Mutational analysis of the cystathionine beta-synthase gene: A splicing mutation, two missense mutations and an insertion in patients with homocystinuria

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G-->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G-->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA. (C) 1998 Wiley-Liss, Inc.

A novel tricopper(II) complex of a polyamine alcohol

The trinuclear copper(II) complex of a new polyamino alcohol ligand has been isolated; it exhibits a structure similar to that found at the active site of ascorbate oxidase.

Role of generalized and episode specific memories in the word frequency effect in recognition

Frequency, recency, and type of prior exposure to very low-and high-frequency words were manipulated in a 3-phase (i.e., familiarization training, study, and test) design. Increasing the frequency with which a definition for a very low-frequency word was provided during familiarization facilitated the word's recognition in both yes-no (Experiment 1) and forced-choice paradigms (Experiment 2). Recognition of very low-frequency words not accompanied by a definition during familiarization first increased, then decreased as familiarization frequency increased (Experiment I). Reasons for these differences were investigated in Experiment 3 using judgments of recency and frequency. Results suggested that prior familiarization of a very low-frequency word with its definition may allow a more adequate episodic representation of the word to be formed during a subsequent study trial. Theoretical implications of these results for current models of memory are discussed.

Chronic ethanol treatment leads to increased ornithine decarboxylase activity: Implications for a role of polyamines in ethanol dependence and withdrawal

Recent research has focused on the N-methyl-D-aspartate receptor system as a major site of ethanol action in the brain and specifically on compensatory changes in the expression of the polyamine-sensitive NR2B subunit. Therefore, we examined the effects of chronic ethanol treatment on polyamine homeostasis in the rat brain. Wistar rats were made dependent by ethanol vapor inhalation. This caused a rise in hippocampal ornithine decarboxylase (ODC) activity that was correlated with the appearance of physiological dependence. ODC activity returned to control levels within 3 days of ethanol withdrawal. Enzyme activity also increased in the cerebral cortex, striatum, and cerebellum of the ethanol-dependent rats. The concentration of the polyamines (putrescine, spermidine, and spermine) in the hippocampus was increased in ethanol-dependent rats. Injection of the ODC inhibitor, gamma-difluoromethylornithine (500 mg/kg) at the onset of withdrawal resulted in a significant reduction in the severity of withdrawal behaviors. The level of ODC activity and the severity of withdrawal behaviors were positively correlated. Perturbed polyamine homeostasis may represent an important molecular component in the initiation of ethanol withdrawal behaviors in the ethanol-dependent rat.

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli. We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gets. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE(22-88) fragment forms hetero-oligomers with MinE(+) when the proteins are co-expressed. In contrast, the MinE(36-88) fragment does not form MinE(+)/MinE(36-88) hetero-oligomers, although MinE36-88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE(+) in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of mini-celling by expression of MinE(36-88) in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE(36-88),nd the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.

Temperature-induced bleaching of corals begins with impairment of the CO2 fixation mechanism in zooxanthellae

The early effects of heat stress on the photosynthesis of symbiotic dinoflagellates (zooxanthellae) within the tissues of a reef-building coral were examined using pulse-amplitude-modulated (PAM) chlorophyll fluorescence and photorespirometry. Exposure of Stylophora pistillata to 33 and 34 degrees C for 4 h resulted in (1) the development of strong non-photochemical quenching (qN) of the chlorophyll fluorescence signal, (2) marked decreases in photosynthetic oxygen evolution, and (3) decreases in optimal quantum yield (F-v/F-m) of photosystern II (PSII), Quantum yield decreased to a greater extent on the illuminated surfaces of coral branches than on lower (shaded) surfaces, and also when high irradiance intensities were combined with elevated temperature (33 degrees C as opposed to 28 degrees C), qN collapsed in heat-stressed samples when quenching analysis was conducted in the absence of oxygen, Collectively, these observations are interpreted as the initiation of photoprotective dissipation of excess absorbed energy as heat (qN) and O-2-dependent electron flow through the Mehler-Ascorbate-Peroxidase cycle (MAP-cycle) following the point at which the rate of light-driven electron transport exceeds the capacity of the Calvin cycle. A model for coral bleaching is proposed whereby the primary site of heat damage in S, pistillata is carboxylation within the Calvin cycle, as has been observed during heat damage in higher plants, Damage to PSII and a reduction in F-v/F-m (i.e. photoinhibition) are secondary effects following the overwhelming of photoprotective mechanisms by light. This secondary factor increases the effect of the primary variable, temperature. Potential restrictions of electron flow in heat-stressed zooxanthellae are discussed with respect to Calvin cycle enzymes and the unusual status of the dinoflagellate Rubisco, Significant features of our model are that (1) damage to PSII is not the initial step in the sequence of heat stress in zooxanthellae, acid (2) light plays a key secondary role in the initiation of the bleaching phenomena.

Structural basis of autoregulation of phenylalanine hydroxylase

Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an alpha-beta sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.

Molecular recognition of macrocyclic peptidomimetic inhibitors by HIV-1 protease

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a P-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 Angstrom N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba(67,95)]HIVPR and [Lys(7),Ile(33),Aba(67,95)]- HIVPR used in this work were shown to have very similar crystal structures.

PAM chlorophyll fluorometry: a new in situ technique for stress assessment in scleractinian corals, used to examine the effects of cyanide from cyanide fishing

Sodium cyanide is being used on reefs in the Asia-Pacific region to capture live fish for the aquarium industry, and to supply a rapidly growing, restaurant-based demand, The effects of cyanide on reef biota have not been fully explored. To investigate its effect on hard corals, we exposed small branch lips of Stylophora pistillata and Acropora aspera to cyanide concentrations estimated to occur during cyanide fishing. Pulse amplitude modulation (PAM) chlorophyll fluorescence techniques were used to examine photoinhibition and photosynthetic electron transport in the symbiotic algae (zooxanthellae) in the tissues of the corals, These measurements were made in situ and in real time using a recently developed submersible PAM fluorometer. In S. pistillata. exposure to cyanide resulted in an almost complete cessation in photosynthetic electron transport rate. Both species displayed marked decreases in the ratio of variable fluorescence (F-v) to maximal fluorescence (F-m) (dark-adapted F-v/F-m), following exposure to cyanide, signifying a decrease in photochemical efficiency. Dark-adapted F-v/F-m recovered to normal levels in similar to 6 d, although intense tissue discolouration, a phenomenon well-recognised as coral 'bleaching' was observed during this period, Bleaching was caused by loss of zooxanthellae from the coral tissues, a well-recognised sub-lethal stress response of corals. Using the technique of chlorophyll fluorescence quenching analysis, corals exposed to cyanide did not show light activation of Calvin cycle enzymes and developed high levels of non-photochemical quenching (q(N)), signifying the photoprotective dissipation of excess light as heat, These features are symptomatic of the known properties of cyanide as an inhibitor of enzymes of the Calvin cycle. The results of this in situ study show that an impairment of zooxanthellar photosynthesis is; the site of cyanide-mediated toxicity, and is the cue that causes corals to release their symbiotic zooxanthellac following cyanide exposure. This study demonstrates the efficacy of PBM fluorometry as a new tool for in situ stress assessment in zooxanthellate scleractinian corals. (C) 1999 Elsevier Science Ltd. All rights reserved.

Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that Of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity:for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not,necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within I h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

The binding of closantel to ovine serum albumin, and homogenate fractions of Haemonchus contortus

Closantel binds to the serum proteins of the host and affects blood sucking parasites when they ingest the brood of treated hosts. Closantel binds specifically to ovine serum albumin (K-a of 9.3 x 10(6)M(-1)) at site I, the warfarin/phenylbutazone binding site of albumin Closantel also binds to invertebrate haemocyanin and haemolymph. The strongest binding of closantel in homogenates of H. contortus is found in fractions containing soluble proteins. This binding is of low affinity and, because the site itself is not fully denaturable, it may not be proteinaceous. There is no detectable difference in binding affinity between homogenate fractions from closantel susceptible and resistant isolates of adult or larval worms suggesting that closantel resistance is not due to changes in the closantel receptor or carrier. (C) 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Sporadic and familial pheochromocytomas are associated with loss of at least two discrete intervals on chromosome 1p

Pheochromocytomas are tumors of the adrenal medulla originating in the chromaffin cells derived from the neural crest. Ten % of these tumors are associated with the familial cancer syndromes multiple endocrine neoplasia type 2, von Hippel-Lindau disease (VHL), and rarely, neurofibromatosis type 1, in which germ-line mutations have been identified in RET, VHL, and NF1, respectively. In both the sporadic and familial forms of pheochromocytoma, allelic loss at 1p, 3p, 17p, and 22q has been reported, yet the molecular pathogenesis of these tumors is largely unknown. Allelic loss at chromosome 1p has also been reported in other endocrine tumors, such as medullary thyroid cancer and tumors of the parathyroid gland, as well as in tumors of neural crest origin including neuroblastoma and malignant melanoma, In this study, we performed fine structure mapping of deletions at chromosome 1p in familial and sporadic pheochromocytomas to identify discrete regions likely housing tumor suppressor genes involved in the development of these tumors. Ten microsatellite markers spanning a region of similar to 70 cM (Ipter to 1p34.3) were used to screen 20 pheochromocytomas from 19 unrelated patients for loss of heterozygosity (LOH). LOH was detected at five or more loci in 8 of 13 (61%)sporadic samples and at five or more loci in four of five (80%) tumor samples from patients with multiple endocrine neoplasia type 2. No LOH at 1p was detected in pheochromocytomas from two VHL patients, Analysis of the combined sporadic and familial tumor data suggested three possible regions of common somatic loss, designated as PCI (D1S243 to D1S244), PC2 (D1S228 to D1S507), and PC3 (D1S507 toward the centromere). We propose that chromosome Ip may be the site of at least three putative tumor suppressor loci involved in the tumorigenesis of pheochromocytomas. At least one of these loci, PC2 spanning an interval of <3.8 cM, is Likely to have a broader role in the development of endocrine malignancies.

Epidural anaesthesia and analgesia and outcome of major surgery: a randomised trial

Background Epidural block is widely used to manage major abdominal surgery and postoperative analgesia, but its risks. and benefits are uncertain. We compared adverse outcomes in high-risk patients managed for major surgery with epidural block or alternative analgesic regimens with general anaesthesia in a multicentre randomised trial. Methods 915 patients undergoing major abdominal surgery with one of nine defined comorbid states to identify high-risk status were randomly assigned intraoperative epidural anaesthesia and postoperative epidural analgesia for 72 h with general anaesthesia (site of epidural selected to provide optimum block) or control. The primary endpoint was death at 30 days or major postsurgical morbidity. Analysis by intention to treat involved 447 patients assigned epidural and 441 control. Findings 255 patients (57.1%) in the epidural group and 268 (60.7%) in the control group had at least one morbidity endpoint or died (p=0.29). Mortality at 30 days was low in both groups (epidural 23 [5.1%], control 19 [4.3%], p=0.67). Only one of eight categories of morbid endpoints in individual systems (respiratory failure) occurred less frequently in patients managed with epidural techniques (23% vs 30%, p=0.02). Postoperative epidural analgesia was associated with lower pain scores during the first 3 postoperative days. There were no major adverse consequences of epidural-catheter insertion. Interpretation Most adverse morbid outcomes in high-risk patients undergoing major abdominal surgery are not reduced by use of combined epidural and general anaesthesia and postoperative epidural analgesia. However, the improvement in analgesia, reduction in respiratory failure, and the low risk of serious adverse consequences suggest that many high-risk patients undergoing major intra-abdominal surgery will receive substantial benefit from combined general and epidural anaesthesia intraoperatively with continuing postoperative epidural analgesia.

ERP 'old/new' effects: memory strength and decisional factor(s)

Event-related potentials (ERPs) were recorded while subjects made old/new recognition judgments on new unstudied words and old words which had been presented at study either once ('weak') or three times ('strong'). The probability of an 'old' response was significantly higher for strong than weak words and significantly higher for weak than new words. Comparisons were made initially between ERPs to new, weak and strong words, and subsequently between ERPs associated with six strength-by-response conditions. The N400 component was found to be modulated by memory trace strength in a graded manner. Its amplitude was most negative in new word ERPs and most positive in strong word ERPs. This 'N400 strength effect' was largest at the left parietal electrode (in ear-referenced ERPs). The amplitude of the late positive complex (LPC) effect was sensitive to decision accuracy (and perhaps confidence). Its amplitude was larger in ERPs evoked by words attracting correct versus incorrect recognition decisions. The LPC effect had a left > right, centro-parietal scalp topography (in ear-referenced ERPs). Hence, whereas, the majority of previous ERP studies of episodic recognition have interpreted results from the perspective of dual-process models, we provide alternative interpretations of N400 and LPC old/new effects in terms of memory strength and decisional factor(s). (C) 2002 Elsevier Science Ltd. All rights reserved.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: Phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (K-i 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.