TCR engagement of peptide-MHC class II ligands involves specific contacts between the TCR and residues on both the MHC and peptide molecules. We have used molecular modeling and assays of peptide binding and T cell function to characterize these interactions for a CD4+ Th1 cell clone, ESL4.34, which recognizes a peptide epitope of the herpes simplex type 2 virus virion protein, VP16 393-405, in the context of several HLA-DR alleles. This clone responded to VP16 393-405 in proliferation and cytotoxicity assays when presented by DRB1*0402, DRB1*1102, and DRB1*1301, which share a common amino acid sequence, ILEDE, at residues 67-71 in the alpha-helical portion of the DRbeta polypeptide, but not when presented by other DR4, DR11, and DR13 alleles that are negative for this sequence. Using a panel of APCs expressing DR4 molecules that were mutagenized in vitro at individual residues within this shared epitope and using peptide analogues with single amino acid substitutions of predicted MHC and TCR contact residues, a unit of recognition was identified dependent on DRbeta residues 67-71 and relative position 4 (P4) of the VP16 393-405 peptide. The interactions of this portion of the peptide-DR ligand with the ESL4.34 TCR support a structural model for MHC-biased recognition in some Ag-specific and alloreactive T cell responses and suggest a possible mechanism for autoreactive T cell selection in rheumatoid arthritis.

phi 6 is an enveloped dsRNA bacterial virus. Its segmented genome resides inside the virion associated polymerase complex which is formed by four proteins (P1, P2, P4 and P7) encoded by the viral L segment. Complete and incomplete polymerase complex particles can be produced using cDNA copies of this largest genome segment. We have analysed the capacity of the different purified particles to (1) package phi 6 (+) sense genomic precursors and unspecific RNA, (2) synthesize (-) and (+) strands and (3) bind phi 6 specific and unspecific RNAs. Both (-) and (+) strand synthesis polymerase activities were found to be associated with protein P2. In addition to complete particles, particles lacking protein P2 were found to package and protect genomic precursor ssRNAs. Protein P7 was needed for efficient packaging. Regulation and specificity of the packaging were found to be independent of P2. Particles composed of proteins P1 and P4 did not package or protect RNA but did bind phi 6 genomic (+) strand RNAs. The three phi 6 (+) strands bound in equal amounts to the particles when tested alone in a filter binding assay. In competition experiments they competed each other for binding, indicating that individual binding sites for the three genomic (+) strands do not exist. Differences in RNA binding competition among the four particles were observed, suggesting that packaging specificity is achieved by complex interactions of proteins and genomic (+) strand RNAs during the advancement of the packaging process after the initial binding events.

The ontogeny of expression of the CRF gene in the paraventricular hypothalamic nucleus and POMC gene in the pituitary was studied in rats using in situ hybridization histochemistry and Northern blotting techniques, respectively. CRF mRNA was first detected on day 17 of gestation (E17) in the paraventricular nucleus of the hypothalamus. The levels of hypothalamic CRF mRNA increased progressively from E17 to E19-E20, decreased during the perinatal period, and increased thereafter. The levels of POMC mRNA in the pituitary paralleled the variations in hypothalamic CRF mRNA, showing a peak on E20-E21. POMC mRNA levels in the anterior pituitary were decreased on days 4-7 after birth (P4-P7) and increased steadily thereafter. In contrast to levels in the anterior pituitary, POMC mRNA levels increased steadily from P1 to P21 in the neurointermediate lobe of the pituitary. These data indicate that the expression of both the CRF and POMC genes in the paraventricular nucleus and anterior pituitary, respectively, are reduced during the first week of life, i.e. within the so-called stress nonresponsive period. Our observations suggest that an impaired regulation of ACTH and CRF synthesis due to an immature neuronal pathway within the brain or increased glucocorticoid feedback may account for the stress nonresponsive period.

Near nanomolar concentrations of substance P induce production of IL-1 or an IL-1-like activity in the mouse macrophage cell line P388D1. Moreover, this could be accomplished with the carboxyl-terminal octapeptide substance P4-11, and could be inhibited with the substance P antagonist [D-Pro2, D-Trp7,9]-substance P. Two other mammalian neurokinins, neurokinin A and neurokinin B, were also found to induce secretion of IL-1-like activity in P388D1 cells. These findings suggest that activation of immune cells by neuromodulators can contribute to the maintenance of the chronic inflammatory state and the immunopathology observed in arthritic disease mediated by IL-1. The results also suggest that one approach to the treatment of rheumatoid arthritis might be to attempt to inhibit the local effects of immuno-modulatory neuropeptides, specifically the neurokinins, in affected joints.

More than 170 million people worldwide are affected by the hepatitis C virus (HCV). The disease has been described as a "silent epidemic" and "a serious global health crisis". HCV infection is a leading cause of chronic liver disease such as cirrhosis, carcinoma, or liver failure. The current pegylated interferon and ribavirin combination therapy is effective in only 50% of patients. Its moderate efficacy and apparent side effects underscore the need for safer and more effective treatments. The nonstructural NS3 protease of the virus plays a vital role in the replication of the HCV virus. The development of small molecule inhibitors of NS3 protease as antiviral agents has been intensively pursued as a viable strategy to eradicate HCV infection. However, it is a daunting task. The protease has a shallow and solvent-exposed substrate binding region, and the inhibitor binding energy is mainly derived from weak lipophilic and electrostatic interactions. Moreover, lack of a robust in vitro cell culture system and the absence of a convenient small animal model have hampered the assessment of both in vitro and in vivo efficacy of any antiviral compounds. Despite the tremendous challenges, with access to a recently developed cell-based replicon system, major progress has been made toward a more effective small molecule HCV drug. In our HCV program, facing no leads from our screening effort, a structure-based drug design approach was carried out. An alpha-ketoamide-type electrofile was designed to trap the serine hydroxyl of the protease. Early ketoamide inhibitors mimicked the structures of the peptide substrates. With the aid of X-ray structures, we successfully truncated the undecapeptide lead that had a molecular weight of 1265 Da stepwise to a tripeptide with a molecular weight of 500 Da. In an attempt to depeptidize the inhibitors, various strategies such as hydrazine urea replacement of amide bonds and P2 to P4 and P1 to P3 macrocyclizations were examined. Further optimization of the tripeptide inhibitors led to the identification of the best moieties for each site: primary ketoamide at P', cyclobutylalanine at P1, gem-dimethylcyclopropylproline at P2, tert-leucine at P3, and tert-butyl urea as capping agent. The combination of these led to the discovery of compound 8 (SCH 503034, boceprevir), our clinical candidate. It is a potent inhibitor in both enzyme assay (Ki* = 14 nM) and cell-based replicon assay (EC 90 = 0.35 microM). It is highly selective (2200x) against human neutrophil elastase (HNE). Boceprevir is well tolerated in humans and demonstrated antiviral activity in phase I clinical trials. It is currently in phase II trials. This Account details the complexity and challenges encountered in the drug discovery process.

Bacteriophage phi 6 contains three segments of double-stranded RNA within a nucleocapsid. Plasmids containing cDNA copies of the large genomic segment direct the synthesis of viral proteins that assemble into procapsids in Escherichia coli or Pseudomonas phaseolicola. These structures are dodecahedral assemblages of proteins P1, P2, P4, and P7. We report in this paper that these particles are capable of packaging viral single-stranded plus-sense RNA in vitro. The packaging reaction requires the presence of ATP or dATP. Synthesis of minus strands takes place within this filled procapsid in the presence of all four nucleoside triphosphates. Packaged ssRNA is found to be protected from added ribonuclease.

Proprotein convertases (PCs) are an important class of host-cell serine endoproteases implicated in many physiological and pathological processes. Owing to their expanding roles in the proteolytic events required for generating infectious microbial pathogens and for tumor growth and invasiveness, there is increasing interest in identifying endogenous PC inhibitors. Here we report the identification of Spn4A, a previously uncharacterized secretory pathway serine protease inhibitor (serpin) from Drosophila melanogaster that contains a consensus furin cleavage site, -Arg(P4)-Arg-Lys-Arg(P1) downsream-, in its reactive site loop (RSL). Our biochemical and kinetics analysis revealed that recombinant Spn4A inhibits human furin (K(i), 13 pM; k(ass), 3.2 x 10(7) M(-1) x s(-1)) and Drosophila PC2 (K(i), 3.5 nM; k(ass), 9.2 x 10(4) M(-1) x s(-1)) by a slow-binding mechanism characteristic of serpin molecules and forms a kinetically trapped SDS-stable complex with each enzyme. For both PCs, the stoichiometry of inhibition by Spn4A is nearly 1, which is characteristic of known physiological serpin-protease interactions. Mass analysis of furin-Spn4A reaction products identified the actual reactive site center of Spn4A to be -Arg(P4)-Arg-Lys-Arg(P1)-downstream-. Moreover, we demonstrate that Spn4A's highly effective PC inhibition properties are critically dependent on the unusual length of its RSL, which is composed of 18 aa instead of the typical 17-residue RSL found in most other inhibitory serpins. The identification of Spn4A, the most potent and effective natural serpin of PCs identified to date, suggests that Spn4A could be a prototype of endogenous serpins involved in the precise regulation of PC-dependent proteolytic cleavage events in the secretory pathway of eukaryotic cells.

Stimulation of human platelets by thrombin leads to rises of both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) within 10 s. The mass of Ins(1,4,5)P3 was measured in platelet extracts after conversion to [3-32P]Ins(1,3,4,5)P4 with Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Basal levels were equivalent to 0.2 microM and rose to 1 microM within 10 s of stimulation by thrombin. The mass of Ins(1,3,4)P3 was more than 10-fold greater than that of Ins(1,4,5)P3 between 10 and 60 s of thrombin stimulation. These results indicate that the majority of InsP3 liberated by phospholipase C in stimulated platelets must be the non-cyclic Ins(1,4,5)P3 in order to allow rapid phosphorylation by Ins(1,4,5)P3 3-kinase to Ins(1,3,4,5)P4 and then dephosphorylation to Ins(1,3,4)P3 by 5-phosphomonoesterase. A significant proportion of the InsP3 extracted from thrombin-stimulated platelets under neutral conditions is resistant to Ins(1,4,5)P3 3-kinase but susceptible after acid treatment, implying the presence of inositol 1,2-cyclic 4,5-trisphosphate (Ins(1,2cyc4,5)P3. The relative proportion of Ins(1,2cyc4,5)P3 increases with time. We suggest that such gradual accumulation is attributable to the relative insensitivity of this compound to hydrolytic and phosphorylating enzymes. Therefore, early Ca2+ mobilization in platelets is more likely to be effected by Ins(1,4,5)P3 than by Ins(1,2cyc4,5)P3.

Three major autophosphorylation sites are located near the C-terminus of the epidermal growth factor receptor, but a fourth site is repeatedly detected. We report here the purification and sequencing of a tryptic peptide containing this site, Tyr-1086. Furthermore, we demonstrate that additional phosphopeptides are observed following both partial digestion and overdigestion. Finally, we show that Tyr-1086 can be phosphorylated in intact cells.

Between August 1971 and August 1982, 197 consecutive patients underwent single stage radical cystectomy with pelvic lymph node dissection and urinary diversion as definitive management of high grade, invasive bladder cancer. In 100 patients 1,600 rad of radiation therapy were given for 4 days preoperatively and 97 patients underwent an operation only. Although not constituting a prospective randomized study, an analysis of these 2 groups of patients managed during an 11-year period by the same surgical team, using identical surgical technique, provides useful information that questions the benefit of preoperative radiation therapy in the management of high grade, invasive bladder cancer. Other factors, such as improved surgical technique with meticulous pelvic node dissection as well as better preoperative and postoperative care, may be responsible for survival results of contemporary surgery only that equal those reported following combination therapy protocols using preoperative radiation therapy. Contemporary surgery with or without preoperative radiation therapy yielded a 5-year survival rate free of tumor of 75 per cent for patients with pathologic stages P2 and P3A disease, 44 per cent with P3A and P3B disease, and 36 per cent with P4 disease and positive pelvic nodes.

In humans, microcephaly (small head for body size) is a common feature of fetal alcohol syndrome. An analogous measure, termed microencephaly (small brain for body size), can be used for evaluating the detrimental effects of the differential timing of alcohol exposure on brain development in animal model systems. Timed-pregnant rats were exposed to binge-like alcohol during either the first 10 days (first trimester equivalent) or second 10 days of gestation (second trimester equivalent), or the combination of first and second trimesters equivalent for prenatal treatments. Offspring from some of the animals exposed to alcohol during the combined first and second trimesters equivalent were raised artificially from postnatal day (P) 4 through P9 (part of the third trimester equivalent), and also received binge-like alcohol during this period, producing animals that were exposed to alcohol during all three trimesters equivalent. Offspring from untreated dams were also raised artificially and received alcohol only from P4 to P9, thus creating animals that were exposed to alcohol only during part of the third trimester equivalent. All pups were perfused on P10. Appropriate controls (nutritional and normally reared) were used for every alcohol treatment combination. Peak blood alcohol concentrations were not different among the treatment groups for a given sampling time. Significant somatic growth deficits occurred in offspring exposed to alcohol for the equivalent of all three trimesters, compared with offspring exposed to alcohol during other periods. Brain growth in offspring also was significantly affected by the timing of alcohol exposure. The whole brain, forebrain, and cerebellum to body weight ratios of pups exposed to alcohol during the third trimester had more significant brain growth deficits than pups in groups exposed to alcohol during other times of brain development. Although alcohol exposure during the third trimester had a significant detrimental impact on overall brain growth, significant differences in temporal vulnerability were also found for the brainstem to body weight ratios. Offspring of dams exposed to alcohol during the first trimester had the same magnitude of deficit as those exposed to alcohol during the third trimester, and those two groups were significantly deficient compared with the groups exposed to alcohol at other times, suggesting some differential vulnerability of this region to alcohol-induced injury at different times of development. This study is the first thorough examination of microencephaly and gross regional vulnerability of the developing brain as related to temporal factors of alcohol exposure in an animal model system, and the results support and expand on the findings of the available clinical literature. Furthermore, our results substantiate claims that the cessation of alcohol before the third trimester can lessen the severity of some alcohol-related birth deficits.

The enveloped dsRNA bacteriophages phi6 and phi8 are the two most distantly related members of the Cystoviridae family. Their structure and function are similar to that of the Reoviridae but their assembly can be conveniently studied in vitro. Electron cryomicroscopy and three-dimensional icosahedral reconstruction were used to determine the structures of the phi6 virion (14 A resolution), phi8 virion (18 A resolution), and phi8 core (8.5 A resolution). Spikes protrude 2 nm from the membrane bilayer in phi6 and 7 nm in phi8. In the phi6 nucleocapsid, 600 copies of P8 and 72 copies of P4 interact with the membrane, whereas in phi8 it is only P4 and 60 copies of a minor protein. The major polymerase complex protein P1 forms a dodecahedral shell from 60 asymmetric dimers in both viruses, but the alpha-helical fold has apparently diverged. These structural differences reflect the different host ranges and entry and assembly mechanisms of the two viruses.

Exogenous NAD utilization or pyridine nucleotide cycle metabolism is used by many bacteria to maintain NAD turnover and to limit energy-dependent de novo NAD synthesis. The genus Haemophilus includes several important pathogenic bacterial species that require NAD as an essential growth factor. The molecular mechanisms of NAD uptake and processing are understood only in part for Haemophilus. In this report, we present data showing that the outer membrane lipoprotein e(P4), encoded by the hel gene, and an exported 5'-nucleotidase (HI0206), assigned as nadN, are necessary for NAD and NADP utilization. Lipoprotein e(P4) is characterized as an acid phosphatase that uses NADP as substrate. Its phosphatase activity is inhibited by compounds such as adenosine or NMN. The nadN gene product was characterized as an NAD-nucleotidase, responsible for the hydrolysis of NAD. H. influenzae hel and nadN mutants had defined growth deficiencies. For growth, the uptake and processing of the essential cofactors NADP and NAD required e(P4) and 5'-nucleotidase. In addition, adenosine was identified as a potent growth inhibitor of wild-type H. influenzae strains, when NADP was used as the sole source of nicotinamide-ribosyl.

Environmental endocrine disrupting compounds (EDCs) are a wide variety of chemicals that typically exert effects, either directly or indirectly, through receptor-mediated processes, thus mimicking endogenous hormones and/or inhibiting normal hormone activities and metabolism. Little is known about the effects of EDCs on echinoderm physiology, reproduction and development. We exposed developing sea urchin embryos (Strongylocentrotus purpuratus and Lytechinus anamesus) to two known EDCs (4-octylphenol (OCT), bisphenol A (BisA)) and to natural and synthetic reproductive hormones (17beta-estradiol (E2), estrone (E1), estriol (E3), progesterone (P4) and 17alpha-ethynylestradiol (EE2)). In addition, we studied two non-estrogenic EDCs, tributyltin (TBT) and o,p-DDD. Successful development to the pluteus larval stage (96 h post-fertilization) was used to define EDC concentration-response relationships. The order of compound potency based on EC50 values for a reduction in normal development was as follows: TBT(L. anamesus>>OCT>TBT(S. purpuratus>>)E2>EE2>DDD>)BisA>P4)E1>)E3. The effect of TBT was pronounced even at concentrations substantially lower than those commonly reported in heavily contaminated areas, but the response was significantly different in the two model species. Sea urchin embryos were generally more sensitive to estrogenic EDCs and TBT than most other invertebrate larvae. Stage-specific exposure experiments were conducted to determine the most sensitive developmental periods using blastula, gastrula and post-gastrula (pluteus) stages. The stage most sensitive to E2, OCT and TBT was the blastula stage with less overall sensitivity in the gastrula stage, regardless of concentration. Selective estrogen receptor modulators (SERMs) were added to the experiments individually and in combination with estrogenic EDCs to interfere with potential receptor-mediated actions. Tamoxifen, a partial ER agonist, alone inhibited development at concentrations as low as 0.02 ng/ml and was effective at this concentration in decreasing the sensitivities of the embryos to estradiol and estrogenic EDCs. The complete antagonist ICI 182,780 inhibited development at concentrations as low as 0.03 ng/ml but increased embryo sensitivity to estradiol and estrogenic EDCs. Estradiol and estrogenic EDCs all cause developmental toxicity in sea urchins through a TAM-sensitive but an ICI-insensitive mechanism. It remains to be demonstrated whether this mechanism involves an estrogen-responsive nuclear receptor (NR), a membrane receptor (NR or non-NR-related) or a completely different mechanism of toxicity. However, early embryo sensitivity and the differential response to SERM co-incubation further suggests more than one mode of EDC action in the developing sea urchin embryo.

OBJECTIVE

This study was undertaken to determine whether progestational agents can prevent inflammation-induced preterm parturition and fetal demise.

METHODS

The activation of contractile and inflammatory pathways in response to localized intrauterine inflammation was investigated by using quantitative polymerase chain reaction (PCR). Serum progesterone (P4) levels and alterations in progesterone receptor-B (PR-B) were determined with radioimmunoassay and quantitative PCR, respectively. With our in vivo model of intrauterine inflammation, animals were randomly assigned to pretreatment with P4 or medroxyprogesterone acetate (MPA) before intrauterine lipopolysaccharide (LPS). Animals were observed for preterm delivery. The number of live pups 48 hours after intrauterine LPS was recorded for each treatment group. The ability of MPA to alter signal transduction pathways leading to preterm parturition were investigated by quantitative PCR and histochemical studies.

RESULTS

Intrauterine inflammation is associated with decreased serum progesterone levels and decreased transcription of PR-B. Preterm delivery rates were 100% for LPS alone, 63% for LPS+P4, and 0% for LPS+MPA. No live pups remained at 48 hours in the LPS or LPS+P4 groups. Pretreatment with MPA significantly preserved fetal viability. MPA suppressed activation of contraction-associated genes and inflammatory mediators and prevented cervical ripening in response to intrauterine inflammation.

CONCLUSIONS

MPA, with its progestational and anti-inflammatory properties, prevented inflammation-induced preterm parturition and significantly preserved fetal viability.

The homologue of the phosphoprotein PII phosphatase PphA from Thermosynechococcus elongatus, termed tPphA, was identified and its structure was resolved in two different space groups, C222(1) and P4(1)2(1)2, at a resolution of 1.28 and 3.05 A, respectively. tPphA belongs to a large and widely distributed subfamily of Mg(2+)/Mn(2+)-dependent phosphatases of the PPM superfamily characterized by the lack of catalytic and regulatory domains. The core structure of tPphA shows a high degree of similarity to the two PPM structures identified so far. In contrast to human PP2C, but similar to Mycobacterium tuberculosis phosphatase PstP, the catalytic centre exhibits a third metal ion in addition to the dinuclear metal centre universally conserved in all PPM members. The fact that the third metal is only liganded by amino acids, which are universally conserved in all PPM members, implies that the third metal could be general for all members of this family. As a specific feature of tPphA, a flexible subdomain, previously recognized as a flap domain, could be revealed. Comparison of different structural isomers of tPphA as well as site-specific mutagenesis implied that the flap domain is involved in substrate binding and catalytic activity. The structural arrangement of the flap domain was accompanied by a large side-chain movement of an Arg residue (Arg169) at the basis of the flap. Mutation of this residue strongly impaired protein stability as well as catalytic activity, emphasizing the importance of this amino acid for the regional polysterism of the flap subdomain and confirming the assumption that flap domain flexibility is involved in catalysis.

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.

The hepatitis delta virus (HDV), an infectious human pathogen affecting millions of people worldwide, leads to intensified disease symptoms, including progression to liver cirrhosis upon coinfection with its helper virus, HBV. Both the circular RNA genome of HDV and its complementary antigenome contain a common cis-cleaving catalytic RNA motif, the HDV ribozyme, which plays a crucial role in viral replication. Previously, the crystal structure of the product form of the cis-acting genomic HDV ribozyme has been determined, and the precursor form has been suggested to be structurally similar. In contrast, solution studies by fluorescence resonance energy transfer (FRET) on a trans-cleaving form of the ribozyme have shown significant global conformational changes upon catalysis, while 2-aminopurine (AP) fluorescence assays have detected concomitant local conformational changes in the catalytic core. Here, we augment these studies by using terbium(III) to probe the structure of the trans-acting HDV ribozyme at nucleotide resolution. We observe significant structural differences between the precursor and product forms, especially in the P1.1 helix and the trefoil turn in the single-stranded region connecting P4 and P2 (termed J4/2) of the catalytic core. We show, using terbium(III) footprinting and sensitized luminescence spectroscopy as well as steady-state, time-resolved, and gel-mobility FRET assays on a systematic set of substrates, that the substrate sequence immediately 5' to the cleavage site significantly modulates these local as well as resultant global structural differences. Our results suggest a structural basis for the previously observed impact of the 5' substrate sequence on catalytic activity.

The transient occipital cortical component of the pyramidal tract which we previously had identified during the postnatal development of the rat (Stanfield et al., '82) has been studied with anterograde as well as retrograde techniques. A continuous band of retrogradely labeled layer V neurons which spans the entire cortex including the occipital cortex is seen following injections of the fluorescent marker Fast Blue into the pyramidal decussation during the first postnatal week. No labeled cells are found in the occipital cortex following similar injections made on postnatal day 20 (P20), although such injections label many neurons in the more rostral cortical fields. However, if the Fast Blue injection is made on P2 and the animal is allowed to survive until P25 a large number of Fast Blue-labeled layer V neurons is found in the occipital cortex, even though an acute, second injection of the retrograde tracer Nuclear Yellow made into the pyramidal decussation shortly before the animal is killed results in no occipital cortical labeling. When Fast Blue injections confined to the mid- or upper-cervical spinal cord are made on P4 and the animals are killed on P9, again many retrogradely labeled neurons are found in the occipital cortex. Further, when injections of 3H-proline or wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) confined to the occipital cortex are made during the first 2 postnatal weeks, anterogradely transported label is seen within the pyramidal tract. At all stages examined the amount of such label and its caudal extent are less than that seen following similar injections into the parietal or frontal cortex. The greatest extent of the labeled occipital cortical fibers is reached at about the end of the first postnatal week and the number of these fibers seems to peak at about this same time. At this stage many of these labeled axons extend for a considerable distance down the spinal cord with some reaching as far caudal as lower lumbar levels, and at this stage some of these labeled occipital corticospinal fibers enter into the spinal gray. Over the next week the number of occipital cortical fibers in the pyramidal tract rapidly decreases and by P17 occipital cortical injections of 3H-proline or WGA-HRP result in virtually no transported label caudal to the pons.(ABSTRACT TRUNCATED AT 400 WORDS)

A clone overproducing diadenosine tetraphosphatase (diadenosine 5', 5'''-P1, P4-tetraphosphate pyrophosphohydrolase) activity was isolated from an Escherichia coli cosmid library. Localization of the DNA region responsible for stimulation of this activity was achieved by deletion mapping and subcloning in various vectors. Maxicell experiments and immunological assays demonstrated that a 3.5-kilobase-pair DNA fragment carried the structural gene apaH encoding the E. coli diadenosine tetraphosphatase. The DNA coding strand was determined by cloning this fragment in both orientations in pUC plasmids. It was also shown that the overproduction of diadenosine tetraphosphatase decreased the dinucleoside tetraphosphate concentration in E. coli by a factor of 10.

The purified high molecular weight form of HeLa cell DNA polymerase alpha (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) was shown to associate tightly with several aminoacyl-tRNA synthetase activities. Fractionation of the high molecular weight enzyme on hexylagarose followed by gel filtration, chromatography on phosphocellulose, or polyacrylamide gel electrophoresis under nondenaturing conditions demonstrated copurification of only tryptophanyl-tRNA synthetase [L-tryptophan:tRNATrp ligase (AMP-forming), EC 6.1.1.2] along with DNA polymerase alpha. The high molecular weight (660,000) and low molecular weight (145,000) forms of DNA polymerase alpha were shown to possess a highly specific, noncovalent, diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding activity. The dissociation constants were determined to be 16 and 22 microM, respectively, by utilization of a charcoal adsorption procedure. No high-affinity binding of ATP could be detected. These findings suggest a link between the amino acid activation process and DNA replication in mammalian cells.

We show that Yersinia pestis and pesticin-sensitive isolates of Y. pseudotuberculosis possess a common 34 kbp DNA region that has all the hallmarks of a pathogenicity island and is inserted into different asparaginyl tRNA genes at different chromosomal locations in each species. This pathogenicity island (YP-HPI) is marked by IS100, has a G + C content different from its host, is flanked by 24 bp direct repeats, encodes a putative, P4-like integrase and contains the iron uptake virulence genes from the pgm locus of Y. pestis. These findings indicate independent horizontal acquisition of this island by Y. pestis and Y. pseudotuberculosis. The two YP-HPI locations and their possession of an integrase gene support a model of site-specific integration of the YP-HPI into these bacteria.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.

A novel type of GABA receptor is present on rod-driven (H4) horizontal cells of the white perch retina (Qian and Dowling, 1993a). These receptors have been tentatively termed GA-BAC receptors. In this study, the pharmacological properties of these receptors were further investigated by applying several conformationally restricted GABAA receptor agonists, GABAA antagonists, and a GABAB agonist to the H4 horizontal cells. GABA analogs locked in a partially folded conformation had a variety of effects. Isonipecotic acid had no effect on these receptors, whereas isoguvacine activated them but with low potency (EC50 = 137 microM). THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) acted as a competitive antagonist on these receptors with an inhibition constant of 82.5 microM. P4S (piperidine-4-sulfonic acid) activated the receptors at high concentrations >> 1 mM), but at lower concentrations it was a competitive antagonist with an inhibition constant of 80.9 microM. 14AA (imidazole-4-acetic acid), a GABA analog with an extended conformation, potently inhibited the GABA responses on H4 horizontal cells with an inhibition constant of 1.67 microM. Muscimol, which can assume both partially folded and extended conformations, acted as a mixed agonist-antagonist. The GABA responses on H4 horizontal cells were resistant to several competitive GABAA receptor antagonists including bicuculline, hydrastine, and SR-95531, but they were very sensitive to picrotoxin (IC50 = 237 nM). The inhibition by picrotoxin was both competitive and noncompetitive in nature. On the other hand, TBPS (tert-butyl-bicyclophosphorothionate), another GABAA receptor channel blocker, had minimal effects on these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)