temporal decay in Portrat et al.’s study. We conclude that currently there exists no direct evidence for temporal decay in the complex-span paradigm. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Liver is not essential for solute transport during peritoneal dialysis

Based on theoretical calculations on solute exchange capacities of various peritoneal tissues, the liver has been predicted to account for up to 43% of the permeability-surface area product (PS) of the entire peritoneal "membrane" for a small solute (sucrose) during peritoneal dialysis (PD). In these calculations, the abdominal wall and the diaphragm were found to contribute only approximately 10 to 15% of the total PS. However, evisceration has in previous studies been shown to affect the PS characteristics during PD only marginally (10 to 30%). In such evisceration experiments the liver was usually not removed, and therefore it has been suggested that an intact liver might have significantly contributed to the solute exchange under these premises. We assessed the peritoneal PS of 51Cr-EDTA (constantly infused intravenously) and the plasma-to-peritoneal clearance (ClB-->P) of 125I-human serum albumin (RISA) (given as an i.v. bolus) in Wistar rats during acute PD. In one group of rats the liver surface was sealed off using Histoacrylate glue (N = 6) and in another group a 90% hepatectomy was performed, the remaining portion of the liver, usually the right lower lobe, being sealed off by glue (N = 6). A third group was sham operated to serve as control (N = 12). The PS for 51Cr-EDTA was 0.32 +/- 0.03(+/- SE) ml. min-1 (N = 12) during control, 0.32 +/- 0.04 ml.min-1 after "sealing" of the liver surface (N = 6, P > 0.1) and 0.40 +/- 0.03 after hepatectomy (N = 6, P > 0.1), that is, remained unchanged after experimental intervention. The CIB-->P of RISA during control was 5.88 +/- 1.0 microliter.min-1 (N = 10), and was not altered after hepatectomy, 6.17 +/- 0.48 microliters.min-1 (N = 5, P > 0.1), but slightly increased after liver surface sealing (9.69 +/- 1.09 microliters.min-1, N = 5, P < 0.05). In conclusion, the present experiments indicate that the liver does not play an essential role in the overall solute exchange between the plasma and the peritoneal cavity (PC) during PD.     

Inositol-1,4,5-trisphosphate receptor-mediated Ca mobilization is not required for cerebellar long-term depression in reduced preparations

Inositol-1,4,5-trisphosphate receptor-mediated Ca mobilization is not required for cerebellar long-term depression in reduced preparations. J. Neurophysiol. 80: 2963-2974, 1998. Cerebellar long-term depression (LTD) is a cellular model system of information storage in which coincident parallel fiber and climbing fiber activation of a Purkinje neuron (PN) gives rise to a sustained attenuation of parallel fiber-PN synaptic strength. Climbing fiber and parallel fiber inputs may be replaced by direct depolarization of the PN and exogenous glutamate pulses, respectively. The parallel fiber-PN synapse has a high-density of mGluR1 receptors that are coupled to phosphoinositide turnover. Several lines of evidence indicated that activation of mGluR1 by parallel fiber stimulation is necessary for the induction of cerebellar LTD. Because phosphoinositide hydrolysis has two initial products, 1, 2-diacylglycerol and inositol-1,4,5-trisphosphate (IP3), we wished to determine whether IP3 signaling via IP3 receptors and consequent Ca mobilization were necessary for the induction of cerebellar LTD. First, ratiometric imaging of free cytosolic Ca was performed on both acutely dissociated and cultured PNs. It was determined that the threshold for glutamate pulses to contribute to LTD induction was below the threshold for producing a Ca transient. Furthermore, the Ca transients produced by depolarization alone and glutamate plus depolarization were not significantly different. Second, the potent and selective IP3 receptor channel blocker xestospongin C was not found to affect the induction of LTD in either acutely dissociated or cultured PNs at a concentration that was sufficient to block mGluR1-evoked Ca mobilization. Third, replacement of mGluR activation by exogenous synthetic diacylglycerol in an LTD induction protocol was successful. Taken together, these results suggest that activation of an IP3 signaling cascade is not required for induction of cerebellar LTD in reduced preparations.     

No evidence for radiation-induced clastogenic factors after in vitro or in vivo exposure of human blood

Experiments were performed with human plasma irradiated in vitro or in vivo in order to evaluate the extent to which clastogenic factors might disturb the adaptive response to DNA-damaging factors currently studied in our laboratory. The studies were carried out with plasma isolated from whole blood given 4 Gy of X-rays in vitro and with plasma from people receiving local radiotherapy at a total dose of about 60 Gy gamma rays. Addition of irradiated plasma to culture medium did not result in a statistically significant increase in structural aberrations in chromosomes of non-irradiated normal blood.     

GTP hydrolysis is not important for Ypt1 GTPase function in vesicular transport

GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37 degrees C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.     

How good a marker is insulin level for insulin resistance?

Epidemiologic studies have correlated fasting and postload insulin levels with the risk of coronary heart disease, assuming that insulin levels are reliable markers of insulin resistance. However, this assumption has not been systematically studied. The author measured insulin response to an oral glucose load and quantitated insulin resistance using the euglycemic hyperinsulinemic clamp technique to evaluate the correlation between insulin level and the degree of insulin resistance in individuals with varying degrees of glucose tolerance. Subjects were randomly selected from previous population studies done in 1987-1989 at the Department of Medicine of the University of Kuopio in east Finland. Altogether, 50 subjects with normal glucose tolerance, 28 with impaired glucose tolerance, and 54 with non-insulin-dependent diabetes mellitus were studied. Correlations of insulin resistance (whole-body glucose uptake in clamp studies) with fasting or postload insulin levels were remarkably consistent, ranging from -0.58 to -0.74 (p < 0.01) in subjects with normoglycemia. In contrast, corresponding correlations were substantially weaker in subjects with impaired glucose tolerance and non-insulin-dependent diabetes. Among these subjects, only the fasting insulin level correlated significantly with insulin resistance (-0.47, p < 0.05 and -0.48, p < 0.01, respectively). The authors conclude that in population studies, only the fasting insulin level should be used as a marker of insulin resistance, particularly in subjects with abnormal glucose tolerance.     

SNAP-23 is not cleaved by botulinum neurotoxin E and can replace SNAP-25 in the process of insulin secretion

The synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in neurons and endocrine cells. It has been shown to play an important role in the release mechanism of neurotransmitters and peptide hormones, including insulin. Thus, when insulin-secreting cells are permeabilized and treated with botulinum neurotoxin E (BoNT/E), SNAP-25 is hydrolyzed, and insulin secretion is inhibited. Recently SNAP-23, a more generally expressed isoform of SNAP-25, has been described. The functional role of SNAP-23 has not been investigated to date. It is now shown that SNAP-23 is resistant to cleavage by BoNT/E. It was therefore possible to test whether transfection of HIT (transformed pancreatic B-) cells with SNAP-23 reconstitutes insulin release from BoNT/E treated cells, in which SNAP-25 is inactivated by the toxin. The results show that SNAP-23 is able to replace SNAP-25 when it is overexpressed. While these results demonstrate that SNAP-23 is a functional homologue of SNAP-25, able to function in regulated exocytosis, they indicate that SNAP-23 may be inefficient in this process. This suggests that both isoforms may have their own specific binding partners and discrete, albeit mechanistically similar, functional roles within the cell.     

Functional evidence for UDP-galactose transporter in Saccharomyces cerevisiae through the in vivo galactosylation and in vitro transport assay

The oligosaccharide profiles in glycoproteins are determined by a series of processing reactions catalyzed by Golgi glycosyltransferases and glycosidases. Recently in vivo galactose incorporation in Saccharomyces cerevisiae has been demonstrated through the expression of human beta-1,4-galactosyltransferase in an alg1 mutant, suggesting the presence of a UDP-galactose transporter in S. cerevisiae (Schwientek, T., Narimatsu, H., and Ernst, J. F. (1996) J. Biol. Chem. 271, 3398-3405). However, this is quite unexpected, because S. cerevisiae does not have galactose residues in its glycoproteins. To address this question we have constructed S. cerevisiae mnn1 mutant strains expressing Schizosaccharomyces pombe alpha-1,2-galactosyltransferase. The mnn1 mutant of S. cerevisiae provides endogenous acceptors for galactose transfer by the expressed alpha-1,2-galactosyltransferase. We present here three lines of evidences for the existence of UDP-galactose transporter in S. cerevisiae. (i) About 15-20% of the total transformed mnn1 cells grown in a galactose medium were stained with fluorescein isothiocyanate-conjugated alpha-galactose-specific lectin, indicating the presence of alpha-galactose residues on the cell surface. (ii) Galactomannan proteins can be precipitated with agarose-immobilized alpha-galactose-specific lectin from a whole cell lysate prepared from transformed mnn1 cells grown in a galactose medium. (iii) The presence of UDP-galactose transporter was demonstrated by direct transport assay. This transport in S. cerevisiae is dependent on time, temperature, and protein concentration and is inhibited by nucleotide monophosphate and Triton X-100. The overall UDP-galactose transport in S. cerevisiae is comparable with that in S. pombe, indicating a more or less similar reaction velocity, while the rate of GDP-mannose transport is higher in S. pombe than in S. cerevisiae.     

Deficits in other-race face recognition: No evidence for encoding-based effects.

The other-race effect (ORE) in face recognition is typically observed in tasks which require long-term memory. Several studies, however, have found the effect early in face encoding (Lindsay, Jack, & Christian, 1991; Walker & Hewstone, 2006). In 6 experiments, with over 300 participants, we found no evidence that the recognition deficit associated with the ORE reflects deficits in immediate encoding. In Experiment 1, with a study-to-test retention interval of 4 min, participants were better able to recognise White faces, relative to Asian faces. Experiment 1 also validated the use of computer-generated faces in subsequent experiments. In Experiments 2 through 4, performance was virtually identical to Asian and White faces in match-to-sample, immediate recognition. In Experiment 5, decreasing target-foil similarity and disrupting the retention interval with trivia questions elicited a re-emergence of the ORE. Experiments 6A and 6B replicated this effect, and showed that memory for Asian faces was particularly susceptible to distraction; White faces were recognised equally well, regardless of trivia questions during the retention interval. The recognition deficit in the ORE apparently emerges from retention or retrieval deficits, not differences in immediate perceptual processing. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Expectancy challenge and drinking reduction: Experimental evidence for a mediational process.

Substantial correlational evidence supports a causal (mediational) interpretation of alcohol expectancy operation, but definitive support requires a true experimental test. Thus, moderately to heavily drinking male college students were randomly assigned to 1 of 3 conditions in a pre–post design: expectancy challenge (designed to manipulate expectancy levels), "traditional" information, and assessment-only control. Expectancy challenge produced significant drinking decreases, compared with the other 2 groups. Decreases in measured expectancies paralleled drinking decreases in the challenge condition. Significant increases in alcohol knowledge in the traditional program were not associated with decreased drinking. These experimental findings support a causal (mediational) interpretation of expectancy operation. The implications for a cognitive (memory) model of expectancies and for prevention and intervention programs for problem drinking and alcoholism are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

No evidence for menstrual synchrony in lesbian couples

Menstrual synchrony was investigated in a sample of 29 cohabiting lesbian couples, ranging in age from 22 to 48 years. One or both partners kept prospective daily records of variables including menses onset dates, intimate contact, and sexual activity. All women reported daily intimate interaction with their partners; none reported intimate interaction with men. Despite these potentially optimal conditions for the manifestation of synchrony, the differences between dyad members in menses onset dates were distributed randomly, and there was no evidence of convergence. In fact, most dyads exhibited divergence of onset dates. Reasons for lack of synchrony in this sample are discussed; one conclusion is that there is no solid evidence that menstrual synchrony is a stable attribute of past or contemporary human populations.     

Trehalose is not relevant for in vivo activity of sigmaS-containing RNA polymerase in Escherichia coli

The sigmaS- and sigma70-associated forms of RNA polymerase core enzyme (E) of Escherichia coli have very similar promoter recognition specificities in vitro. Nevertheless, the in vivo expression of many stress response genes is strongly dependent on sigmaS. Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of EsigmaS and thereby contributes to promoter selectivity (S. Kusano and A. Ishihama, J. Bacteriol. 179:3649-3654, 1997). However, we demonstrate here that a trehalose-free otsA mutant exhibits growth phase-related and osmotic induction of various sigmaS-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced). We conclude that in vivo trehalose does not play a role in the expression of sigmaS-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.     

The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo

We produced defined isogenic Helicobacter pylori ureI mutants to investigate the function of UreI, the product of one of the genes of the urease cluster. The insertion of a cat cassette had a strong polar effect on the expression of the downstream urease genes, resulting in very weak urease activity. Urease activity, measured in vitro, was normal in a strain in which ureI was almost completely deleted and replaced with a nonpolar cassette. In contrast to previous reports, we thus found that the product of ureI was not necessary for the synthesis of active urease. Experiments with the mouse-adapted H. pylori SS1 strain carrying the nonpolar ureI deletion showed that UreI is essential for H. pylori survival in vivo and/or colonization of the mouse stomach. The replacement of ureI with the nonpolar cassette strongly reduced H. pylori survival in acidic conditions (1-h incubation in phosphate-buffered saline solution at pH 2.2) in the presence of 10 mM urea. UreI is predicted to be an integral membrane protein and may therefore be involved in a transport process essential for H. pylori survival in vivo.