Toxic effect of valproic acid on tryptophan binding to rat hepatic nuclei

This study evaluated whether valproic acid, a branched-chain fatty acid which has been used in the treatment of seizures, would influence the binding Of L-tryptophan to rat hepatic nuclei. Previous studies have indicated that binding of L-tryptophan to hepatic nuclear envelope protein was saturable, stereospecific, and of high affinity. In this study, we investigated whether valproic acid, which under certain conditions is heptatoxic, would influence L-tryptophan binding to rat hepatic nuclei as assayed by in vitro L-(5-3H)tryptophan binding. Our results indicate that the addition of valproic acid to hepatic nuclei or nuclear envelopes in vitro has little influence on their L-(5-3H)tryptophan binding. On the other hand, when valproic acid (80 mg/100 g body weight) is tube-fed 2 h before killing, the isolated nuclei show decreased specific L-tryptophan binding (total binding minus non-specific binding using unlabeled L-tryptophan (10(-4)M), at 2000-fold excess) compared with controls. Other fatty acids (oleic, palmitic or linoleic acid at 10(-4)M) when added with excess, unlabeled L-tryptophan (10(-4)M) in vitro to hepatic nuclei revealed some (but less than with valproic acid) decreased specific binding compared with controls. At high doses, valproic acid (80 mg/100 g body weight) appears to decrease tryptophan-induced stimulation of hepatic protein synthesis, probably in a hepatotoxic manner.     

DNase I hypersensitive sites far upstream of the rat tryptophan oxygenase gene direct developmentally regulated transcription in livers of transgenic mice

An experimental study on the rat sciatic nerve was performed to evaluate nerve regeneration through a collagen guide and to study the effects of alpha-melanocytic stimulating hormone (alpha-MSH) and basic fibroblast growth factor (b-FGF) in accelerating axonal elongation. After transection, nerves were repaired over a 7 mm gap using a placental collagen type IV guide. The channel was filled with either a b-FGF solution or an alpha-MSH solution or was produced with b-FGF incorporated into the guide. Four weeks later, only groups in which b-FGF had been injected or incorporated displayed a significant somatosensory evoked potential response. Histological and quantitative analysis of nerve fibres confirmed the existence of nerve continuity in groups receiving an alpha-MSH solution or a channel containing b-FGF. These results demonstrate that alpha-MSH in solution and b-FGF incorporated into a collagen type IV channel enhance peripheral nerve regeneration. However, at 4 weeks, only b-FGF (3 ng) restores functional activity.     

Histone deacetylase. A key enzyme for the binding of regulatory proteins to chromatin

Stone and urine composition were analysed in 75 men and 40 women with recurrent calcium oxalate stone disease (group R) and in 48 men and 19 women who had formed only one calcium-oxalate-containing stone (group S). Patients who had developed stones with a large fraction of calcium phosphate were significantly more frequent in group R than in group S. There was furthermore a higher excretion of calcium and higher calcium oxalate supersaturation levels in patients with stones containing more than 25% calcium phosphate. It was concluded from these observations that the calcium phosphate content of renal stones might be a useful factor in predicting the future course of the disease.     

Morphological studies on Mn-SOD, NOS and calcium binding proteins in the rat hippocampus

Head movement propensity-the pattern of head saccades dependent on methods of target presentation-varies among individuals. The present group of 9 young adults was previously ranked in a visual saccadic task according to this propensity. The present report examines how and why this propensity changes if the saccades are made to auditory targets. 1) Spatially identical, interleaved, auditorily and visually elicited horizontal saccadic gaze shifts (jumps) differed in amplitude and in starting and/or ending position. The jumps were executed in two head movement modes: first, the non-aligned mode was a standard reaction-time single gaze step between two points. Second, the head-aligned mode required alignment of the head with the fixation (starting) point; thereafter both modes were identical. All results in the auditory task are expressed relative to the visual results. 2) In the non-aligned mode, head movement amplitudes were increased on average by 15% (for example, an 80 degrees jump elicited a 12 degrees larger head movement), and velocity decreased by 12%, reflecting the increased demands of the auditory task. More importantly, the differences between subjects was narrowed; that is, head movement propensity was homogenized in the auditory task. In the visual task, head-movers willingly move their heads off and across the midline, whereas non-movers are unwilling to leave the midline from eccentric starting points or to eccentric ending points. This is called the midline attraction effect and was previously linked to spatial reference frames. The homogenization in the auditory task was characterized by head-movers increasing, and non-movers decreasing, their midline attraction, suggesting altered spatial reference frames. 3) For heuristic purposes, the ideal head-mover is defined by a gain of 1.0 in the visual task, and by external earth-fixed reference frames. Similarly, the ideal non-mover has a gain of 0.0 and has a bias toward body (or some par of the body)-fixed reference frames. In the auditory task these gains (and reference frames) in head movers and non-movers are homogenized (close to 0.5), either by the participation of the head (movement of the ears in space) in sensory acquisition or by differences in central nervous processing of the two modalities, or both.     

Structural determinants present in the C-terminal binding protein binding site of adenovirus early region 1A proteins

The C-terminal binding protein (CtBP) has previously been shown to bind to a highly conserved six-amino acid motif very close to the C terminus of adenovirus early region 1A (Ad E1A) proteins. We have developed an enzyme-linked immunosorbent assay that has facilitated the screening of synthetic peptides identical or similar to the binding site on Ad E1A for their ability to bind CtBP and thus inhibit its interaction with Ad12 E1A. It has been shown that amino acids both C-terminal and N-terminal to the original proposed binding site contribute to the interaction of peptides with CtBP. Single amino acid substitutions across the binding site appreciably alter the Kd of the peptide for CtBP, indicative of a marked reduction in the affinity of the peptide for CtBP. The solution structures of synthetic peptides equivalent to the C termini of both Ad5 and Ad12 E1A and two substituted forms of these have been determined by proton NMR spectroscopy. Both the Ad12 and Ad5 peptides dissolved in trifluoroethanol/water mixtures were found to adopt regular secondary structural conformations seen as a series of beta-turns. An Ad12 peptide bearing a substitution that resulted in only very weak binding to CtBP (Ad12 L258G) was found to be random coil in solution. However, a second mutant (Ad12 V256K), which bound to CtBP rather more strongly (although not as well as the wild type), adopted a conformation similar to that of the wild type. We conclude that secondary structure (beta-turns) and an appropriate series of amino acid side chains are necessary for recognition by CtBP.     

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding

To examine a role for the medullary nucleus paragigantocellularis (PGi) in mediation of the symptomatology of opioid withdrawal, bilateral electrical stimulation of the PGi was performed in conscious, unrestrained, opioid naive (nondependent) rats. A characteristic series of behaviors was elicited during each 30-min session of PGi stimulation. The profile of these behaviors resembled qualitatively, but was not quantitatively identical with those seen during precipitated withdrawal from opioid dependence. This behavioral syndrome has been termed, opioid withdrawal-like behavior. The opioid withdrawal-like behaviors were voltage-, but not frequency-, dependent. Tolerance to repeated stimulation of the PGi did not develop following a series of 30-min runs of stimulation over 3.5 h. Intracerebroventricular (i.c.v.) injections of the nonselective opioid antagonist, naloxone, significantly decreased (by 40-50%) the intensity of stimulation-induced behavioral responses, as did injections of either the mu-selective (beta-funaltrexamine, beta-FNA) or the delta-selective (naltrindole, NTI) opioid antagonists. In contrast, similar i.c.v. injections of the kappa-selective antagonist, nor-binaltorphimine (nor-BNI), did not block behavioral responses to PGi stimulation. The results indicate that activation of the PGi by electrical stimulation can elicit behaviors similar to those observed during opioid withdrawal. Endogenous opioids, acting through mu- and delta-, but not kappa-opioid receptors, participate in mediating opioid withdrawal-like behaviors induced by PGi stimulation.     

Analysis of proteins that interact with the IL-2 regulatory region in patients with rheumatic diseases

Bone marrow transplantation (BMT) involves conditioning with cyclophosphamide and, for leukemic patients, total body irradiation (TBI). Based on the concern that this may lead to later neuropsychologic impairment in children, a longitudinal study was conducted. Thirty pediatric bone marrow transplant recipients, treated for leukemia or severe aplastic anemia (SAA), and their sibling donors, were given a neuropsychological examination in 1986 and 1988. A third follow-up study of patients treated before 12 years of age was undertaken in 1990-91. We present longitudinal data on patients treated with BMT when 3-11 (n = 15) and 12-17 (n = 11) years old. No neuropsychological deficits were found in the older group, or among non-irradiated SAA patients. In the first follow-up, children treated with BMT, including TBI at 3-11 years of age, performed less well than donors on tasks involving perceptual and fine-motor speed. In the second follow-up, this group of patients also demonstrated a slight deficit in non-verbal problem solving. An additional relative decline in verbal reasoning was noted in the third follow-up, 5.5-10 years after treatment. Alertness to signs of developmental difficulties in children treated with BMT, including TBI, is recommended.     

Nuclear detection of cellular retinoic acid binding proteins I and II with new antibodies

Apart from the retinoic acid nuclear receptor family, there are two low molecular weight (15 kD) cellular retinoic acid binding proteins, named CRABPI and II. Mouse monoclonal and rabbit polyclonal antibodies were raised against these proteins by using as antigens either synthetic peptides corresponding to amino acid sequences unique to CRABPI or CRABPII, or purified CRABP proteins expressed in E. coli. Antibodies specific for mouse and/or human CRABPI and CRABPII were obtained and characterized by immunocytochemistry and immunoblotting. They allowed the detection not only of CRABPI but also of CRABPII in both nuclear and cytosolic extracts from transfected COS-1 cells, mouse embryos, and various cell lines.     

Multiple transitions to non-B-DNA structures occur in the distal regulatory region of the rat prolactin gene

The developmentally regulated rat prolactin (rPRL) gene presents a promising model system toward understanding the biological role of non-B-DNA structural elements. Two predominantly alternating purine-pyrimidine (APP) (dA-dC)n.(dG-dT)n repeats of 58 and 178 base-pairs flank the (A + T)-rich distal regulatory region. We have characterized several transitions to non-B-DNA structures within this region in negatively supercoiled plasmids by utilizing high resolution chemical probing. Each repeat undergoes a full-length conversion to a novel left-handed helical structure via the stepwise nucleation and propagation of discrete "segments". These segments are delimited by out-of-alternation bases that are susceptible to attack by potassium permanganate and thus appear to be significantly unstacked within the left-handed helices. Moreover, the spatial order of successive right- to left-handed DNA transitions within each repeat exhibits a clear polarity toward the distal regulatory region of the rPRL gene. An additional transition involving the long-range unpairing of (A + T)-rich sequences establishes a directional propagation toward the regulatory region. These data demonstrate a complex series of quasi-independent transitions to non-B-DNA structures that impinge upon a known regulatory control region.     

Evidence for the covalent binding of hydroxyethyl radicals to rat liver microsomal proteins

It is well known that acetaldehyde is capable of covalent binding to liver proteins. However, in experiments using liver microsomes prepared from chronically ethanol-fed rats we have observed that the addition of EDTA-iron complex to the microsomes increases by about 4-5 fold both the spin trapping of hydroxyethyl radicals and the covalent binding of 14C-ethanol to proteins, while it only doubles acetaldehyde formation. Conversely, the presence of GSH strongly decreases the trapping of hydroxyethyl radicals and completely inhibits the covalent binding, without affecting acetaldehyde production. Furthermore, the spin trapping agent 4-pyridyl-N-oxide-t-butyl nitrone (4-POBN), previously employed for the detection of hydroxyethyl radicals, decreases by about 70% the covalent binding of 14C-ethanol to microsomal proteins. 4-POBN does not affect acetaldehyde production by liver microsomes, nor does it interfere with the covalent binding of acetaldehyde produced by ADH-mediated oxidation of ethanol. The results obtained indicate that hydroxyethyl radicals generated during ethanol oxidation by cytochrome P-450 play an important role in the alkylation of microsomal proteins consequent to ethanol metabolism.     

Role of the hypervariable region in streptococcal M proteins: binding of a human complement inhibitor

Antigenic variation allows pathogenic microorganisms to evade the immune system of the infected host. The variable structure must play an important role in pathogenesis, but its function is in most cases unknown. Here, we identify a function for the surface-exposed hypervariable region of streptococcal M5 protein, a virulence factor that inhibits phagocytosis. The hypervariable region of M5 was found to bind the human complement inhibitor FHL-1 (factor H-like protein 1), a 42-kDa plasma protein. Plasma absorption experiments with M5-expressing bacteria showed that the interaction with FHL-1 occurs also under physiologic conditions. Studies of another extensively characterized M protein, M6, indicated that this protein also has a binding site for FHL-1 in the hypervariable region. The complement-inhibitory function of FHL-1 was retained after binding to streptococci, suggesting that bound FHL-1 protects bacteria against complement attack. All available data now indicate that FHL-1, or another human complement inhibitor, binds to the hypervariable region of M proteins. These findings provide insights into the forces that drive antigenic variation and may explain why the hypervariable region of M protein is essential for phagocytosis resistance. Moreover, these data add to a growing body of evidence that human complement inhibitors are major targets for pathogenic microorganisms.     

Photoaffinity labeling of peroxisome proliferator binding proteins in rat hepatocytes; dehydroepiandrosterone sulfate- and bezafibrate-binding proteins

Neuronal nitric oxide synthase produces nitric oxide, a radical involved in neurotransmission as well as in cytotoxicity during stroke and neurodegenerative diseases. In the adult Wistar rat neuronal nitric oxide synthase-positive neurons are inhomogenously distributed along defined cortical areas, with highest densities (18 cells/mm2) in cingular area 1, piriform cortex, frontal motor area Fr 2 and in the medial visual association area Oc 2MM. A medium packing density of neuronal nitric oxide synthase neurons (10/mm2) characterizes primary sensory areas, whereas retrosplenial cortices contain lowest cell numbers (3-5/mm2). The data suggest that functions of certain cortical areas are more dependent on intracortically produced nitric oxide than others, and that cortical injury may cause more severe nitric oxide related cytotoxicity in areas with higher numbers of neuronal nitric oxide synthase-positive neurons.