Epidemiological study of latent and recent infection by Toxoplasma gondii in pregnant women from a regional population in the U.K

The last two amino acids in the nascent peptide influence translation termination in E. coli (Mottagui-Tabar et al., 1994; Bj?rnsson et al., 1996). We have compared the effects on termination in Escherichia coli, Bacillus subtilis and Salmonella typhimurium obtained by varying the -1 and -2 codons upstream of the weak UGAA stop signal. The peptide effect from the penultimate amino acid on translation termination in B. subtilis is similar to that seen in E. coli (with 66.5% RF-2 amino acid sequence similarity), whereas the influence in S. typhimurium (with 95.3% similarity to E. coli) is weaker. The effect of changing the -1 codon (P-site) is weaker in S. typhimurium as compared to those in E. coli and B. subtilis. RF-2s from E. coli and S. typhimurium have a threonine or alanine at position 246, respectively. This amino acid exchange in RF-2 can explain the difference in efficiency and toxicity during overexpression when E. coli and S. typhimurium are compared (Uno et al., 1996). However, B. subtilis RF-2 also has an alanine at that position, yet the sensitivity to the nascent peptide is similar to that in E. coli. Thus, the amino acid difference at position 246 in the RF-2 sequences cannot explain why termination in E. coli and B. subtilis is similar in peptide sensitivity while being different from that in S. typhimurium. Sequence alignments of RF-2 from the three bacteria show other regions of the molecule that could be involved in the functional interactions with the C-terminal end of the nascent peptide.     

Differential stimulation of c-fos expression in hypothalamic nuclei of the rat brain during short-term heat acclimation and mild dehydration

Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).     

The role of methionine-192 of the chymotrypsin active site in the binding and catalysis of mono(amino acid) and peptide substrates

We find that specific oxidation for the Met-192 residue in delta-chymotrypsin to methionine sulfoxide results in a twofold increase in Km(app) and unchanged kcat in the hydrolysis of N-acetyl mono(amino acid) amide substrates. However, the catalyzed hydrolyses of N-acetyl dipeptide amide substrates by (methionine sulfoxide)-192-delta-chymotrypsin (MS-delta-Cht) shows a four- to fivefold decrease in kcat and unchanged Km(app) with respect to delta-chymotrypsin. Hydrolysis of alpha-casein by MS-delta-Cht shows a similar 4.2-fold decrease in kcat. These results imply that the Met-192 acts differently with substrates that bind only in the primary, S1, binding site (i.e., AcPheNH2) from those that bind to more extended regions of the enzyme active site. In the binding of c+AcPheNH2 and AcTrpNH2, the results support a mechanism in which the Met-192 acts to slow the rate of sustrate dissociation from the Michaelis complex to free substrate and enzyme. This is in agreement with the x-ray crystallographic structure of dioxane inhibited alpha-chymotrypsin (Steitz, T., et al. (1969), J. Mol. Biol. 46, 337). However, this mechanism is not apparent when peptide and protein substrates bind. The decrease in kcat on Met-192 modification of approximately fivefold in the hydrolysis of polypeptide substrates show a small, but significant, catalytic contribution of the Met-192 toward the lowering of the energy of activation polypeptide substrate hydrolysis by chymotrypsin. This may support the crystallographic model of Fersht et al. (Fersht, A., et al. (1973), Biochemistry 12, 2035) in which it is proposed that the Met-192 participates in the distortion of bound polypeptide substrates toward the reaction transition-state configuration and, thus, plays a role in catalysis. However, if this mechanism occurs, the effect is small, only contributing about 1 kcal/mol to the lowering of the reaction activation energy.     

The incidence of large venous emboli during total knee arthroplasty without pneumatic tourniquet use

The T4 phage capsid accessory protein genes soc and hoc have recently been developed for display of peptides and protein domains at high copy number (Ren et al., 1996. Protein Science 5, 1833-1843; Ren et al., 1997. Gene 195, 303-311). That biologically active and full-length foreign proteins can be displayed by fusion to SOC and HOC on the T4 capsid is demonstrated in this report. A 271-residue heavy and light chain fused IgG anti-EWL (egg white lysozyme) antibody was displayed in active form attached to the COOH-terminus of the SOC capsid protein, as demonstrated by lysozyme-agarose affinity chromatography (>100-fold increase in specific titer). HOC with NH2-terminal fused HIV-I CD4 receptor of 183 amino acids can be detected on the T4 outer capsid surface with human CD4 domain 1 and 2 monoclonal antibodies. The number of molecules of each protein (10-40) bound per phage and their activity suggest that proteins can fold to native conformation and be displayed by HOC and SOC to allow binding and protein-protein interactions on the capsid.     

CFTR: domains, structure, and function

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) (Collins, 1992). Over 500 naturally occurring mutations have been identified in CF gene which are located in all of the domains of the protein (Kerem et al., 1990; Mercier et al., 1993; Ghanem et al., 1994; Fanen et al., 1992; Ferec et al., 1992; Cutting et al., 1990). Early studies by several investigators characterized CFTR as a chloride channel (Anderson et al.; 1991b,c; Bear et al., 1991). The complex secondary structure of the protein suggested that CFTR might possess other functions in addition to being a chloride channel. Studies have established that the CFTR functions not only as a chloride channel but is indeed a regulator of sodium channels (Stutts et al., 1995), outwardly rectifying chloride channels (ORCC) (Gray et al., 1989; Garber et al., 1992; Egan et al., 1992; Hwang et al., 1989; Schwiebert et al., 1995) and also the transport of ATP (Schwiebert et al., 1995; Reisin et al., 1994). This mini-review deals with the studies which elucidate the functions of the various domains of CFTR, namely the transmembrane domains, TMD1 and TMD2, the two cytoplasmic nucleotide binding domains, NBD1 and NBD2, and the regulatory, R, domain.     

A physical comparison of chromosome III in six strains of Saccharomyces cerevisiae

We have tested the clones used in the European Yeast Chromosome III Sequencing Programme for possible artefacts that might have been introduced during cloning or passage through Escherichia coli. Southern analysis was performed to compare the BamHI, EcoRI, HindIII and PstI restriction pattern for each clone with that of the corresponding locus on chromosome III in the parental yeast strain. In addition, further enzymes were used to compare the restriction maps of most clones with the map predicted by the nucleotide sequence (Oliver et al., 1992). Only four of 506 6-bp restriction sites predicted by the sequence were not observed experimentally. No significant cloning artefacts appear to disrupt the published sequence of chromosome III. The restriction patterns of six yeast strains have also been compared. In addition to two previously identified sites of Ty integration on chromosome III (Warmington et al., 1986; Stucka et al., 1989; Newlon et al., 1991), a new polymorphic site involving Ty retrotransposition (the Far Right-Arm transposition Hot-Spot, FRAHS) has been identified close to CRY1. On the basis of simple restriction polymorphisms, the strains S288C, AB972 and W303-1b are closely related, while XJ24-24a and J178 are more distant relatives of S288C. A polyploid distillery yeast is heterozygous for many polymorphisms, particularly on the right arm of the chromosome.     

Cyclooxygenases and the central nervous system

Prostaglandins (PGs) were first described in the brain by Samuelsson over 30 years ago (Samuelsson, 1964). Since then a large number of studies have shown that PGs are formed in regions of the brain and spinal cord in response to a variety of stimuli. The recent identification of two forms of cyclooxygenase (COX; Kujubu et al., 1991; Xie et al., 1991; Smith and DeWitt, 1996), both of which are expressed in the brain, along with superior tools for mapping COX distribution, has spurred a resurgence of interest in the role of PGs in the central nervous system (CNS). In this review we will describe new data in this area, focusing on the distribution and potential role of the COX isoforms in brain function and disease.     

Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons

Astrocytes exhibit a form of excitability and communication on the basis of intracellular Ca2+ variations (Cornell-Bell et al., 1990; Charles et al., 1991) that can be initiated by neuronal activity (Dani et al., 1992; Porter and McCarthy, 1996). A Ca2+ elevation in astrocytes induces the release of glutamate (Parpura et al., 1994; Pasti et al., 1997; Araque et al., 1998;Bezzi et al., 1998), which evokes a slow inward current in neurons and modulates action potential-evoked synaptic transmission between cultured hippocampal cells (Araque et al., 1998), suggesting that astrocytes and neurons may function as a network with bidirectional communication. Here we show that a Ca2+ elevation in astrocytes increases the frequency of excitatory as well as inhibitory miniature postsynaptic currents (mPSCs), without modifying their amplitudes. Thapsigargin incubation, microinjection of the Ca2+ chelator BAPTA, and photolysis of the Ca2+ cage NP-EGTA demonstrate that a Ca2+ elevation in astrocytes is both necessary and sufficient to modulate spontaneous transmitter release. This Ca2+-dependent release of glutamate from astrocytes enhances mPSC frequency by acting on NMDA glutamate receptors, because it is antagonized by D-2-amino-5-phosphonopentanoic acid (AP5) or extracellular Mg2+. These NMDA receptors are located extrasynaptically, because blockage specifically of synaptic NMDA receptors by synaptic activation in the presence of the open channel blocker MK-801 did not impair the AP5-sensitive astrocyte-induced increase of mPSC frequency. Therefore, astrocytes modulate spontaneous excitatory and inhibitory synaptic transmission by increasing the probability of transmitter release via the activation of NMDA receptors.     

1H NMR studies of the high-affinity Rev binding site of the Rev responsive element of HIV-1 mRNA: base pairing in the core binding element

1H NMR studies of a 30-nucleotide RNA oligonucleotide (RBE3), which contains a high-affinity binding site for Rev of the HIV-1 Rev responsive element (RRE), two derivatives of RBE3 (RBE3AA and RBE3-A), and the complex of RBE3 with peptides derived from the RNA binding domain of HIV-1 Rev, are presented. The high-affinity binding site of the RRE consists of an asymmetric internal loop and surrounding Watson-Crick base pairs. In the wild-type RRE, one of the stems is closed by a loop; this is replaced in REB3 by the stable UUCG tetraloop. NOE data suggest that the internal loop of the free RNA contains structural features that have been predicted on the basis of in vitro selection experiments [Bartel, D.P., et al. (1991) Cell 67, 529-536]. The structural features include a Gsyn.Ganti base pair, a Ganti.Aanti base pair, and a looped out U. When the Rev peptide is bound to the RNA, the base pairs in the internal loop appear to be stabilized, although the RNA chemical shifts indicate that the RNA conformation undergoes some changes when bound by Rev peptide.     

Sperm motility is a major determinant of pregnancy outcome following intrauterine insemination

The presence and the distribution of neuroendocrine cells in the gastrointestinal tract of adult wild boar were investigated. The endocrine cells have been identified by means of immunocytochemical techniques using antibodies against serotonin, gastrin, somatostatin, cholecystokinin (CCK), met-enkephalin (MET-ENK), gastric-inhibitory peptide (GIP) and glucagon. The number of positive cells for each antiserum in each region was evaluated. Results were compared with data present in the literature and obtained previously by us and other authors in swine and domestic mammals (Ceccarelli et al., 1985, 1987, 1990, 1991, 1995; Capella and Solcia, 1972; Domeneghini and Castaldo, 1981; Peranzi and Lehy, 1984; Krause et al., 1985).     

Staurosporine inhibits interferon alpha-induced gene expression in Friend erythroleukemia cells through a PKC independent pathway

Diabetes mellitus is associated with typical patterns of long term vascular complications which vary with the organ involved. The microvascular kidney disease (Olgemoller and Schleicher, 1993) is characterized by thickening of the capillary basement membranes and increased deposition of extracellular matrix components (ECM), while loss of microvessels with subsequent neovascularisation is predominant in the eye and peripheral nerves. On the other hand macrovascular disease is characterized by accelerated atherosclerosis. These complications are dependent on long term hyperglycemia. Specific biochemical pathways linking hyperglycaemia to microvascular changes were proposed: the polyol pathway (Greene et al., 1987), non-enzymatic glycation of proteins (Brownlee et al., 1988), glucose autooxidation and oxidative stress (Hunt et al., 1990), hyperglycemic pseudohypoxia (Williamson et al., 1993) enhanced activation of protein kinase C by de novo-synthesis of diacyl glycerol (Lee et al., 1989; DeRubertis and Craven 1994) and others. These pathways are not mutually exclusive (Larkins and Dunlop, 1992; Pfeiffer and Schatz, 1992). They may be linked to alterations in the synthesis of growth factors particularly since atherosclerosis and angioneogenesis are associated with increased proliferation of endothelial and smooth muscle cells. Increased synthesis of ECM components is stimulated by growth factors like transforming growth factor beta (TGF beta) (Derynck et al., 1984) and insulin-like growth factor I (IGF-I) (Moran et al., 1991). This review will summarize some of the recent evidence for an involvement of growth factors in diabetic vascular complications and will attempt to assign their emergence in the sequence of events leading to vascular complications.     

Toxicology and pharmacology of the chemical warfare agent sulfur mustard

There have been reports of chemical attacks in which sulfur mustard might have been used (a) on Iranian soldiers and civilians during the Gulf War in 1984 and 1985 and (b) in an Iraqi chemical attack on the Iranian-occupied village of Halbja in 1988, resulting in many civilian casualties. Heavy use of chemical warfare in Afghanistan by the Soviet military is a recent innovation in military tactics that has been highly successful and may ensure further use of chemical agents in future military conflicts and terrorist attacks as a profitable adjunct to conventional military arms. Mustard is a poisonous chemical agent that exerts a local action on the eyes, skin, and respiratory tissue, with subsequent systemic action on the nervous, cardiac, and digestive systems in humans and laboratory animals, causing lacrimation, malaise, anorexia, salivation, respiratory distress, vomiting, hyperexcitability, and cardiac distress. Under extreme circumstances, dependent upon the dose and length of exposure to the agent, necrosis of the skin and mucous membranes of the respiratory system, bronchitis, bronchopneumonia, intestinal lesions, hemoconcentration, leucopenia, convulsions with systemic distress, and death occur. Severe mustard poisoning in humans is associated with systemic injury, which is manifested as headache, epigastric distresses, anorexia, diarrhea, and cachexia and is usually observed at mustard doses of 1000 mg/min/m3 with damage to hematopoietic tissues and progressive leucopenia. Sulfur mustard is a cell poison that causes disruption and impairment of a variety of cellular activities that are dependent upon a very specific integral relationship. These cytotoxic effects are manifested in widespread metabolic disturbances whose variable characteristics are observed in enzymatic deficiencies, vesicant action, abnormal mitotic activity and cell division, bone marrow disruption, disturbances in hematopoietic activity, and systemic poisoning. Indeed, mustard gas readily combines with various components of the cell such as amino acids, amines, and proteins. Although evidence of an association between lung cancer and mustard gas encountered on the battlefields of World War I is at best suggestive if not problematical (Case and Lea, 1955; Beebe, 1960; Norman, 1975), the epidemiological data accumulated from the poison gas factories in Japan (Yamada et al., 1953; Wada et al., 1968; Inada et al., 1978; Shigenobu, 1980; Nishimoto et al., 1983; Hirono et al., 1984; Takuoka et al., 1986), in Germany (Weiss, 1958; Hellmann, 1970a; Weiss and Weiss, 1975; Klehr, 1984) and in England (Manning et al., 1981; Easton et al., 1988) are substantial (International Agency for Research on Cancer, 1975). Unfortunately, attempts to seek confirmatory and substantial evidence in laboratory animals such as mice (Boyland and Horning, 1949; Heston, 1950; Heston, 1953a; McNamara et al., 1975) and rats (Griffin et al., 1951; McNamara et al., 1975; Sasser et al., 1996) have not been consistent. Sulfur mustard has been shown to be mutagenic in a variety of different species using many different laboratory techniques from fruit flies, microorganisms and mammalian cell cultures (Fox and Scott, 1980). Evidence is slowly accumulating from human data (Hellmann, 1970a; Lohs, 1975; Wulf et al., 1985). Evidence for the teratogenicity of mustard has been negative in assessment of fetotoxicity and adverse effects of mustard on the reproductive potential of both human and animal studies. Indeed, investigations of women adversely affected by mustard are minimal because most of the studies have been performed on former men employees of poison gas factories and have been negative or questionable. We have recently emphasized the need to assess the affect of a suspected teratogen on maternal toxicity in laboratory animals before any conclusions can be made.(ABSTRACT TRUNCATED)     

FGF and EGF are mitogens for immortalized neural progenitors

Individual neural progenitors, derived from the external germinal layer of neonatal murine cerebellum, were previously immortalized by the retrovirus-mediated transduction of avian myc (v-myc). C17-2 is one of those clonal multipotent progenitor cell lines (Snyder et al., 1992, Cell 68: 33-51; Ryder et al., 1990, J. Neurobiol. 21:356-375). When transplanted into newborn mouse cerebellum (CB), the cells participate in normal CB development; they engraft in a cytoarchitecturally appropriate, nontumorigenic manner and differentiate into multiple CB cell types (neuronal and glial) similar to endogenous progenitors (Snyder et al., 1992, as above). They also appear to engraft and participate in the development of multiple other structures along the neural axis and at multiple other stages (Snyder et al., 1993, Soc. Neurosci. Abstr. 19). Thus conclusions regarding these immortalized progenitors may be applicable to endogenous neural progenitors in vivo. To help identify and analyze factors that promote differentiation of endogenous progenitors, we first investigated the ability to maintain C17-2 cells in a defined, serum-free medium (N2). The cells survive in vitro in N2 but undergo mitosis at a very low rate. Addition of epidermal growth factor (EGF), however, either from mouse submaxillary gland or the human recombinant protein, appreciably stimulates thymidine incorporation and cell division approximately threefold. Basic fibroblast growth factor (bFGF) is an even more potent mitogen, promoting thymidine incorporation, cell division, and a net increase in cell number equal to that in serum. Both EGF and bFGF are active at very low nanomolar concentrations, suggesting that they interact with their respective receptors rather than a homologous receptor system. The findings demonstrate that C17-2 cells can be maintained and propagated in a fully defined medium, providing the basis for analysis of other growth and differentiation factors. That EGF and particularly bFGF are mitogenic for these cells is in accord with recent observations on primary neural tissue (Reynolds and Weiss, 1992, Science 255:1707-1710; Kilpatrick and Bartlett, 1993, Neuron 10:255-265; Ray et al., 1993, Proc. Natl. Acad. Sci. USA 90:3602-3606) suggesting that bFGF and EGF responsiveness may be fundamental properties of neural progenitors.     

Genetic organization and sequence analysis of the hypha-specific cell wall protein gene HWP1 of Candida albicans

A previously isolated partial cDNA encoding a cell wall protein antigen found on hyphal surfaces of the opportunistic fungal pathogen, Candida albicans (Staab et al., 1996) was used to clone the complete hyphal wall protein 1 gene (HWP1). Hyphal forms of C. albicans invade mucosal surfaces of immunocompromised patients such as those with AIDS. HWP1 consisted of an open reading frame predicting an acidic protein (pI of 3.37) with a calculated molecular size of 61,122. The antigenic domain was located in the N-terminal third of the protein. The remainder of the protein contained abundant hydroxy amino acids, and terminated with a string of 15 amino acids typical of sequences specifying post-translational modification with glycosylphosphatidylinositol (6PI). The analyses suggested that Hwp1 is a glucan-linked protein with serine/threonine-rich regions that are predicted to function in extending a ligand-binding domain into the extracellular space.     

Characteristics of age-related behavioral changes in senescence-accelerated mouse SAMP8 and SAMP10

Senescence-Accelerated Mouse (SAM), a murine model of accelerated senescence, has been established by Takeda et al. (1981). SAM consists of senescence-accelerated-prone mouse (SAMP) and senescence-accelerated-resistant mouse (SAMR), the latter of which shows normal aging characteristics. In 1991 there were eight different substrains in the P-series, which commonly exhibited accelerated aging with a shortened life span (Takeda et al., 1991). Among the P-series, we have found that SAMP8 mice show significant impairments in a variety of learning tasks when compared with SAMR1 mice (Miyamoto et al., 1986). Further studies suggest that SAMP8 exhibits an age-related emotional disorder characterized by reduced anxiety-like behavior (Miyamoto et al., 1992). On the other hand, it has been shown that SAMP10 exhibits brain atrophy and learning impairments in an avoidance task (Shimada et al., 1992, 1993). Here, characteristics of age-related deficits in learning and memory, changes in emotional behavior, and abnormality of circadian rhythms in SAMP8 and SAMP10 mice are described. In the experiments, SAMP8/Ta (SAMP8), SAMP10/(/)Ta (SAMP10) and SAMR1TA (SAMR1) reared under specific pathogen-free conditions at Takeda Chemical Industries were used.     

Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda:Rhabditida)

We have calculated the curvature of 504 eukaryotic promoters predicted by the bent A-tract model of Bolshoy et al. (Proc. Natl. Acad. Sci. USA, 88(6), pp. 2312-16) and the bent non-A-tract models of Calladine et al. (J. Mol. Biol., 201, pp. 127-37) and Satchwell et al. (J. Mol. Biol., 191, pp. 659-75) and found in each case a correlation between TBP binding sites and DNA curvature. Characterizing the TBP binding sites revealed that in addition to the classical TATA box (TATAAA) five more elements occur significantly often in the promoters, nearly all of them being one point mutations of the classical TATA box element. Separate curvature calculations for promoters with canonical and non-canonical TATA boxes have shown that in both cases the strong curvature of the helix axes in the domain of the binding sites is maintained (classical TBP binding sites: + 64-135%, non-classical TBP binding sites: + 27-49%). These results support the proposition that beside DNA flexibility and DNA-protein interactions intrinsic curvature of DNA is one further important criterion for the recognition of different DNA elements by TBP.     

Behavioral and neural effects of chromatic isoluminance in the primate visual motion system

We have previously reported that the responses of individual neurons in macaque visual area MT elicited by movement of contrast-reversing heterochromatic red/green borders are largest when the two hues are "balanced" or isoluminant (Dobkins & Albright, 1994). This "neural" isoluminant point was found to vary somewhat across the sample of neurons. Here, we compare the average neural isoluminant point in area MT to a behavioral measure of isoluminance, obtained using a modification of an oculomotor procedure developed by Chaudhuri and Albright (1992). These behavioral estimates of isoluminance closely parallel the neuronal data obtained from area MT. In accordance with previous evidence (e.g. Lee et al., 1988; Kaiser et al., 1990; Valberg et al., 1992), this correlation suggests that activity within the dorsal/magnocellular stream underlies behavioral expression of chromatic isoluminance.     

The medullary reticular formation is a site of muscle relaxant action of diazepam on deep back and neck muscles in the female rat

We tested the hypothesis that the effect of systemic injections of diazepam (DZ, 125 mg/kg) to reduce the quality of the reproductive behavior, lordosis, and to reduce the EMG of lumbar back muscles involved in lordosis (Schwartz-Giblin et al., 1984) is exerted through a reticulospinal pathway with cells of origin in the nucleus gigantocellularis that excites lumbar motoneurons indirectly (Robbins et al., 1990, Robbins et al., 1992). In contrast, DZ facilitates lordosis behavior when infused into the midbrain central gray (McCarthy et al., 1995). Direct deposits of crystalline mixtures of DZ (20-80 ng) in dextrose were delivered to the medullary reticular formation (MRF) by diffusion from a cannula inserted through a guide to which a bipolar stimulating electrode was attached. The multiunit EMG response evoked by 20 (300 ms long) stimulus trains was recorded in back and neck muscles, lateral longissimus and splenius before and 5, 15, 30 and 60 min after local DZ deposits. There was a significant reduction in EMG response over this time period when stimulus intensities were within the range of 1.2-1.5 times threshold (Friedman two-way non-parametric test, P < 0.002). Large amplitude motor units that provide large tensions were the most sensitive to DZ-induced inhibition. Control deposits of dextrose had no significant effect. Systemic injections of progesterone (1 mg, i.p.) 60 min after DZ deposits, but not after dextrose deposits, further reduced the MRF-evoked EMG responses over the course of 1 h. As predicted, DZ infusions into the midbrain central gray did not reduce the reticulospinal-evoked axial muscle response, consistent with the facilitatory effect of midbrain central gray infusions of DZ on the lordosis quotient. The results suggest that benzodiazepine agonists (if endogenous) acting at sites in the MRF would be effective muscle relaxants during pregnancy, prior to the fall in progesterone that precedes labor.     

A 1.4 A crystal structure for the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi, etiologic agent of Chagas' disease, was cocrystallized with the inosine analogue Formycin B (FmB) and the structure determined to 1.4 A resolution. This is the highest resolution structure yet reported for a phosphoribosyltransferase (PRT), and the asymmetric unit of the crystal contains a dimer of closely associated, nearly identical subunits. A conserved nonproline cis peptide in one active-site loop exposes the main-chain nitrogen to the enzyme active site, while the adjacent lysine side chain interacts with the other subunit of the dimer, thereby providing a possible mechanism for communication between the subunits and their active sites. The three-dimensional coordinates for the invariant Ser103-Tyr104 dipeptide are reported here for the first time. These are the only highly conserved residues in a second active-site loop, termed the long flexible loop, which is predicted to close over the active site of HPRTs to protect a labile transition state [Eads et al. (1994) Cell 78, 325-334]. This structure represents a major step forward in efforts to design/discover potent selective inhibitors of the HPRT of T. cruzi.