Lung macro- and microvascular endothelial cells exhibit unique functional attributes, including signal transduction and barrier properties. We therefore sought to identify structural and functional features of endothelial cells that discriminate their phenotypes in the fully differentiated lung. Rat lung macro- (PAEC) and microvascular (PMVEC) endothelial cells each exhibited expression of typical markers. Screening for reactivity with nine different lectins revealed that Glycine max and Griffonia (Bandeiraea) simplicifolia preferentially bound microvascular endothelia whereas Helix pomatia preferentially bound macrovascular endothelia. Apposition between the apical plasmalemma and endoplasmic reticulum was closer in PAECs (8 nm) than in PMVECs (87 nm), implicating this coupling distance in the larger store operated calcium entry responses observed in macrovascular cells. PMVECs exhibited a faster growth rate than did PAECs and, once a growth program was initiated by serum, PMVECs sustained growth in the absence of serum. Thus, PAECs and PMVECs differ in their structure and function, even under similar environmental conditions.

The expression of osteopontin (OPN), CD44 variants, and integrins has been correlated with tumorigenesis and metastasis. Here we show that these proteins cooperate to enhance cell motility. First, we demonstrate that several different CD44 variants bind to OPN in an arginine-glycineaspartic acid-independent manner, but that the standard form of CD44 does not. These CD44 variants bind to both the amino- and COOH-terminal portions of OPN independently of the arginine-glycine-aspartic acid sequence, suggesting that multiple domains on OPN can be bound by the CD44 variants. Antibodies directed against the integrin beta1 subunit are able to inhibit this binding. The binding of CD44 variants to OPN is significantly augmented by both anti-CD44s and anti-CD44v antibodies. This augmentation by anti-CD44 antibodies is OPN specific and, again, can be blocked by anti-beta1 antibodies. Finally, we show that OPN binding by CD44 variants/beta1-containing integrins promotes cell spreading, motility, and chemotactic behavior.

We have expressed glycine receptor (GlyR) alpha and beta subunit cDNAs in HEK-293 cells to study the functional properties of homo- versus hetero-oligomeric GlyR channels. Dose-response curves of whole-cell currents in cells expressing alpha 1 subunits revealed an average Hill coefficient of h = 4.2. Co-expression with the beta subunit markedly increased glycine-gated whole-cell currents, which now exhibited a mean Hill coefficient of only h = 2.5. For alpha 1, alpha 2 and alpha 3 homo-oligomers, the main-state single-channel conductances were 86, 111 and 105 pS, respectively, recorded at symmetrical Cl- concentrations of 145 mM. The mutant alpha 1 G221A gave rise to a main-state of 107 pS. This indicates that the main-state of alpha homo-oligomers depends on residue 221 which is located within transmembrane segment M2. Importantly, the main-state conductances of alpha 1/beta, alpha 2/beta and alpha 3/beta hetero-oligomers were only 44, 54 and 48 pS, respectively. The latter values are similar to those found in spinal neurons, suggesting that native GlyRs are predominantly alpha/beta hetero-oligomers. Co-expression of alpha 1 with mutant beta subunits revealed that residues within and close to segment M2 of the beta subunit determine the conductance differences between homo- and hetero-oligomers.

BACKGROUND

D-serine, a selective full agonist at the glycine site of N-methyl-D-aspartate glutamate receptor, might presently be the compound of choice for counteracting the hypothesized dysfunction of this receptor class in schizophrenia. Studies performed with Taiwanese patients indicate that D-serine significantly improves schizophrenia symptoms when used as adjuvant to conventional neuroleptics but not to clozapine. We assessed the efficacy and safety of D-serine adjuvant treatment for Occidental schizophrenia patients treated with newer atypical antipsychotics.

METHODS

Thirty-nine risperidone- or olanzapine-treated schizophrenia patients participated in a double-blind, placebo-controlled, 6-week crossover trial with 30 mg/kg/day D-serine added to their antipsychotic medication. Measures of clinical efficacy and side effects were determined biweekly throughout the study. Clinical laboratory parameters and amino acid serum levels were monitored.

RESULTS

D-serine administration induced increased serine serum levels (p < .001) and resulted in significant (p < .001) improvements in negative, positive, cognitive, and depression symptoms, as measured by the Positive and Negative Syndrome Scale. For approximately one third of the sample, D-serine treatment resulted in significant (>20%) reductions in Brief Psychiatric Rating Scale total scores. D-serine was well tolerated, and no detrimental changes in clinical laboratory parameters were noted.

CONCLUSIONS

These findings 1) indicate that risperidone and olanzapine efficacy might be augmented with D-serine adjuvant treatment; 2) confirm D-serine efficacy against main schizophrenia symptom domains; and 3) warrant the assessment of D-serine antipsychotic monotherapy for this illness.

Electrospinning of type I collagen in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to fabricate a biomimetic nanofibrous extracellular matrix for tissue engineering was investigated. The average diameter of collagen nanofibers electrospun from 8% collagen solution in HFIP was 460 nm (range of 100-1200 nm). The as-spun collagen nanofibrous matrix was chemically cross-linked by glutaraldehyde vapor with a saturated aqueous solution and then treated with aqueous 0.1m glycine to block unreacted aldehyde groups. With vapor phase cross-linking for 12h, porosity of the collagen matrix decreased from 89% to 71%. The collagen nanofibrous matrix showed good tensile strength, even in aqueous solution. Effects on cytocompatibility, cell behavior, cell and collagen nanofiber interactions, and open wound healing in rats were examined. Relatively low cell adhesion was observed on uncoated collagen nanofibers, whereas collagen nanofibrous matrices treated with type I collagen or laminin were functionally active in responses in normal human keratinocytes. Collagen nanofibrous matrices were very effective as wound-healing accelerators in early-stage wound healing. Our results indicate that cross-linked collagen nanofibers coated with ECM proteins, particularly type I collagen, may be a good candidate for biomedical applications, such as wound dressing and scaffolds for tissue engineering.

Bacillus subtilis was reported to produce the catecholic siderophore itoic acid (2,3-dihydroxybenzoate (DHB)-glycine) in response to iron deprivation. However, by inspecting the DNA sequences of the genes dhbE, dhbB, and dhbF as annotated by the B. subtilis genome project to encode the synthetase complex for the siderophore assembly, various sequence errors within the dhbF gene were predicted and confirmed by re-sequencing. According to the corrected sequence, dhbF encodes a dimodular instead of a monomodular nonribosomal peptide synthetase. We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DhbB, DhbE, and DhbF. DhbE, a stand-alone adenylation domain of 59.9 kDa, activates, in an ATP-dependent reaction, DHB, which is subsequently transferred to the free thiol group of the cofactor phosphopantetheine of the bifunctional isochorismate lyase/aryl carrier protein DhbB. The third synthetase, DhbF, is a dimodular nonribosomal peptide synthetase of 264 kDa that specifically adenylates threonine and, to a lesser extent, glycine and that covalently loads both amino acids onto their corresponding peptidyl carrier domains. To functionally link the dhb gene cluster to siderophore synthesis, we have disrupted the dhbF gene. Comparative mass spectrometric analysis of culture extracts from both the wild type and the dhbF mutant led to the identification of a mass peak at m/z 881 ([M-H](1-)) that corresponds to a cyclic trimeric ester of DHB-glycine-threonine.

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.

The protein coding regions of plastid mRNAs in higher plants are generally flanked by 3' inverted repeat sequences. In spinach chloroplast mRNAs, these inverted repeat sequences can fold into stem-loop structures and serve as signals for the correct processing of the mature mRNA 3' ends. The inverted repeat sequences are also required to stabilize 5' upstream mRNA segments, and interact with chloroplast protein in vitro. To dissect the molecular components involved in chloroplast mRNA 3' end processing and stability, a spinach chloroplast protein extract containing mRNA 3' end processing activity was fractionated by FPLC and RNA affinity chromatography. The purified fraction consisted of several proteins and was capable of processing the 3' ends of the psbA, rbcL, petD and rps14 mRNAs. This protein fraction was enriched for a 28 kd RNA-binding protein (28RNP) which interacts with both the precursor and mature 3' ends of the four mRNAs. Using specific antibodies to this protein, the poly(A) RNA-derived cDNA for the 28RNP was cloned and sequenced. The predicted amino acid sequence for the 28RNP reveals two conserved RNA-binding domains, including the consensus sequences RNP-CS1 and CS2, and a novel acidic and glycine-rich N-terminal domain. The accumulation of the nuclear-encoded 28RNP mRNA and protein are developmentally regulated in spinach cotyledons, leaves, root and stem, and are enhanced during light-dependent chloroplast development. The general correlation between accumulation of the 28RNP and plastid mRNA during development, together with the result that depletion of the 28RNP from the chloroplast protein extract interferes with the correct 3' end processing of several chloroplast mRNAs, suggests that the 28RNP is required for plastid mRNA 3' end processing and/or stability.

The structure of present-day ferredoxin, with its simple, inorganic active site and its functions basic to photon-energy utilization, suggests the incorporation of its prototype into metabolism very early during biochemical evolution, even before complex proteins and the complete modern genetic code existed. The information in the amino acid sequence of ferredoxin enables us to propose a detailed reconstruction of its evolutionary history. Ferredoxin has evolved by doubling a shorter protein, which may have contained only eight of the simplest amino acids. This shorter ancestor in turn developed from a repeating sequence of the amino acids alanine, aspartic acid or proline, serine, and glycine. We explain the persistence of living relics of this primordial structure by invoking a conservative principle in evolutionary biochemistry: The processes of natural selection severely inhibit any change a well-adapted system on which several other essential components depend.

The rate of formation of intramolecular interactions in unfolded proteins determines how fast conformational space can be explored during folding. Characterization of the dynamics of unfolded proteins is therefore essential for the understanding of the earliest steps in protein folding. We used triplet-triplet energy transfer to measure formation of intrachain contacts in different unfolded polypeptide chains. The time constants (1/k) for contact formation over short distances are almost independent of chain length, with a maximum value of about 5 ns for flexible glycine-rich chains and of 12 ns for stiffer chains. The rates of contact formation over longer distances decrease with increasing chain length, indicating different rate-limiting steps for motions over short and long chain segments. The effect of the amino acid sequence on local chain dynamics was probed by using a series of host-guest peptides. Formation of local contacts is only sixfold slower around the stiffest amino acid (proline) compared to the most flexible amino acid (glycine). Good solvents for polypeptide chains like EtOH, GdmCl and urea were found to slow intrachain diffusion and to decrease chain stiffness. These data allow us to determine the time constants for formation of the earliest intrachain contacts during protein folding.

A mutant (gltB) of Escherichia coli lacking glutamate synthase (GOGAT) was unable to utilize a wide variety of compounds as sole nitrogen source (e.g., arginine, proline, gamma-aminobutyrate, and glycine). Among revertants of these Asm- strains selected on one of these compounds (e.g., arginine, proline, or gamma-aminobutyrate) were those that produce glutamine synthetase (GS) constitutively (GlnC phenotype). These revertants had a pleiotropically restored ability to grow on compounds that are metabolized to glutamate. This suggested that the expression of the genes responsible for the metabolism of these nitrogen sources was regulated by GS. An examination of the regulation of proline oxidase confirmed this hypothesis. The differential sensitivities of GlnC and wild-type strains to low concentrations (0.1 mM) of the glutamine analog L-methionine-DL-sulfoximine supported the conclusion that the synthesis of a glutamine permease was also positively controlled by GS. During the course of this study we found that the reported position of the locus (gltB) for glutamate synthase is incorrect. We have relocated this gene to be 44% linked to the argG locus by P1 transduction. Further mapping has shown that the locus previously called aspB is in reality the gltB locus and that the "suppressor" of the aspB mutation (A. M. Reiner, J. Bacteriol. 97:1431-1436, 1969) is the locus for glutamate dehydrogenase (gdhA).

It has been shown previously that externally added glycine betaine is accumulated in Escherichia coli in response to the external osmotic strength. Here we have shown, by using nuclear magnetic resonance spectroscopy and radiochemical methods, that E. coli growing in a glucose-mineral medium of elevated osmotic strength generated with NaCl, had the same capacity to accumulate proline betaine and glycine betaine. Its capacity to accumulate gamma-butyrobetaine was, however, 40 to 50% lower. Accordingly, externally added proline betaine and glycine betaine stimulated aerobic growth of osmotically stressed cells equally well, and they were more osmoprotective than gamma-butyrobetaine. In cells grown at an osmotic strength of 0.64, 1.01, or 1.47 osmolal, respectively, the molal cytoplasmic concentration of the two former betaines corresponded to 29, 38, or 58% of the external osmotic strength. Nuclear magnetic resonance spectroscopy revealed that trehalose and glutamic acid were the only species of organic osmolytes accumulated in significant amounts in cells grown under osmotic stress in glucose-mineral medium without betaines. Their combined molal concentration in the cytoplasm of cells grown at 1.01 osmolal corresponded to 27% of the external osmotic strength.

Recent studies have indicated that acute inhibition of nitric oxide biosynthesis in the rat promotes arterial hypertension and renal vasoconstriction. We evaluated the renal and systemic effects of 4-6 weeks of nitric oxide blockade in Munich-Wistar rats receiving the nitric oxide inhibitor nitro-L-arginine orally. Age-matched untreated rats were used as controls. In an additional seven rats, nitric oxide blockade was carried out in conjunction with oral administration of the novel angiotensin II antagonist losartan potassium. Tail-cuff pressure rose progressively in nitro-L-arginine-treated rats, reaching 164 +/- 6 mm Hg at 4-6 weeks, compared with 108 +/- 3 mm Hg in controls. In rats concomitantly receiving losartan, tail-cuff pressure reached 125 +/- 6 mm Hg, still elevated compared with rats receiving losartan alone (98 +/- 3 mm Hg). Nitro-L-arginine-treated rats presented marked renal vasoconstriction and hypoperfusion, as well as a 30% fall in glomerular filtration rate and a 39% increase in filtration fraction. Treatment with Losartan normalized glomerular filtration rate, but not filtration fraction or renal vascular resistance. Plasma renin activity was elevated after nitro-L-arginine treatment. Renal histological examination revealed widespread arteriolar narrowing, focal arteriolar obliteration, and segmental fibrinoid necrosis in the glomeruli. In a separate group of rats, nitro-L-arginine administered for 1 week induced hypertension that was partially reversed by acute L-arginine, but not D-arginine or L-glycine, infusions. We conclude that chronic nitric oxide blockade may constitute a new model of severe arterial hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

The dominant cue for localization of low-frequency sounds are microsecond differences in the time-of-arrival of sounds at the two ears [interaural time difference (ITD)]. In mammals, ITD sensitivity is established in the medial superior olive (MSO) by coincidence detection of excitatory inputs from both ears. Hence the relative delay of the binaural inputs is crucial for adjusting ITD sensitivity in MSO cells. How these delays are constructed is, however, still unknown. Specifically, the question of whether inhibitory inputs are involved in timing the net excitation in MSO cells, and if so how, is controversial. These inhibitory inputs derive from the nuclei of the trapezoid body, which have physiological and structural specializations for high-fidelity temporal transmission, raising the possibility that well timed inhibition is involved in tuning ITD sensitivity. Here, we present physiological and pharmacological data from in vivo extracellular MSO recordings in anesthetized gerbils. Reversible blockade of synaptic inhibition by iontophoretic application of the glycine antagonist strychnine increased firing rates and significantly shifted ITD sensitivity of MSO neurons. This indicates that glycinergic inhibition plays a major role in tuning the delays of binaural excitation. We also tonically applied glycine, which lowered firing rates but also shifted ITD sensitivity in a way analogous to strychnine. Hence tonic glycine application experimentally decoupled the effect of inhibition from the timing of its inputs. We conclude that, for proper ITD processing, not only is inhibition necessary, but it must also be precisely timed.

Ankyrin repeat (AR) proteins mediate innumerable protein-protein interactions in virtually all phyla. This finding suggested the use of AR proteins as designed binding molecules. Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues. We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat. Randomly chosen library members are expressed in soluble form in the cytoplasm of Escherichia coli constituting up to 30% of total cellular protein and show high thermodynamic stability. We determined the crystal structure of one of those library members to 2.0-A resolution, providing insight into the consensus AR fold. Besides the highly complementary hydrophobic repeat-repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the beta-turn and loop regions are noteworthy. Furthermore, all residues found in the turn region of the Ramachandran plot are glycines. Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion. We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues. This scaffold represents an excellent basis for the design of novel binding molecules.

SUMO, a small ubiquitin-related modifier, is known to covalently attach to a number of nuclear regulatory proteins such as p53, IkappaB, promyelocytic leukemia protein and c-Jun. The sumoylation reaction is catalyzed by the SUMO protease, which exposes the C-terminal active glycine residue of the nascent SUMO, the heterodimeric SUMO activating enzyme, the SUMO conjugating enzyme, Ubc9, and SUMO protein ligases, in a manner similar to ubiquitinylation. Identification of SUMO-regulated proteins is hampered by the fact that many sumoylated proteins are present at a level below normal detection limit. This limitation was overcome by either in vivo overexpression of Myc-SUMO or in vitro sumoylation with excess biotin-SUMO and Ubc9. Sumoylated proteins so obtained were affinity purified or isolated by immunoprecipitation. The isolated sumoylated proteins were identified by sequence analysis using mass spectrometric methods. Results reveal that several heterogeneous nuclear ribonucleoproteins (hnRNPs), zinc finger proteins, and nuclear pore complex proteins were sumoylated. The sumoylation of hnRNP A1, hnRNP F, and hnRNP K were confirmed in vivo by coimmunoprecipitation. In view of the facts that hnRNPs have been implicated in RNA splicing, transport, stability, and translation, our findings suggest that sumoylation could play an important role in regulating mRNA metabolism.

Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the alpha-meso heme carbon is due primarily to steric influence of the distal helix.

Spondylocostal dysostosis (SD, MIM 277300) is a group of vertebral malsegmentation syndromes with reduced stature resulting from axial skeletal defects. SD is characterized by multiple hemivertebrae, rib fusions and deletions with a non-progressive kyphoscoliosis. Cases may be sporadic or familial, with both autosomal dominant and autosomal recessive modes of inheritance reported. Autosomal recessive SD maps to a 7.8-cM interval on chromosome 19q13.1-q13.3 that is homologous with a mouse region containing a gene encoding the Notch ligand delta-like 3 (Dll3). Dll3 is mutated in the X-ray-induced mouse mutant pudgy (pu), causing a variety of vertebrocostal defects similar to SD phenotypes. Here we have cloned and sequenced human DLL3 to evaluate it as a candidate gene for SD and identified mutations in three autosomal recessive SD families. Two of the mutations predict truncations within conserved extracellular domains. The third is a missense mutation in a highly conserved glycine residue of the fifth epidermal growth factor (EGF) repeat, which has revealed an important functional role for this domain. These represent the first mutations in a human Delta homologue, thus highlighting the critical role of the Notch signalling pathway and its components in patterning the mammalian axial

Growth factors rapidly induce transcription of a set of genes that encode regulatory proteins, many of which have been identified by cDNA cloning. Here we report the analysis of a cDNA corresponding to a gene induced in mouse 3T3 cells by growth factors and a variety of other extracellular signaling agents. The cDNA encodes a proline-, serine-, and glycine-rich nuclear protein designated Nup475 of 319 amino acids that contains two tandemly repeated cysteine- and histidine-containing sequences (CX8CX5CX3H) suggestive of a novel heavy metal-binding domain. Nup475 produced in Escherichia coli binds zinc. Its mRNA is present in a number of mouse tissues and cell lines, being especially abundant in intestine, thymus, and regenerating liver and in a macrophage cell line stimulated by gamma-interferon. We hypothesize that Nup475 is a regulatory protein with a novel zinc finger structure.

Folate-dependent one-carbon metabolism is required for the synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine. Methionine is subsequently adenylated to S-adenosylmethionine (SAM), a cofactor that methylates DNA, RNA, proteins, and many metabolites. Previous experimental and theoretical modeling studies have indicated that folate cofactors are limiting for cytoplasmic folate-dependent reactions and that the synthesis of DNA precursors competes with SAM synthesis. Each of these studies concluded that SAM synthesis has a higher metabolic priority than dTMP synthesis. The influence of cytoplasmic serine hydroxymethyltransferase (cSHMT) on this competition was examined in MCF-7 cells. Increases in cSHMT expression inhibit SAM concentrations by two proposed mechanisms: (1) cSHMT-catalyzed serine synthesis competes with the enzyme methylenetetrahydrofolate reductase for methylenetetrahydrofolate in a glycine-dependent manner, and (2) cSHMT, a high affinity 5-methyltetrahydrofolate-binding protein, sequesters this cofactor and inhibits methionine synthesis in a glycine-independent manner. Stable isotope tracer studies indicate that cSHMT plays an important role in mediating the flux of one-carbon units between dTMP and SAM syntheses. We conclude that cSHMT has three important functions in the cytoplasm: (1) it preferentially supplies one-carbon units for thymidylate biosynthesis, (2) it depletes methylenetetrahydrofolate pools for SAM synthesis by synthesizing serine, and (3) it sequesters 5-methyltetrahydrofolate and inhibits SAM synthesis. These results indicate that cSHMT is a metabolic switch that, when activated, gives dTMP synthesis higher metabolic priority than SAM synthesis.

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.

1. The interaction of the intravenous general anaesthetic propofol (2,6-diisopropylphenol) with the GABAA receptor has been investigated in voltage-clamped bovine chromaffin cells and rat cortical neurones in cell culture. Additionally, the effects of propofol on the glycine and GABAA receptors of murine spinal neurones were determined. 2. Propofol (1.7-16.8 microM) reversibly and dose-dependently potentiated the amplitude of membrane currents elicited by GABA (100 microM) applied locally to bovine chromaffin cells. Intracellular application of propofol (16.8 microM) was ineffective. In rat cortical neurones and murine spinal neurones, extracellular application of 8.4 microM and 1.7-16.8 microM propofol respectively produced a potentiation of GABA-evoked currents qualitatively similar to that seen in the bovine chromaffin cell. 3. The potentiation by propofol (1.7 microM) was not associated with a change in the reversal potential of the GABA-evoked whole cell current. On outside-out membrane patches isolated from bovine chromaffin cells, propofol (1.7 microM) had little or no effect on the GABA single channel conductances, but greatly increased the probability of the GABA-gated channel being in the conducting state. 4. The potentiation of GABA-evoked whole cell currents by propofol (1.7 microM) was not influenced by the benzodiazepine antagonist flumazenil (0.3 microM). A concentration of propofol (1.7 microM) that substantially potentiated GABA currents had little effect on currents induced by the activation of the GABAA receptor by pentobarbitone (1 mM). 5. Bath application of propofol (8.4-252 microM), to bovine chromaffin cells voltage clamped at -60 mV, induced an inward current associated with an increase in membrane current noise on all cells sensitive to GABA. Intracellular application of propofol (16.8 microM) was ineffective in this respect. Local application of propofol (600 microM) induced whole cell currents with a reversal potential dependent upon the Cl- gradient across the cell membrane. 6. On outside-out membrane patches formed from bovine chromaffin cells, propofol (30 microM) induced single channels with mean chord conductances of 29 and 12 pS. The frequency of propofol channels was greatly reduced by coapplication of 1 microM bicuculline. Under identical ionic conditions, GABA (1 microM) activated single channels with mean chord conductances of 33, 16 and 10pS. 7. Bath applied propofol (0.84-16.8 microM) dose-dependently potentiated strychnine-sensitive currents evoked by glycine (100 microM) in murine spinal neurones. 8. The relevance of the present results to the general anaesthetic action of propofol is discussed.

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin.

In eukaryotic cells, amino acid depletion reduces translation by a mechanism involving phosphorylation of eukaryotic initiation factor-2 (eIF2). Herein we describe that mice lacking the eIF2 kinase, general control nonderepressible 2 (GCN2) fail to alter the phosphorylation of this initiation factor in liver, and are moribund in response to dietary leucine restriction. Wild-type (GCN2(+/+)) and two strains of GCN2 null (GCN2(-/-)) mice were provided a nutritionally complete diet or a diet devoid of leucine or glycine for 1 h or 6 days. In wild-type mice, dietary leucine restriction resulted in loss of body weight and liver mass, yet mice remained healthy. In contrast, a significant proportion of GCN2(-/-) mice died within 6 days of the leucine-deficient diet. Protein synthesis in wild-type livers was decreased concomitant with increased phosphorylation of eIF2 and decreased phosphorylation of 4E-BP1 and S6K1, translation regulators controlled nutritionally by mammalian target of rapamycin. Whereas translation in the liver was decreased independent of GCN2 activity in mice fed a leucine-free diet for 1 h, protein synthesis in GCN2(-/-) mice at day 6 was enhanced to levels measured in mice fed the complete diet. Interestingly, in addition to a block in eIF2 phosphorylation, phosphorylation of 4E-BP1 and S6K1 was not decreased in GCN2(-/-) mice deprived of leucine for 6 days. This suggests that GCN2 activity can also contribute to nutritional regulation of the mammalian target of rapamycin pathway. As a result of the absence of these translation inhibitory signals, liver weights were preserved and instead, skeletal muscle mass was reduced in GCN2(-/-) mice fed a leucine-free diet. This study indicates that loss of GCN2 eIF2 kinase activity shifts the normal maintenance of protein mass away from skeletal muscle to provide substrate for continued hepatic translation.