CORRELATION OF PLASMA AND BRAIN AMINO ACID AND PUTATIVE NEUROTRANSMITTER ALTERATIONS DURING ACUTE HEPATIC COMA IN THE RAT

During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.     

TRYPTOPHAN TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER DURING ACUTE HEPATIC FAILURE

Abstract— Tryptophan transport across the blood-brain barrier was studied using a single injection dual isotope label technique, in the following three conditions: normal rats, rats with portacaval shunts, and rats with portacaval shunts followed 65 h later by hepatic artery ligation. In both normal rats and those with acute hepatic failure the tryptophan transport system was found to be comprised of two kinetically distinct components. One component was saturable and obeyed Michaelis-Menten kinetics (normal: V_max= 19.5 nmol.min^?1.g^?1. K_m= 113 μM; hepatic failure: V_max, = 33.8 nmol.min^?1.g^?1, K_m= 108 μM), and the second was a high capacity system which transported tryptophan in direct proportion to concentration over the range tested (normal: K= 0.026 ml.min^?1.g^?1; hepatic failure: K= 0.067 ml.min^?1.g^?1). Since the saturable low capacity component transports several neutral amino acids, and their collective plasma concentration is high in relation to the individual K_ms, tryptophan transport by this component is reduced by competitive inhibition under physiological conditions. Thus it was calculated that in normal rats approx 40% of tryptophan influx occurs via the high capacity system. During acute hepatic failure transport via both components was increased substantially, approximately doubling the rate of tryptophan penetration of the blood-brain barrier at all concentrations tested. The contribution by the high capacity component became even more significant than in normal rats, accounting for about 75% of all tryptophan passage from plasma to brain. Brain tryptophan content was 29.9 nmol/g in normal rats and rose to 45.2 nmol/g in rats with portacaval shunts and 50.5 nmol/g in those with acute hepatic failure, correlating with the increased rate of tryptophan transport. In a previous study we found that plasma competing amino acids were greatly increased during acute hepatic failure. Calculations predict that these increased concentrations would cause a reduction in tryptophan transport by the low capacity system. However, because of the increase in the rate of transport by the high capacity component, net tryptophan entry across the blood-brain barrier was actually increased. This increased rate of transport clearly contributes to the increased content of brain tryptophan found during hepatic failure.     

A single nonpolar residue in the deep pore of related K+ channels acts as a K+:Rb+ conductance switch.

K+ and Rb+ conductances (GK+ and GRb+) were investigated in two delayed rectifier K+ channels (Kv2.1 and Kv3.1) cloned from rat brain and a chimera (CHM) of the two channels formed by replacing the putative pore region of Kv2.1 with that of Kv3.1. CHM displayed ion conduction properties which resembled Kv3.1. In CHM, GK+ was three times greater than that of Kv2.1 and GRb+/GK+ = 0.3 (compared with 1.5 and 0.7, respectively, in Kv2.1 and Kv3.1). A point mutation in CHM L374V, which restored 374 to its Kv2.1 identity, switched the K+/Rb+ conductance profiles so that GK+ was reduced fourfold, GRb+ was increased twofold, and GRb+/GK+ = 2.8. Quantitative restoration of the Kv2.1 K+/Rb+ profiles, however, required simultaneous point mutations at three nonadjacent residues suggesting the possibility of interactions between residues within the pore. The importance of leucine at position 374 was verified when reciprocal changes in K+/Rb+ conductances were produced by the mutation of V374L in Kv2.1 (GK+ was increased threefold, GRb+ was decreased threefold, and GRb+/GK+ = 0.2). We conclude that position 374 is responsible for differences in GK+ and GRb+ between Kv2.1 and Kv3.1 and, given its location near residues critical for block by internal tetraethylammonium, may be part of a cation binding site deep within the pore.     

Plant dihydroxyacetone phosphate reductases : purification, characterization, and localization

A cytosolic form of dihydroxyacetone phosphate (DHAP) reductase was purified 200,000-fold from spinach (Spinacia oleracea L.) leaves to apparent electrophoretic homogeneity. The purification procedure included anion-exchange chromatography, gel filtration, hydrophobic chromatography, and dye-ligand chromatography on Green-A and Red-A agaroses. The enzyme, prepared in an overall yield of 14%, had a final specific activity of about 500 μmol of DHAP reduced min⁻¹ mg⁻¹ protein, a subunit molecular mass of 38 kD, and a native molecular mass of 75 kD. A chloroplastic isoform of DHAP reductase was separated from the cytosolic form by anion-exchange chromatography and partially purified 56,000-fold to a specific activity of 135 μmol min⁻¹ mg⁻¹ protein. Antibodies generated in rabbits against the cytosolic form did not cross-react with the chloroplastic isoform. The two reductases were specific for NADH and DHAP. Although they exhibited some dissimilarities, both isoforms were severely inhibited by higher molecular weight fatty acyl coenzyme A esters and phosphohydroxypyruvate and moderately inhibited by nucleotides. In contrast to previous reports, the partially purified chloroplastic enzyme was not stimulated by dithiothreitol or thioredoxin, nor was the purified cytosolic enzyme stimulated by fructose 2,6-bisphosphate. A third DHAP reductase isoform was isolated from spinach leaf peroxisomes that had been prepared by isopycnic sucrose density gradient centrifugation. The peroxisomal DHAP reductase was sensitive to antibodies raised against the cytosolic enzyme and had a slightly smaller subunit molecular weight than the cytosolic isoform.     

Neuraxin corresponds to a C-terminal fragment of microtubule-associated protein 5 (MAP5)

From cloned DNA, neuraxin has been identified as a tubulin binding protein of predicted molecular weight of 94 kDa. The deduced sequence of the rat protein exhibits high homology to the C-terminal region of mouse microtubule-associated protein 5 (MAP5). Here, we show that different neuraxin antibodies recognize MAP5, but fail to detect a protein of 94 kDa, in subcellular and microtubular fractions of the rat central nervous system. Furthermore, tubulin binding by neuraxin was found to be dependent on taxol. These data are consistent with neuraxin corresponding to a C-terminal fragment of MAP5 that contains a low-affinity tubulin binding site.     

Expression of subregion and haplotype preference for H-2Kk restricted cytotoxic T-cell responses in vivo and at the level of the precursor frequencies

Several strains of mice bearing the H-2K^k allele were found to generate in vivo strong CTL responses against TNP-haptenated syngeneic cells, while several other strains of mice were found to generate comparably weak or no responses. C3H × DBA/2)F1 mice (H-2^k × H-2^d) and A/J mice with the recombinant haplotype $\text{H-2}{}^{\mathrm{<mtext>k</mtext><mtext>d</mtext>}}$ generated CTL responses in vivo that were completely restricted toward the H-2^k haplotype or the K end of the $\text{H-2}{}^{\mathrm{<mtext>k</mtext><mtext>d</mtext>}}$ haplotype, respectively. The CTL activity of C3H × DBA/2)F1 and A/J mice against haptenated H-2^k targets was found to be more than 25-fold higher than the CTL activity on H-2^d targets. The CTL responses in vitro under macroculture conditions showed, on the other hand, only a 3- to 6-fold higher cytotoxic activity against the haptenated H-2^k targets as compared with haptenated allogeneic or H-2^d targets; and limiting dilution experiments in microcultures revealed that the CTL precursor frequencies were only 2- to 3-fold smaller for TNP-haptenated H-2^d or haptenated allogeneic targets than for haptenated H-2^k target cells. This indicated that sufficient numbers of H-2^d-restricted and allorestricted CTL precursors were actually present in these strains, but did not develop detectable cytotoxic activity in vivo. The exceptional property of the H-2^k haplotype is, therefore, only partly determined by a difference in the CTL precursor frequencies, and to the larger extent determined at the level of the activation of the CTL response.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.     

Process‐based functions for seed retention on animals: a test of improved descriptions of dispersal using multiple data sets

Studies of external seed transport on animals usually assume that the probability of detachment is constant, so that seed retention should show a simple exponential relationship with time. This assumption has not been tested explicitly, and may lead to inaccurate representation of long distance seed dispersal by animals. We test the assumption by comparing the fit to empirical data of simple, two‐parameter functions. Fifty‐two data sets were obtained from five published studies, describing seed retention of 32 plant species on sheep, cattle, deer, goats and mice. Model selection suggested a simple exponential function was adequate for data sets in which seed retention was followed for short periods ( <48 h). The data gathered over longer periods (49–219 days) were best described by the power exponential function, a form of the stretched exponential which allows a changing dropping rate. In these cases the power exponential showed that seed dropping rate decreased with time, suggesting that seeds vary in attachment, with some seeds becoming deeply buried or wound up in the animal's coat. Comparison of fitted parameters across all the data sets also confirmed that seeds with adhesive structures have lower dropping rates than those without. We conclude that the seed dropping rate often changes with time during external transport on animals and that the power exponential is an effective function to describe this change. We advise that, to analyse seed dropping rates adequately, retention should be measured over reasonable time periods – until most seeds are dropped – and both the simple and power exponential functions should be fitted to the resulting data. To increase its utility, we provide functions describing the seed dropping rate and the dispersal kernel resulting from the power exponential relationship.