Immunobiology, 1996 Jul, 195(2), 220 - 30 Anti-candidial activity of natural killer (NK) and lymphokine activated killer (LAK) lymphocytes in vitro; Gulay Z et al.; The natural cytotoxic effects of peripheral blood lymphocytes (PBL) on Candida stellatoidea and several other Candida species were examined by a colony forming inhibition (CFI) assay . Peripheral blood mononuclear cells (PBMC), were incubated with C . stellatoidea yeast cells . After the incubation period the colony-forming ability of the yeast was significantly reduced . In similar experiments, six different Candida species (C . albicans, C . krusei, C . stellatoidea, C . tropicalis, C . pseudotropicalis, C . guillermondii) were used as target cells . There was no statistically significant difference in the anticandidial activities of PBL against the Candida species used . It was demonstrated that a fraction of lymphocytes, natural killer cells (NK), had the major natural anti-candidial activity by using anti-Leu M1 (CD 15) and anti-Leu 11b (CD 16) monoclonal antibodies (mAbs) plus complement (C') . It was observed that inhibition of colony-forming ability of C . stellatoidea was significantly (78-96%) reduced when anti-Leu 11b plus C' were used . In addition, the colony formation inhibition capacity of NK cells was increased by recombinant human interleukin-2 (rhIL-2) while anti-interferon-gamma (IFN-gamma) had no effect . Besides the fact that NK cells are among those responsible for natural immunity against Candida species, this colony-forming inhibition assay performed with C . stellatoidea yeast cells as target and monocyte-depleted PBMC as effector cells, is a simple method to assess NK cell activity. J Med Vet Mycol, 1996 Jul-Aug, 34(4), 293 - 7 Random amplified polymorphic DNA analysis of culture collection strains of Candida species; Zeng S et al.; Random amplified polymorphic DNA (RAPD) profiles, obtained when screening culture collection strains of Candida spp., showed several species to be highly heterogeneous . The RAPD-defined groups were as follows: C . catenulata, two groups among five strains; Debaryomyces hansenii (anamorph C . famata), three groups among five strains; C . sake, three groups among three strains; C . intermedia, two groups among two strains; C . rugosa, two groups among three strains and C . parapsilosis, three groups among four strains . The five strains of Issatchenkia orientalis (anamorph C . krusei) belonged to a single RAPD-defined group . The two strains of the teleomorphic yeast Arxiozyma telluris had distinct RAPD profiles but neither resembled profiles of its anamorph, C . pintolopesii . The two varieties of C . pintolopesii had different RAPD profiles . Researchers are cautioned that organisms with the same name may not be closely related. Bull Cancer, 1996 Jul, 83(7), 527 - 34 {Apropos of the studies of Lewis, Nusslein-Volhard and Wieschaus, 1995 Nobel prize winners, on the genetic mechanisms of embryonic development of drosophila . A model for human cancer progression}; Cillo C; EB Lewis, C Nusslein-Volhard and E Wieschaus were the winners of the Nobel prize in 1995 for the discovery of genes controling the embryonic development in drosophila . Drosophila development is dependent on sequential activities of three types of genes: the maternal genes, the segmentation genes, and the homeotic genes which are responsible for the segment identity and finally for the building of the body . Mutations of these genes are spectacular because they affect the body structure formed from individual segments . Therefore, the molecular processes regulating the development of inferior organisms such as yeast or more complex as the vertebrates were elucidated by these three researchers . These early biological mechanisms regulate the cell life through interactions with neighbouring cells . We speculate that any alteration of these processes might be implicated in cancer . Understanding of these molecular mechanisms which control cell interactions in cancer constitutes a basis for definition of new prognostic markers and putatively novel therapeutic approaches. Mol Biochem Parasitol, 1996 Jul, 79(1), 1 - 12 Current status of the Plasmodium falciparum genome project; Dame JB et al.; The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures . Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasite's genes and to prepare an ordered set of overlapping clones spanning each of the parasite's 14 chromosomes . Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasite's genes, and approximately 5% of the parasite's genes are tentatively identified from similarity searches of entries in the international sequence databases . A total of > 0.5 Mb of P . falciparum sequence tag data is available . The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome . Routes of access to project information and services are described. Int J Pept Protein Res, 1996 Jul, 48(1), 31 - 47 Chemical synthesis and purification of proteins: a methodology; Ball HL et al.; Classical stepwise solid-phase peptide synthesis (SPPS) has been used successfully for the synthesis of proteins up to 150 residues in length, although usually with poor yields and homogeneity . The major limitation has been the inability to separate chromatographically similar deletion and truncated impurities from the target sequence . We have developed a highly effective protocol for stepwise SPPS and 'one-step' purification of small proteins . to demonstrate the effectiveness of the methodology we synthesised the 101 residue chaperonin 10 protein from Rattus norvegicus (Rat Cpn 10) using three different chemical protocols . Highly homogeneous Rat Cpn10 was obtained using an optimised synthetic strategy and one-step purification procedure (method C), involving (i) HBTU/HOBt activation, (ii) N-(2-chlorobenzyloxycarbonyloxy)succinimide as capping agent and (iii) the incorporation of a reversible Fmoc-based chromatographic probe, derivatised with a lipophilic group for fast one-step RP purification, to give an overall yield of 9.6% . Analysis by ESI-MS indicated that the product was virtually free of deletion impurities, while RP-HPLC under four different conditions and CZE indicated that the protein was 100 and 84% pure, respectively . The spontaneous folding of Rat Cpn10 into its biologically active form was found to correlate well with the degree of purity as assessed by chromatography, ESI-MS and sequencing, since 29 (A), 55 (B) and 81% (C) of correctly folded heptameric structure was obtained . The degree of homogeneity was also reflected in the ability of purified Rat Cpn10 to facilitate the refolding of yeast enolase. J Rheumatol, 1996 Jul, 23(7), 1233 - 6 Generation of inorganic pyrophosphate from extracellular adenosine triphosphate by human serum and plasma; Park W et al.; OBJECTIVE: To quantify inorganic pyrophosphate (PPi) production from extracellular adenosine triphosphate (ATP) by human serum or plasma . METHODS: Serial measurements of ATP hydrolysis (t1/2) were performed by the luciferase method from a starting concentration of 1 microM in serum or platelet-poor plasma incubated under physiologic conditions . ATP was then pumped into another sample of each specimen using the rate constant derived from the ATP t1/2 of that specimen . Trace (32P) gamma ATP was added at the start of the infusion; conversion to (32P) inorganic orthophosphate (Pi) and to (32P) PPi was determined by precipitation of Pi as reduced phosphomolybdate before and after treatment with yeast pyrophosphatase . RESULTS: ATP was hydrolyzed by all serum and plasma specimens; the rate of hydrolysis in serum and plasma from the same blood sample was nearly identical . PPi was the major product, averaging 71% . CONCLUSION: PPi is the major product of ATP catabolism in serum and platelet-poor plasma. Fam Med, 1996 Jul-Aug, 28(7), 493 - 5 The use of over-the-counter antifungal vaginitis preparations by college students; Lipsky MS et al.; BACKGROUND AND OBJECTIVES: This study examined college students' perceptions of the over-the-counter (OTC) availability of vaginal antifungal preparations . METHODS: A cross-sectional survey of 258 college students participated in the study over a 3-month period . Students were surveyed about their use of OTC antifungal preparations and their experiences with product effectiveness, benefits, and risks . RESULTS: Among 157 women who experienced a yeast infection, 110 (71%) had used OTC antifungal agents . Approximately 92% reported that the products cured their infection . Most students believed the OTC agents were a good idea because of the convenience and cost savings . CONCLUSIONS: The consumer group, which consisted of young female college students, felt they had assimilated the introduction of OTC antifungal preparations into their personal health care safely and effectively. Br J Pharmacol, 1996 Jul, 118(5), 1183 - 91 The inhibition of antigen-induced eosinophilia and bronchoconstriction by CDP840, a novel stereo-selective inhibitor of phosphodiesterase type 4; Hughes B et al.; 1 . The novel tri-aryl ethane CDP840, is a potent and selective inhibitor of cyclic AMP phosphodiesterase type 4 (PDE 4) extracted from tissues or recombinant PDE 4 isoforms expressed in yeast (IC50S: 4-45 nM) . CDP840 is stereo-selective since its S enantiomer (CT 1731) is 10-50 times less active against all forms of PDE 4 tested while both enantiomers are inactive (IC50S: > 100 microM) against PDE types 1, 2, 3 and 5 . 2 . Oral administration of CDP840 caused a dose-dependent reduction of interleukin-5 (IL-5)-induced pleural eosinophilia in rats (ED50 = 0.03 mg kg-1) . The eosinophils in pleural exudates from CDP840-treated animals contained higher levels of eosinophil peroxidase (EPO) than cells from control animals, suggesting a stabilizing effect on eosinophil degranulation . CDP840 was approximately equi-active with the steroid dexamethasone in this model and was 10-100 times more potent than the known PDE 4-selective inhibitors rolipram and RP73401 . The activity of CDP840 was not influenced by adrenalectomy, beta-sympathomimetics or beta-sympatholytics . 3 . Antigen-induced pulmonary eosinophilia in sensitized guinea-pigs was reduced dose-dependently by CDP840 (0.01-1 mg kg-1, i.p.) and intracellular EPO levels were significantly higher . CDP840 was more potent in these activities than CT1731 or rolipram and comparable in potency to RP73401 . 4 . Rolipram or CDP840 were less active than dexamethasone in preventing neutrophil accumulation, or exudate formation in carrageenan-induced pleurisy in rats and thus do not exhibit general anti-inflammatory activity . 5 . In sensitized guinea-pigs, aerosols of the antigen ovalbumin caused a dose-dependent bronchoconstriction demonstrated by an increase in pulmonary inflation pressure . Administration of CDP840 (0.001-1.0 mg kg-1, i.p.), 1 h before antigen challenge, resulted in dose-dependent reduction in response to antigen . This activity was not due to bronchodilatation since higher doses of CDP840 (3 mg kg-1) did not significantly change the bronchoconstrictor response to histamine . Rolipram was approximately 10 times less active than CDP840 in preventing antigen-induced bronchoconstriction . 6 . These results confirm the observations that selective PDE 4 inhibitors reduce antigen-induced bronchoconstriction and pulmonary eosinophilic inflammation . CDP840 is more potent than rolipram in inhibiting native or recombinant PDE 4 . Unlike the recently described potent PDE 4 inhibitor RP73401, CDP840 is more active than rolipram in the rat IL-5 model following oral administration . The novel series of tri-aryl ethanes, of which CDP840 is the lead compound, could be the basis of an orally active prophylactic treatment for human asthma. J Neurobiol, 1996 Jul, 30(3), 349 - 58 Expression of a developmentally regulated gene, Mng10, in identified neurosecretory cells in the CNS of Manduca sexta; Meszaros M et al.; We are interested in the molecular events underlying the development of the nervous system of Manduca sexta during the final 24 h of the pupal molt . In this article we describe a gene, Mng10, that is expressed in the abdominal nervous system of M . sexta and is developmentally regulated over this 24-h period . In situ hybridization analysis shows that the transcript is localized predominantly to a single pair of uniquely identifiable neurosecretory neurons, the NS-L1 cells in the abdominal ganglia . Mng10 is a single copy gene encoding a 229 amino acid protein with a predicted molecular mass of 26 kDa . At the amino acid level the protein shows 34% identity to the yeast transcription unit, Yer082 . Northern blot analysis shows that the transcript of Mng10 is very rare, comprising about 0.001% of the poly (A)+ RNA from the CNS and is detectable at 4 h but not 24 h prior to pupal ecdysis . One of the physiological events that develops over the final 24 h of the pupal molt is the ability of the nervous system to respond to the neuropeptide eclosion hormone . In this context, it is interesting to note that the NS-L1 cells are members of the group of 50 neurons that show increased cGMP immunoreactivity when the nervous system is exposed to the neuropeptide eclosion hormone. Curr Biol, 1996 Jul 1, 6(7), 848 - 54 HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs; Fritz CC et al.; BACKGROUND: The structural proteins of human immunodeficiency virus type 1 (HIV-1) are encoded by intron-containing mRNAs that normally are retained in the nucleus . A viral regulatory protein, Rev, specifically induces the accumulation of these transcripts in the cytoplasm . Rev is an RNA-binding protein that also contains an 'effector' domain . The Rev effector domain has recently been shown to function as an autonomous nuclear export signal (NES) that, when fused to a foreign protein, will cause its rapid nuclear export . We and others have recently reported the cloning of a human protein (hRIP/Rab), that specifically interacts with the effector domain of Rev . RESULTS: Here we show that the NESs contained within two cellular proteins, PKI and I kappa B, which are not involved in RNA metabolism, also interact with hRIP . Fusion of these cellular sequences to the Rev RNA-binding domain reconstitutes a functional Rev protein . In addition to hRIP, these NESs also bind to several nuclear pore complex (NPC) . We show that this protein export pathway is highly conserved by demonstrating that mammalian NESs also function in yeast . CONCLUSIONS: Our results indicate that the HIV-1 Rev protein evolved to take advantage of a cellular protein export pathway in order to allow the nucleocytoplasmic transport of unspliced viral RNA . Our data suggest a model in which the export substrate is translocated through the NPC by sequential interactions with different nucleoporins . Finally, our experiment suggests a mechanism by which I kappa B can downregulate nuclear NF kappa B activity by causing its rapid export from the nucleus. Biosci Biotechnol Biochem, 1996 Jul, 60(7), 1193 - 4 Conversion of glutathione into cadystins and their analogs catalyzed by carboxypeptidase Y; Imai K et al.; Cadystins induced in a fission yeast treated with Cd2+ are the higher homologs of glutathione . In the present work, glutathione was incubated with Carboxypeptidase Y at a high substrate concentration . The reaction afforded not only the degraded product, but also cadystins and their analogs . A possible transformation pathway for glutathione by this enzyme is proposed. J Struct Biol, 1996 Jul-Aug, 117(1), 1 - 15 Intermediate filament protein polymerization: molecular analysis of Drosophila nuclear lamin head-to-tail binding; Stuurman N et al.; Polymerization of intermediate filament proteins results from interactions among several distinct binding sites on the constituent proteins . Nuclear lamin head-to-tail polymers arise from one such interaction . We studied this binding using Drosophila lamin Dm0-derived fragments containing either the NH2-terminal or COOH-terminal binding site with a combination of co-immunoprecipitation, yeast two-hybrid, analytical ultracentrifugation, and electron microscopic assays . Fragment binding and full-length lamin head-to-tail polymerization were similar to each other in morphology, buffer requirements, and inhibition after phosphorylation with cdc2 kinase . Deletion analysis localized the binding sites to the ends of the rod domain that are highly conserved among all intermediate filament proteins . Point mutants, defective in binding, were isolated . Two were identical to point mutations in specific human keratin genes known to affect keratin assembly and to cause genetic skin diseases . Results further indicate that the binding sites only function in specific sequence contexts and that binding can be modulated by elements outside the binding sites (like the cdc2 kinase phosphorylation site) . Our data indicate that one type of interaction in intermediate filament protein polymerization is the longitudinal binding of dimers via the conserved end segments of the coiled-coil rod domain. FEBS Lett, 1996 Jul 1, 389(2), 153 - 6 Kinetic and spectral properties of pea cytosolic ascorbate peroxidase; Marquez LA et al.; Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties . Its rate constant for compound I formation from reaction with H2O2 is 4.O x 10(7) M-1 s-1, somewhat faster than is typical for peroxidases . Compound I has the typical optical spectrum of an iron(IV)-porphyrin-pi-cation radical, despite considerable homology with yeast cytochrome c peroxidase . The rate constant for compound I reduction by ascorbate is extremely fast (8.0 x 10(7) M-1 S-1 at pH 7.8), again in marked contrast to the behavior of the yeast enzyme . The pH-rate profile for compound I formation indicates a pKa value of 5.0 for a group affecting the active site reaction. Eur J Immunol, 1996 Jul, 26(7), 1613 - 20 The mouse immunoglobulin kappa locus contains about 140 variable gene segments; Kirschbaum T et al.; In continuation of our efforts to elucidate the immunoglobulin kappa locus of the mouse we analyzed 46 yeast artificial chromosomes (YACs) containing V kappa, J kappa and C kappa genes . The YACs, which were derived from DNA of C57BL/6 and C3H mice, ranged from 0.3-1.9 Mb in size . On the basis of hybridization with probes specific for the V kappa gene families a group of 13 YACs was selected for detailed analysis . The V kappa genes of the YACs were then characterized by hybridization to the family-specific probes and by the sizes of the EcoRI fragments on which they were found . This way evidence was obtained for 140 different V kappa gene signals on the YACs . Of these 63 had been characterized before on clones from a cosmid library of total mouse DNA (I . Zocher et al., Eur . J . Immunol . 1995 . 25: 3326-3331) and 22 others were found now on cosmid clones derived from the YACs . Six V kappa genes of the previous study which were not found on the YACs are probably located outside of the kappa locus . The YACs were arrayed in a unique order establishing a YACs panel which most likely contains the whole kappa locus . The cosmid contigs and solitary cosmid clones which contain the 63 plus 22 V kappa gene signals mentioned above comprise about 2.0 Mb . Assuming that the remaining 55 V kappa genes are spaced at the same average distance of 24 kb, one may extrapolate to a locus size of 3.3 Mb. Physiol Rev, 1996 Jul, 76(3), 651 - 85 Regulation of protein transport to the nucleus: central role of phosphorylation; Jans DA et al.; Nuclear protein transport is integral to eukaryotic cell processes such as differentiation, transformation, and the control of gene expression . Although the targeting role of nuclear localization signals (NLSs) has been known for some time, more recent results indicate that NLS-dependent nuclear protein import is precisely regulated . Phosphorylation appears to be the main mechanism controlling the nuclear transport of a number of proteins, including transcription factors such as NFkappaB, c-rel, dorsal, and SWI5 from yeast . Cytoplasmic retention factors, intra- and intermolecular NLS masking, and NLS masking by phosphorylation are some of the mechanisms by which phosphorylation specifically regulates nuclear transport . Even nuclear localization of the archetypal NLS-containing simian virus 40 large tumor antigen (T-ag) is regulated, namely by the "CcN motif," which comprises the T-ag NLS ("N") determining ultimate subcellular destination, a casein kinase II site ("C") 13 amino acids NH2-terminal to the NLS modulating the rate of nuclear import, and a cyclin-dependent kinase site ("c") adjacent to the NLS regulating the maximal level of nuclear accumulation . The CcN motif appears to be a special form of phosphorylation-regulated NLS (prNLS), where phosphorylation at site(s) close to the NLS specifically regulates NLS function . The regulation of nuclear transport through phosphorylation and prNLSs appears to be common in eukaryotic cells from yeast and plants to higher mammals. Microbiology, 1996 Jul, 142 ( Pt 7), 1597 - 604 Cell wall protein and glycoprotein constituents of Aspergillus fumigatus that bind to polystyrene may be responsible for the cell surface hydrophobicity of the mycelium; Penalver MC et al.; Cell surface hydrophobicity (CSH) of Aspergillus fumigatus grown both in complex medium (yeast extract/peptone/dextrose; YPD) and minimal (Vogel's N) medium was monitored by assessing attachment of polystyrene microspheres to the cell surface . It was found that mature mycelium was hydrophobic . Treatment of intact mycelium with beta-mercaptoethanol (beta ME) abolished binding of the microspheres to hyphal elements, and coating of the microspheres with beta ME extracts from mycelium inhibited their attachment to intact mycelial cells . A . fumigatus mycelium was tagged in vivo with biotin and treated with beta ME . The beta ME extracts were analysed by SDS-PAGE and Western blotting with both peroxidase-conjugated-ExtrAvidin and concanavalin A (ConA) . This procedure allowed identification of cell wall surface proteins and glycoproteins . Rabbit polyclonal antisera were raised against beta ME extracts obtained from cells grown in YPD and Vogel's N media . These antisera defined some major cell-wall-bound antigens . SDS-PAGE and Western blotting analysis of the cell wall material released by beta ME and adsorbed on polystyrene microspheres revealed about 19 protein species with apparent molecular masses ranging from 20 to 70 kDa, and two high-molecular-mass glycoproteins of 115 and 210 kDa . Treatment of cells grown in YPD, but not those grown in Vogel's N medium, with beta ME released a 55 kDa polypeptide able to adsorb to polystyrene microspheres that was detectable with the antisera . The ability to bind to polystyrene particles exhibited by several protein and glycoprotein species released by beta ME treatment suggested that these cell wall moieties possess exposed hydrophobic domains that could be responsible for the CSH of mycelium. Br J Haematol, 1996 Jul, 94(1), 105 - 11 The der(21)t(12;21) chromosome is always formed in a 12;21 translocation associated with childhood acute lymphoblastic leukaemia; Kobayashi H et al.; We studied 116 patients (93 children and 23 adults) with acute lymphoblastic leukaemia (ALL) using fluorescence in situ hybridization (FISH) with the yeast artificial chromosome (YAC) clone, 964c10, which includes the recently described ETS-like gene, TEL, on 12p13 . FISH revealed that nine of the patients had a t(12;21), which had not been previously detected . The nine patients were all children, seven boys and two girls, aged 1-10 years (median 3 years), had an early B immunophenotype, and achieved complete remission, although two of them experienced haematological relapse . In addition to the t(12;21), FISH also revealed that three of the nine had a del(12p) in the other homolog of chromosome 12 or in the der(12) chromosome itself, and that two others had 12p translocations in the other chromosome 12 homolog . Although chromosomal rearrangements associated with the t(12;21) were heterogenous and complex, fusion of the sequences from chromosomes 12 and 21 on the der(21)t(12;21) chromosomes was consistent, suggesting that the TEL-AML1 gene fusion on the der(21) chromosome may be critical in leukaemogenesis and that FISH or reverse transcriptase-polymerase chain reaction (RT-PCR) targeted to the chimaeric sequences on the der(21) will be most useful in detecting the t(12;21) or following a patient with the t(12;21), which is one of the most frequent chromosomal rearrangements in both Caucasian and Asian childhood ALL. Appl Biochem Biotechnol, 1996 Jul, 60(1), 41 - 8 Biotransformation of uridine monophosphate (UMP) and glucose to uridine diphosphate-glucose (UDPG) by Candida saitoana KCTC7249 cells; Ko JH et al.; The present study investigates the biotransformation of glucose with uridine monophosphate (UMP) to obtain sugar nucleotide, UDP-glucose (UDPG), by the dried cells of Candida saitoana KCTC7249 . The biotransformation was optimized by varying the concentrations of substrates and phosphate ion . UDPG (24 mM) was biotransformed from 200 mM glucose and 37.5 mM UMP by dried cells of C . saitoana . The glucose yields about 64% UDP-glucose, based on UMP concentration . The addition of glucose-1-phosphate to the reaction mixture accelerated the formation of UDPG from a concentration of UMP . The structure of UDP-glucose obtained was determined with 13C NMR and FAB mass spectra . These results indicate that the yeast-dried cells could be used for the production of nucleotide sugars for donor molecules of complex carbohydrate synthesis. Infect Immun, 1996 Jul, 64(7), 2716 - 23 Natural immune response to the C-terminal 19-kilodalton domain of Plasmodium falciparum merozoite surface protein 1; Shi YP et al.; We have characterized the natural immune responses to the 19-kDa domain of merozoite surface protein 1 in individuals from an area of western Kenya in which malaria is holoendemic . We used the three known natural variant forms of the yeast-expressed recombinant 19-kDa fragment that are referred to as the E-KNG, Q-KNG, and E-TSR antigens . T-cell proliferative responses in individuals older than 15 years and the profile of immunoglobulin G (IgG) antibody isotypes in individuals from 2 to 74 years old were determined . Positive proliferative responses to the Q-KNG antigen were observed for 54% of the individuals, and 37 and 35% of the individuals responded to the E-KNG and E-TSR constructs, respectively . Considerable heterogeneity in the T-cell proliferative responses to these three variant antigens was observed in different individuals, suggesting that the 19-kDa antigen may contain variant-specific T epitopes . Among responses of the different isotypes of the IgG antibody, IgG1 and IgG3 isotype responses were predominant, and the prevalence and levels of the responses increased with age . We also found that a higher level of IgG1 antibody response correlated with lower parasite density among young age groups, suggesting that IgG1 antibody response may play a role in protection against malaria . However, there was no correlation between the IgG3 antibody level and protection . Furthermore, we observed that although the natural antibodies cross-reacted with all three variant 19-kDa antigens, IgG3 antibodies in 12 plasma samples recognized only the E-KNG and Q-KNG constructs and not the E-TSR antigen . This result suggests that the fine specificity of IgG3 antibodies differentiates among variant-specific natural B-cell determinants in the second epidermal growth factor domain (KNG and TSR) of the antigen. Nucleic Acids Res, 1996 Jul 1, 24(13), 2551 - 9 XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes; van der Spek PJ et al.; The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER) . The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al . (1994) EMBO J . 8, 1831-1843) . Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC . In contrast, we cannot detect any bound HHR23A . Thus the HHR23 proteins may have an additional function independent of XPC . The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction . Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation . Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex . However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex . Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus. Arch Environ Contam Toxicol, 1996 Jul, 31(1), 120 - 7 Toxicity and oxidative stress of different forms of organic selenium and dietary protein in mallard ducklings; Hoffman DJ et al.; Concentrations of over 100 ppm (mg/kg) selenium (Se) have been found in aquatic plants and insects associated with irrigation drainwater and toxicity to fish and wildlife . Composition of diet for wild ducklings can vary in selenium-contaminated environments . Earlier studies have compared toxicities and oxidative stress of Se as selenite to those of seleno-DL-methionine (DL) in mallards (Anas platyrhynchos) . This study compares DL, seleno-L-methionine (L), selenized yeast (Y) and selenized wheat (W) . Day-old mallard ducklings received an untreated diet (controls) containing 75% wheat (22% protein) or the same diet containing 15 or 30 ppm Se in the above forms except for 30 ppm Se as W . After 2 weeks, blood and liver samples were collected for biochemical assays and Se analysis . All forms of selenium caused significant increases in plasma and hepatic glutathione peroxidase activities . Se as L at 30 ppm in the diet was the most toxic form, resulting in high mortality (64%) and impaired growth (>50%) in survivors and the greatest increase in ratio of oxidized to reduced hepatic glutathione (GSH) . Se as both L and DL decreased the concentrations of hepatic GSH and total thiols . Se as Y accumulated the least in liver (approximately 50% of other forms) and had less effect on GSH and total thiols . In a second experiment, in which the basal diet was a commercial duck feed (22% protein), survival was not affected by 30 ppm Se as DL, L, or Y and oxidative effects on GSH metabolism were less pronounced than with the wheat diet. Hum Genet, 1996 Jul, 98(1), 7 - 11 Identification and characterization of NF1-related loci on human chromosomes 22, 14 and 2; Hulsebos TJ et al.; Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder . The disease is characterized by a very high mutation rate (up to 1/10000 gametes per generation) . NF1-related loci in the human genome have been implicated in the high mutation rate by hypothesizing that these carry disease-causing mutations, which can be transferred to the functional NF1 gene on chromosome arm 17q by interchromosomal gene conversion . To test this hypothesis, we want to identify and characterize the NF1-related loci in the human genome . In this study, we have localized an NF1-related locus in the most centromeric region of the long arm of chromosome 22 . We demonstrate that this locus contains sequences homologous to cDNAs that include the GAP-related domain of the functional NF1 gene . However, the GAP-related domain itself is not represented in this locus . In addition, cosmids specific to this locus reveal, by in situ hybridization, NF1-related loci in the pericentromeric region of chromosome arm 14q and in chromosomal band 2q21 . These cosmids will enable us to determine whether identified disease-causing mutations are present at the chromosome 22-associated NF1-related locus. Hum Genet, 1996 Jul, 98(1), 16 - 21 Fine mapping of the 1q21 breakpoint of the papillary renal cell carcinoma-associated (X;1) translocation; Weterman MA et al.; A combination of Southern blot analysis on a panel of tumor-derived somatic cell hybrids and fluorescence in situ hybridization (FISH) techniques was used to map a series of DNA markers relative to the 1q21 breakpoint of the renal cell carcinoma (RCC)-associated (X;1)-(p11;q21) translocation . This breakpoint maps between several members of the S100 family which are clustered in the 1q21 region and a conserved region between man and mouse containing the markers SPTA1-CRP-APCS-FcER1A-ATP1A2-APOA2 . The location of the breakpoint coincides with the transition of a region of synteny of human chromosome 1 with mouse chromosomes 3 and 1. Genes Dev, 1996 Jul 1, 10(13), 1683 - 98 A nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5' splice site; Lewis JD et al.; The mechanism by which intron-containing RNAs are recognized by the splicing machinery is only partly understood . A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has previously been shown to play a role in pre-mRNA splicing . Using a combination of splicing complex and psoralen cross-linking analysis we demonstrate that CBC is required for efficient recognition of the 5' splice site by U1 snRNP during formation of E (early) complex on a pre-mRNA containing a single intron . However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron . In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC . These results support models in which the splice sites in a pre-mRNA are originally recognized by interactions spanning exons . The defects in splicing and U1 snRNP binding caused by CBC depletion can be specifically reversed by recombinant CBC . In summary, efficient recognition of the cap-proximal 5' splice site by U1 snRNP is facilitated by CBC in what may be one of the earliest steps in pre-mRNA recognition . Data in Colot et al . (this issue) indicate that this function of CBC is conserved in humans and yeast. J Gerontol A Biol Sci Med Sci, 1996 Jul, 51(4), B280 - 3 Failure to confirm increased longevity in Drosophila melanogaster submitted to a food restriction procedure; Le Bourg E et al.; Several studies have shown that, contrary to what occurs in rodents and in some invertebrate species, food restriction has no positive effect on longevity in Drosophila melanogaster . However, Chippindale et al . (1993) reported that flies subjected to food restriction, by modulating the yeast level, could live longer . In the present study we used the same yeast levels as Chippindale et al . in an attempt to confirm these results . No positive effect of food restriction on longevity could be observed in either sex in mated and virgin flies. J Virol, 1996 Jul, 70(7), 4299 - 310 Binding of the human immunodeficiency virus type 1 Gag polyprotein to cyclophilin A is mediated by the central region of capsid and requires Gag dimerization; Colgan J et al.; The cellular peptidyl-prolyl isomerase cyclophilin A (CyPA) is incorporated into human immunodeficiency virus type 1 (HIV-1) virions via direct contacts with the HIV-1 Gag polyprotein . Disruption of the Gag-CyPA interaction leads to the production of HIV-1 particles lacking CyPA; these virions are noninfectious, indicating that contacts between CyPA and Gag are necessary for HIV-1 replication . Here, we have used the yeast two-hybrid system in conjunction with an in vitro binding assay to identify the minimal domain of Gag required for binding to CyPA . Analysis of a panel of gag deletion mutants in the two-hybrid system indicated that a region spanning the central portion of the capsid (CA) domain was sufficient for interactions with CyPA, but discrepancies between results obtained in different fusion protein contexts suggested that multimerization of Gag might also be necessary for binding to CyPA . Consistent with a requirement for multimerization, the binding of Gag to CyPA in vitro required a region within the nucleocapsid (NC) domain shown previously to be important for Gag self-association . Substitution of a heterologous dimerization motif for the region from NC also promoted specific binding to CyPA, confirming that interactions with CyPA are dependent on Gag multimerization . Fusion of the heterologous dimerization motif to a 100-amino-acid domain from CA was sufficient for binding to CyPA in vitro . These results define the minimal CyPA-binding domain within Gag and provide insight into the mechanism by which CyPA is incorporated into HIV-1 virions. J Virol, 1996 Jul, 70(7), 4228 - 36 A conserved domain of the Epstein-Barr virus nuclear antigens 3A and 3C binds to a discrete domain of Jkappa; Zhao B et al.; EBNA-3C can affect the LMP-1 promoter in both a positive and a negative manner through distinct DNA sequence elements . The viral transactivator EBNA-2 normally binds DNA indirectly via Jkappa to activate transcription, but this activation is prevented in the presence of EBNA-3C . The DNA element recognized by Jkappa is both required and sufficient for this inhibition . Jkappa clones isolated in a yeast two-hybrid screen using EBNA-3C as bait allowed us to delineate the sequences of both proteins mediating the interaction . Two isoforms of Jkappa that differ in exon 1, Jkappa-1 and RBP-2N, interact with EBNA-3C, suggesting that exon 1 is not required for this interaction; indeed, clones with deletion of the N-terminal third of Jkappa interacted as efficiently with EBNA-3C as full-length Jkappa clones . A Jkappa domain as small as 56 amino acids was sufficient to bind to EBNA-3C . A 74-amino-acid domain of EBNA-3C, conserved in all three EBNA-3 family members, was sufficient to interact with Jkappa . A specific mutation in this conserved domain suppressed the ability of EBNA-3C to downregulate transcription . Accordingly, EBNA-3A was also able to interact with Jkappa and downregulate Jkappa-mediated transcription as efficiently as EBNA-3C . The ability of the EBNA-3 proteins to prevent Jkappa from binding to DNA in vitro and suppress transactivation via Jkappa DNA elements suggests that the EBNA-3 proteins act analogously to the Drosophila protein Hairless. EMBO J, 1996 Jul 1, 15(13), 3394 - 402 The hbrm and BRG-1 proteins, components of the human SNF/SWI complex, are phosphorylated and excluded from the condensed chromosomes during mitosis; Muchardt C et al.; In yeast, the SNF/SWI complex is believed to regulate transcription by locally altering the chromatin structure . At the present time, three human homologues of yeast SNF/SWI proteins have been characterized: hbrm and BRG-1, homologues of SNF2/SWI2, and hSNF5, a homologue of SNF5 . We show here that, during mitosis, hbrm and BRG-1 are phosphorylated and excluded from the condensed chromosomes . In this phase of the cell cycle, the level of hbrm protein is also strongly reduced, whereas the level of BRG-1 remains constant . The mitotic phosphorylation of hbrm and BRG-1 is found not to disrupt the association of these proteins with hSNF5 but correlates with a decreased affinity for the nuclear structure in early M phase . We suggest that chromosomal exclusion of the human SNF/SWI complex at the G2-M transition could be part of the mechanism leading to transcriptional arrest during mitosis. EMBO J, 1996 Jul 1, 15(13), 3229 - 37 An essential ubiquitin-conjugating enzyme with tissue and developmental specificity in th nematode Caenorhabditis elegans; Zhen M et al.; The ubc-2 gene in Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme (E2) homologous to yeast UBC4 and UBC5 . UBC4 and UBC5 are individually dispensable class I E2 enzymes involved in the degradation of short-lived and abnormal proteins . Transgenic analysis using ubc-2-lacZ fusions and in situ immunofluorescence indicate that ubc-2 is abundantly expressed in most tissues of embryos and early larvae, but becomes specific to the nervous system in L4 larvae and adults . This suggests that the functions of this type of E2 are developmentally regulated in C.elegans . This hypothesis is supported by antisense analysis, which shows that blocking the expression of ubc-2 has a more severe effect in early developmental stages than in later stages . Through complementation of previously identified essential genes in the vicinity of ubc-2, we demonstrate that ubc-2 corresponds to let-70, a gene essential for C.elegans larval development . One let-70(ubc-2) allele contains a His75-->Tyr substitution, while another has an altered splice donor site. Mol Cell Biol, 1996 Jul, 16(7), 3317 - 26 Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly; Chiara MD et al.; We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly . This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex . Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site . At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes . As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation . With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes . Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site . In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex . The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway. Virology, 1996 Jul 1, 221(1), 44 - 53 The transactivation and DNA binding domains of the BPV-1 E2 protein have different roles in cooperative origin binding with the E1 protein; Winokur PL et al.; The bovine papillomavirus E2 transactivator protein enhances the ability of the E1 protein to bind to the viral origin of replication which contains an E1 binding site flanked by two E2 binding sites . To determine which regions and functions of the E2 protein are important for this cooperative interaction, a series of mutated E2 proteins were assayed for their ability to enhance E1 origin-specific binding . Cooperative origin binding required at least one E2 DNA binding site, an intact functional E2 DNA binding domain, and an intact transactivation domain . The hinge region of the E2 proteins was dispensable for this activity . To further examine the role of the E2 C-terminal domain, a series of chimeric proteins were generated that substituted the yeast GAL4 DNA binding domain for the E2 DNA binding domain . These chimeric proteins were able to cooperatively bind to a hybrid origin that contained GAL4 binding sites in place of the E2 binding sites . These studies indicate that the E2 transactivation domain is sufficient for interaction with the E1 protein and that the E2 DNA binding domain is required for interaction with origin DNA sequences. Genomics, 1996 Jul 1, 35(1), 241 - 3 Gene structure and chromosome localization to 7q21.3 of the human rod photoreceptor transducin gamma-subunit gene (GNGT1); Scherer SW et al.; The transducin gamma-subunit gene (GNGT1) encodes a member (gamma1) of the family of heterotrimeric G-protein gamma-subunits that is specific to rod photoreceptors . In this report we have determined the complete structure of the GNGT1 gene and have localized it to human chromosome 7q21.3 using somatic cell hybrid and yeast artificial chromosome analysis. Genomics, 1996 Jul 1, 35(1), 172 - 81 Characterization and genomic mapping of genes and pseudogenes of a new human protein tyrosine phosphatase; Zhao Z et al.; Previously described protein tyrosine phosphatases (PTPs) are classified into three types according to their sequence homology and structural features . Here we describe the characterization of genes and pseudogenes of a member of a fourth type of PTP, designated protein tyrosine phosphatase 4A (PTP4A) . The 167-amino-acid human PTP4A bears the signature active site of all PTPs, but does not show any other sequence homology to any of the previously described PTPs . Two cDNAs encoding PTP4A that differed in their noncoding regions were isolated . Another cDNA that has a high level of sequence identity with these two cDNAs and a deletion in the coding region was also isolated . Northern analysis using a probe from a common 3'-untranslated region of the cDNAs recognized mRNAs of about 2 and 4 kb . Both species of mRNA were seen in all human adult and fetal tissues tested . Fluorescence in situ hybridization mapping of the corresponding yeast artificial chromosome clones and sequence-tagged site analysis suggested that one of the PTP4A coding genes is located at 1p35 and the other is on chromosome 11 . A processed pseudogene for PTP4A was found in the BRCA1 region of 17q21 and shares 96% sequence identity to one of the PTP4A coding cDNAs . Our studies also suggest the existence of another processed pseudogene on chromosome 11. Genomics, 1996 Jul 1, 35(1), 118 - 28 Physical mapping of the bloom syndrome region by the identification of YAC and P1 clones from human chromosome 15 band q26.1; Straughen J et al.; The gene for Bloom syndrome (BLM) has been mapped to human chromosome 15 band q26.1 by homozygosity mapping . Further refinement of the location of BLM has relied upon linkage-disequilibrium mapping and somatic intragenic recombination . In combination with these mapping approaches and to identify novel DNA markers and probes for the BLM candidate region, a contiguous representation of the 2-Mb region that contains the BLM gene was generated and is presented here . YAC and P1 clones from the region have been identified and ordered by using previously available genetic markers in the region along with newly developed sequence-tagged sites from radiation-reduced hybrids, polymorphic dinucleotide repeat loci, and end sequences of YACs and P1s . A long-range restriction map of the 2-Mb region that allowed estimation of the distance between polymorphic microsatellite loci is also reported . This map and the DNA markers derived from it were instrumental in the recent identification of the BLM gene. Genomics, 1996 Jul 1, 35(1), 109 - 17 Direct cloning of DNA sequences from the common fragile site region at chromosome band 3p14.2; Rassool FV et al.; Despite several lines of evidence suggesting that common chromosomal fragile sites are biologically important as hot spots for recombination, their structure remains unknown . We showed previously that the plasmid pSV2neo preferentially integrates into bands containing fragile sites in cells transfected under conditions of fragile site induction . Here we report the isolation and characterization of the DNA sequences from two such independent integrations into 3p14.2, a common fragile site (FRA3B) . These FRA3B region sequences were shown to lie within a 1330-kb YAC, 850A6, approximately 350 kb telomeric of the breakpoint of t(3;8), a constitutional rearrangement . The two integration sites are 10 kb apart, but each integration is associated with a deletion . We have constructed a partial genomic contig of the integration sites and deleted regions spanning approximately 85 kb . Analysis of the DNA sequences immediately surrounding the plasmid integrations revealed no known coding sequences or repeat structures resembling the (CGG)n motif characteristic of the rare fragile sites . In addition, by Southern blotting analysis, none of the phage clones isolated from the FRA3B region were found to contain CGG repeats . Fluorescence in situ hybridization analysis of genomic clones from this contig to metaphase cells induced to express breaks demonstrated hybridization adjoining the chromosome breaks, and occasionally the hybridization signal spanned the break . The results imply that breakage occurs at variable positions within a large region (at least on the order of 85 kb) . Together, these data suggest that the structure of FRA3B differs from that of rare fragile sites. Genomics, 1996 Jul 1, 35(1), 94 - 100 Physical mapping and genomic structure of the human TNFR2 gene; Beltinger CP et al.; The tumor necrosis factor receptor 2 (TNFR2) gene localizes to 1p36 . 2, a genomic region characteristically deleted in neuroblastomas and other malignancies . In addition, TNFR2 is the principal mediator of the effects of TNF on cellular immunity, and it may cooperate with TNFR1 in the killing of nonlymphoid cells . Therefore, we undertook an analysis of the genomic structure and precise physical mapping of this gene . The TNFR2 gene is contained on 10 exons that span 26 kb . Most of the functional domains of TNFR2 are encoded by separate exons, and each of the repeats of the extracellular cysteine-rich domain is interrupted by an intron . The genomic structure reveals a close relationship to TNFR1, another member of the TNFR superfamily . Based on electrophoretic analysis of yeast artificial chromosomes, TNFR2 maps within 400 kb of the genetic marker D1S434 . In addition, we have identified a new polymorphic dinucleotide repeat within intron 4 of TNFR2 . The genetic sequence information and exon-intron boundaries we have determined will facilitate mutational analysis of this gene to determine its potential role in neuroblastoma, as well as in other cancers with characteristic deletions or rearrangements of 1p36. Genomics, 1996 Jul 1, 35(1), 87 - 93 A 350-kb cosmid contig in 3p14.2 that crosses the t(3;8) hereditary renal cell carcinoma translocation breakpoint and 17 aphidicolin-induced FRA3B breakpoints; Paradee W et al.; The constitutive fragile site at human chromosomal band 3p14.2, FRA3B, has been described as the most active common fragile site in the human genome . FRA3B is cytologically indistinguishable from the chromosome 3 breakpoint observed in the hereditary renal cell carcinoma (hRCC) translocation t(3;8) (p14.2;q24.13) . Previous work demonstrated that a 1330-kb YAC clone, YC850A6, spans both the t(3;8) translocation and FRA3B and also encompasses FRA3B-associated breakpoints induced in hamster-human hybrids . This YAC was used to construct a multi-hit cosmid library . Screening of this library resulted in a 350-kb cosmid contig that extends distally from the t(3;8) translocation breakpoint . Seventeen aphidicolin-induced 3p14 . 2 breakpoints derived from hamster-human hybrids were mapped within this cosmid contig . These breakpoints were found to localize as two distinct clusters, separated by 200 kb, which lie on either side of a region of frequent breakage within FRA3B as defined by FISH analysis using cosmids from the contigs . The most proximal of the breakpoint clusters lies approximately 100 kb distal to the hRCC t(3;8) breakpoint . The distribution of these breakpoints, together with the region of frequent chromosomal breakage mapped by FISH analysis, further confirms the position of FRA3B and helps to define the extent over which its fragility is exerted . These data indicate that FRA3B comprises several hundred kilobases of DNA sequence within 3p14.2 . The 350-kb contig and the cosmid library constructed from YAC YC850A6 will be essential for further characterization of the region surrounding FRA3B and in experiments to determine the molecular basis of the fragility of FRA3B. Genomics, 1996 Jul 1, 35(1), 71 - 8 Efficient construction of a physical map by fiber-FISH of the CLN5 region: refined assignment and long-range contig covering the critical region on 13q22; Klockars T et al.; The variant form of late infantile neuronal ceroid lipofuscinosis (vLINCL, locus definition CLN5) represents a progressive brain disease with autosomal recessive inheritance . We have previously assigned the CLN5 locus to chromosome 13q21.1-q32 between markers D13S160 and D13S162 by linkage analysis in Finnish families . The information on ancient recombination events obtained from linkage disequilibrium provided an efficient tool for further refining the assignment of the CLN5 locus . Isolation of two novel (CA)n markers, COLAC1 and AC224, resulted in a dramatic restriction of the critical DNA region . We utilized the Fiber-FISH technique to orient and order the large DNA clones isolated by STSs and were able to eliminate almost totally the restriction digestion and PFGE step in the construction of the long-range DNA contig . Both linkage disequilibrium data and Fiber-FISH analyses assigned the CLN5 locus to a well-defined 200-kb region . Here we report a complete physical map of about 350 kb covering the critical chromosomal region of CLN5, which will facilitate the final isolation of the CLN5 gene. Genomics, 1996 Jul 1, 35(1), 46 - 54 Physical mapping of chromosome 8p22 markers and their homozygous deletion in a metastatic prostate cancer; Bova GS et al.; Numerous studies have implicated the short arm of chromosome 8 as the site of one or more tumor suppressor genes inactivated in carcinogenesis of the prostate, colon, lung, and liver . Previously, we identified a homozygous deletion on chromosome 8p22 in a metastatic prostate cancer . To map this homozygous deletion physically, long-range restriction mapping was performed using yeast artificial chromosomes (YACs) spanning approximately 2 Mb of chromosome band 8p22 . Subcloned genomic DNA and cDNA probes isolated by hybrid capture from these YACs were mapped in relation to one another, reinforcing map integrity . Mapped single-copy probes from the region were then applied to DNA isolated from a metastatic prostate cancer containing a chromosome 8p22 homozygous deletion and indicated that its deletion spans 730-970 kb . Candidate genes PRLTS (PDGF-receptor beta-like tumor suppressor) and CTSB (cathepsin B) are located outside the region of homozygous deletion . Genethon marker D8S549 is located approximately at the center of this region of homozygous deletion . Two new microsatellite polymorphisms, D8S1991 and D8S1992, also located within the region of homozygous deletion on chromosome 8p22, are described . Physical mapping places cosmid CI8-2644 telomeric to MSR (macrophage scavenger receptor), the reverse of a previously published map, altering the interpretation of published deletion studies . This work should prove helpful in the identification of candidate tumor suppressor genes in this region. Cell, 1996 Jun 28, 85(7), 1077 - 88 Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs; Kiss-Laszlo Z et al.; Eukaryotic cells contain many fibrillarin-associated small nucleolar RNAs (snoRNAs) that possess long complementarities to mature rRNAs . Characterization of 21 novel antisense snoRNAs from human cells followed by genetic depletion and reconstitution studies on yeast U24 snoRNA provides evidence that this class of snoRNAs is required for site-specific 2'-O-methylation of preribosomal RNA (pre-rRNA) . Antisense sno-RNAs function through direct base-pairing interactions with pre-rRNA . The antisense element, together with the D or D' box of the snoRNA, provide the information necessary to select the target nucleotide for the methyltransfer reaction . The conclusion that sno-RNAs function in covalent modification of the sugar moieties of ribonucleotides demonstrates that eukaryotic small nuclear RNAs have a more versatile cellular function than earlier anticipated. J Biol Chem, 1996 Jun 28, 271(26), 15322 - 9 p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues; Miao W et al.; Previous structural studies of RasGAP have failed to clearly localize sites of Ras interaction to individual amino acids . Hypothesizing that sites of interaction with Ras-GTP would be conserved, 11 of the most highly conserved amino acid residues of RasGAP were changed by mutation . Each mutant protein was purified as a glutathione S-transferase catalytic domain fusion and analyzed for protein stability, Ras GTPase stimulating activity, affinity for Ras-GTP, and when possible, secondary structure . The majority of conserved positions were found to be important structurally but with no direct role in Ras interactions . However, Arg786, Lys831, and Arg925 were observed to be essential for binding to Ras-GTP but not for protein structure . RasGAP residues 890-902 (block 3A) were observed to be homologous to residues 1540-1552 of the yeast adenylyl cyclase with amino acid substitutions in both regions resulting in increased affinity for Ras . This is the first example of a conserved Ras interaction motif in distinct Ras effector proteins . Our data are supportive of a model for GAP/Ras-GTP association in which the conserved, positively charged Arg786, Lys831, and Arg925 residues form salt bridges with the conserved, negatively charged residues in the Ras effector loop. Biochem Biophys Res Commun, 1996 Jun 25, 223(3), 729 - 34 Difference in the mechanism of interaction of Raf-1 and B-Raf with H-Ras; Shinkai M et al.; Ras is known to possess multiple cellular targets including Raf-1 . Here, we measured both direct binding of various H-Ras mutants to two representative mammalian Ras targets, Raf-1 and B-Raf, and the activity of the mutants to stimulate Raf-1 and B-Raf, and analysed the difference in their Ras-interaction mechanisms . B-Raf was shown to share almost the same H-Ras binding-specificity with Raf-1 by examining binding of the H-Ras mutants to Raf-1 and B-Raf in the yeast two-hybrid and in vitro binding assays . Mutants, Y32F, A59E, and V45E bound to Raf-1 in Sf9 cells coexpressing them, but failed to activate Raf-1 . On the other hand, Y32F activated B-Raf in a cell-free system which consisted of rat brain cytosol and recombinant MEK . These results suggest that there is a subtle structural difference in requirements for the interaction of Ras with Raf-1 and B-Raf. Biochem Biophys Res Commun, 1996 Jun 25, 223(3), 701 - 5 Molecular interaction between TLE1 and the carboxyl-terminal domain of HES-1 containing the WRPW motif; Grbavec D et al.; Groucho is a protein implicated in Notch signaling and involved in segmentation and neural development in Drosophila . Groucho forms transcription complexes with the basic helix-loop-helix proteins encoded by the hairy/Enhancer of split ("hairy-like") gene family . These interactions are mediated by the carboxyl-terminal WRPW motif of Hairy-like proteins . We are interested in determining whether Groucho and its mammalian homologues, the TLE proteins, perform conserved functions . We show that TLE1 interacts with HES-1, a murine homologue of Drosophila Hairy-like proteins, both in the yeast two-hybrid assay and in an interaction assay based on glutathione S-transferase fusion proteins . These results show that Groucho/TLE proteins and Hairy-like/HES proteins are involved in similar interactions in Drosophila and mammals and further suggest that these proteins perform conserved cellular functions. Oncogene, 1996 Jun 20, 12(12), 2713 - 7 Tissue-specific expression, evolutionary conservation and localization of the cph proto-oncogene on Syrian hamster chromosome X; Velasco JA et al.; Treatment of Syrian hamster embryo fibroblasts with a single dose of 3-methylcholanthrene caused the activation of the transforming potential of cellular sequences (Notario et al, Oncogene 5: 1425-1430, 1990), which were subsequently isolated by cosmid rescue techniques, and further identified as a novel oncogene, termed cph because of its involvement in the carcinogenic progression of hamster embryo cells (Velasco et al, Oncogene 9: 2065-2069, 1994) . We have analysed the expression of the cph proto-oncogene in adult Syrian hamster tissues by northern hybridization using cph-specific genomic probes . The three cph transcripts expressed in normal and neoplastic Syrian hamster embryo cells in culture (5.0, 3.5 and 2.0 kb) were also present in most adult tissues, although different mRNA species, most likely resulting from alternative splicing events, were expressed in testes . The highest steady-state level of cph mRNA was found in kidney, whereas cph expression was nearly undetectable in skin and skeletal muscle . Southern blot analyses of DNAs from other eucaryotic organisms were performed under moderate stringency conditions with a Syrian hamster-specific cph probe . Discrete cph-hybridizing sequences were present in genomes from yeast to mammalian species, including humans, thus demonstrating that cph is a highly conserved gene in eucaryotic evolution . Using fluorescence in situ hybridization (FISH), we have determined also the chromosomal localization of the cph proto-oncogene in the hamster genome . FISH experiments demonstrated that cph is a single copy gene, localized on the euchromatic short arm of the X chromosome, at region Xpa7 . Because chromosome X is frequently involved in structural alterations in neoplastic Syrian hamster cells transformed by chemical carcinogens and oncogenic viruses, the localization of the cph locus on this chromosome supports the notion that the cph oncogene plays a role in the malignant conversion of chemically transformed hamster fibroblasts . The wide range of tissue-specific expression and species-specific distribution of cph strongly suggest that the normal function of the cph protein product(s) may be essential for metabolic processes involved in the regulation of cell proliferation and survival. Oncogene, 1996 Jun 20, 12(12), 2563 - 71 RN-tre identifies a family of tre-related proteins displaying a novel potential protein binding domain; Matoskova B et al.; Eps8 is a recently identified SH3-containing substrate for tyrosine kinase receptors . To understand the role of eps8 in receptor-mediated signaling, we cloned cDNAs encoding proteins that bind to its SH3 domain . One of these cDNAs predicts the synthesis of an 828 amino acid protein with homology to the N-terminal region of the tre oncogene . We designated this protein RN-tre for Related to the N-terminus of tre . RN-tre is ubiquitously expressed and maps to 10p13, a region known to be involved in translocations in various leukemias . In addition, a 10p13 monosomy syndrome, characterized by developmental alterations, has been reported . The regional homology between RN-tre and tre, which is limited to their N-terminal portion, prompted us to investigate the origin of the tre oncogene transcriptional unit . We were able to show that tre is the fusion product of a 5' genetic element, homologous to RN-tre and a 3' element, encoding a de-ubiquinating enzyme . Moreover, we identified, within the N-terminus of RN-tre and tre, a domain (named TrH, for Tre Homology), which is conserved within several proteins from yeast to mammals and has protein-binding properties in vitro. EMBO J, 1996 Jun 17, 15(12), 2997 - 3005 IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs; Hart MJ et al.; Proteins that associate with the GTP-bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches . A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs-GTP and a corresponding cDNA was isolated . Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP-related domain (GRD) contained in IRA, NF-1, SAR1 and rasGAP . p195 was found to be identical to IQGAP1, a protein previously reported to bind ras . Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected . The C-terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, but a smaller fragment containing only the GRD did not . Cdc42 was also co-immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1 . Calmodulin also co-immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain . Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed . In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co-localization with actin was apparent . These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology. Genomics, 1996 Jun 15, 34(3), 422 - 5 Characterization of a translocation-associated deletion defines the candidate region for the gene responsible for branchio-oto-renal syndrome; Kalatzis V et al.; Fluorescence in situ hybridization analysis of an 8q translocation breakpoint, dir ins(8)(q24.11;q13.3;q21.13), carried by an individual presenting with Branchio-Oto-Renal (BOR) syndrome, resulted in the identification of an associated deletion . The generation of a YAC contig and the isolation of overlapping recombinant P1 and lambda phage clones from the region allowed further characterization of this deletion . Its size was estimated to be between 470 and 650 kb, and it was flanked by the two polymorphic markers D8S1060 and D8S1807 . This mapping led us to reevaluate the localization of the gene responsible for BOR syndrome and has now focused the search for the BOR gene to within the limits of this deletion. Genomics, 1996 Jun 15, 34(3), 381 - 8 Genomic structure and precise mapping of a thymic regulatory region on mouse chromosome 17 revealed by a c-myc transgene insertion; Lavenu A et al.; In one transgenic strain harboring a human c-myc proto-oncogene construct, the transgene was actively and exclusively expressed in the thymus, where it contributed to the development of lymphoma that corresponded to CD4(+)CD8(+) cells . Here, we have pursued the analysis of transgene expression in healthy transgenic mice and show that transgene activation occurs in the thymus 3 days before birth, at a time when CD4(+)CD8(+) lymphocytes emerge . In the adult, its expression is restricted to the CD4(+)CD8(+) cells . The region flanking the transgene insertion site was isolated and made it possible to map the preintegration locus, hereafter called Tsil (for thymus-specific integration locus) on chromosome 17 between D17Rp11e and Ras12-3 . A YAC that contains both Tsil and the Pim2 locus, previously shown to be involved in progression of T-cell lymphoma, was isolated . Analysis of Tsil offers a unique opportunity to identify a regulatory region or a gene that might play an important role in T-cell maturation. Biochem J, 1996 Jun 15, 316 ( Pt 3), 723 - 7 Isolation and characterization of a 30 kDa protein with antifungal activity from leaves of Engelmannia pinnatifida; Huynh QK et al.; During the course of screening plants for novel antifungal activity, we found that a high-molecular-mass fraction of an extract from leaves of Engelmannia pinnatifida exhibited potent and broad-spectrum antifungal activity . In this study a 30 kDa protein from E . pinnatifida leaves was purified to homogeneity by ammonium sulphate precipitation, gel filtration, Mono-Q and C18 reverse-phase column chromatographies . The purified protein showed potent antifungal activity against various plant pathogens with as little as 50 ng . The N-terminal amino acid sequence of the purified protein was determined as XXTKFDFFTLALQXPAXF, where X indicates an unidentified residue . This sequence showed 35-50% sequence identity with purified style glycoproteins associated with self-incompatibility from wild tomato, tobacco and petunia, a phosphate-starvation-induced ribonuclease from cultured tomato cells and the SIR 63.4 kDa protein from yeast. Genes Dev, 1996 Jun 15, 10(12), 1503 - 15 Xe-p9, a Xenopus Suc1/Cks homolog, has multiple essential roles in cell cycle control; Patra D et al.; The small Suc1/Cks protein is a ubiquitous subunit of Cdk/cyclin complexes, but its precise function has remained unclear . We have isolated a Xenopus homolog, Xe-p9, of the Suc1/Cks protein by virtue of its ability to rescue a fission yeast mutant that enters mitosis prematurely . To assess its functional role in cell cycle control, we have both overexpressed p9 in Xenopus egg extracts and immunodepleted the protein from these extracts . We found that addition of recombinant His6-p9 to egg extracts results in a pronounced delay of mitosis that can be attributed to an inhibition of the tyrosine dephosphorylation of the inactive Cdc2/cyclin B complex . In immunodepletion studies, we observed that the consequences of removing p9 from egg extracts depend on the stage of the cell cycle . Specifically, in the case of interphase extracts, the removal of p9 abolishes the entry into mitosis as a result of a failure in the activation of the Cdc2/cyclin B complex by tyrosine dephosphorylation . Furthermore, mitotic extracts lacking p9 fail to exit mitosis because of a defect in the destruction of cyclin B . Collectively, these results indicate that p9 has multiple essential roles in the cell cycle by governing the interaction of the Cdc2/cyclin B complex with both positive and negative regulators. Cell, 1996 Jun 14, 85(6), 829 - 39 cul-1 is required for cell cycle exit in C . elegans and identifies a novel gene family; Kipreos ET et al.; The gene cul-1 (formerly lin-19) is a negative regulator of the cell cycle in C . elegans . Null mutations cause hyperplasia of all tissues . cul-1 is required for developmentally programmed transitions from the G1 phase of the cell cycle to the GO phase or the apoptotic pathway . Moreover, the mutant phenotype suggests that G1-to-S phase progression is accelerated, overriding mechanisms for mitotic arrest and producing abnormally small cells . Significantly, diverse aspects of cell fate and differentiation are unaffected in cul-1 mutants . cul-1 represents a conserved family of genes, designated cullins, with at least five members in nematodes, six in humans, and three in budding yeast. J Mol Biol, 1996 Jun 14, 259(3), 337 - 48 hnRNP A1 selectively interacts through its Gly-rich domain with different RNA-binding proteins; Cartegni L et al.; Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant nuclear polypeptides, most likely involved in different steps of pre-mRNA processing . Protein A1 (34 kDa), a prominent member of the hnRNP family, seems to act by modulating the RNA secondary structure and by antagonizing some splicing factors (SR proteins) in splice-site selection and exon skipping/inclusion . A role of A1 in the nucleo-cytoplasmic transport of RNA has also been proposed . These activities might depend not only on the RNA-binding properties of the protein but also on specific protein-protein interactions . Here we report that A1 can indeed selectively interact, in vitro, both with itself and with other hnRNP basic "core" proteins . Such selective binding is mediated exclusively by the Gly-rich C-terminal domain, where a novel protein-binding motif constituted by hydrophobic repeats can be envisaged . The same domain is necessary and sufficient to promote specific interaction in vivo, as assayed by the yeast two-hybrid assay . Moreover, an in vitro interaction with some SR proteins was also observed . These observations suggest that diverse and specific protein-protein interactions might contribute to the different functions of the hnRNP A1 protein in mRNA maturation. J Mol Biol, 1996 Jun 14, 259(3), 317 - 24 Topoisomerase II-mediated DNA cleavage: evidence for distinct regions of enzyme-DNA contacts; Alsner J et al.; To determine the specific interaction sites of topoisomerase II within the DNA region defined by the footprint of the enzyme, we have investigated the cleavage reaction on double-stranded DNA substrates containing nicks and deletions . Topoisomerase II-mediated cleavage of the DNA substrates is suicidal as the enzyme is unable to religate the cleaved DNA due to diffusion of the small nucleotides 5' to the cleavage position . Thus, suicidal cleavage is obtained with substrates having one, two or three nucleotides 5' to the cleavage position . The enzyme requires interaction with three distinct regions of double-stranded DNA for cleavage to occur, one region spanning the eight nucleotides located around the cleavage position and two distal regions each spanning approximately six nucleotides . A model is proposed, where these data are taken to imply that two distinct regions of interactions exist between each topoisomerase II subunit and its DNA substrate . The model is discussed in relation to the recently solved three-dimensional structure of yeast topoisomerase II. Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 6203 - 8 Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers; Nobori T et al.; 5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadeno-sine: ortho-phosphate methylthioribosyltransferase, EC 24.2.28; MTAP) plays a role in purine and polyamine metabolism and in the regulation of transmethylation reactions . MTAP is abundant in normal cells but is deficient in many cancers . Recently, the genes for the cyclin-dependent kinase inhibitors p16 and p15 have been localized to the short arm of human chromosome 9 at band p21, where MTAP and interferon alpha genes (IFNA) also map . Homozygous deletions of p16 and p15 are frequent malignant cell lines . However, the order of the MTAP, p16, p15, and IFNA genes on chromosome 9p is uncertain, and the molecular basis for MTAP deficiency in cancer is unknown . We have cloned the MTAP gene, and have constructed a topologic map of the 9p21 region using yeast artificial chromosome clones, pulse-field gel electrophoresis, and sequence-tagged-site PCR . The MTAP gene consists of eight exons and seven introns . Of 23 malignant cell lines deficient in MTAP protein, all but one had complete or partial deletions . Partial or total deletions of the MTAP gene were found in primary T-cell acute lymphoblastic leukemias (T-ALL) . A deletion breakpoint of partial deletions found in cell lines and primary T-ALL was in intron 4 . Starting from the centromeric end, the gene order on chromosome 9p2l is p15, p16, MTAP, IFNA, and interferon beta gene (IFNB) . These results indicate that MTAP deficiency in cancer is primarily due to codeletion of the MTAP and p16 genes. Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 6048 - 52 Mutations in the rotated abdomen locus affect muscle development and reveal an intrinsic asymmetry in Drosophila; Martin-Blanco E et al.; In bilateral animals, the left and right sides of the body usually present asymmetric structures, the genetic bases of whose generation are still largely unknown {CIBA Foundation (1991) Biological Asymmetry and Handedness, CIBA Foundation Symposium 162 (Wiley, New York), pp . 1-327} . In Drosophila melanogaster, mutations in the rotated abdomen (rt) locus cause a clockwise helical rotation of the body . Even null alleles are viable but exhibit defects in embryonic muscle development, rotation of the whole larval body, and helical staggering of cuticular patterns in abdominal segments of the adult . rotated abdomen is expressed in the embryonic mesoderm and midgut but not in the ectoderm; it encodes a putative integral membrane glycoprotein (homologous to key yeast mannosyltransferases) . Mesodermal cells defective in O-glycosylation lead to an impaired larval muscular system . We propose that the staggering of the adult abdominal segments would be a consequence of the relaxation of intrinsic rotational torque of muscle architecture, preventing the colateral alignment of the segmental histoblast cells during their proliferation at metamorphosis. Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 6043 - 7 A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain; Lee J et al.; Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells . We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification . Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A . The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein . The MEase is highly specific for PP2A . It does not catalyze the demethylation of other protein or peptide methylesters . Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A . From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase . Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation. Hum Gene Ther, 1996 Jun 10, 7(9), 1103 - 9 Gene targeting to the centromeric DNA of a human minichromosome; Raimondi E et al.; A human supernumerary minichromosome (MC), previously identified as a derivative of chromosome 9, has been introduced into Chinese hamster ovary (CHO) cells by means of cell fusion . A hybrid clone containing the MC as the only free human chromosome was isolated . A selectable marker gene (neo) inserted into a yeast artificial chromosome (YAC) has been successfully targeted to the MC centromeric DNA via co-transfection with chromosome-9-specific alpha satellite DNA . In situ hybridization and Southern blotting experiments demonstrated that the intact neo gene was integrated into the MC centromeric DNA . Studies on the clonal distribution and on the stability of the MC either in the presence or in the absence of the selective agent have been carried out . The MC is susceptible to further manipulations and may thus represent a model for the construction of a large-capacity vector for somatic gene therapy. J Biol Chem, 1996 Jun 7, 271(23), 13300 - 3 Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues; Ravichandran V et al.; The specificity of vesicular transport is regulated, in part, by the interaction of a vesicle-associated membrane protein termed synaptobrevin/VAMP with a target compartment membrane protein termed syntaxin . These proteins, together with SNAP-25 (synaptosome-associated protein of 25 kDa), form a complex which serves as a binding site for the general membrane fusion machinery . Synaptobrevin/VAMP and syntaxin are ubiquitously expressed proteins and are believed to be involved in vesicular transport in most (if not all) cells . However, SNAP-25 is present almost exclusively in the brain, suggesting that a ubiquitously expressed homolog of SNAP-25 exists to facilitate transport vesicle/target membrane fusion in other tissues . Using the yeast two-hybrid system, we have identified a 23-kDa protein from human B lymphocytes (termed SNAP-23) that binds tightly to multiple syntaxins and synaptobrevins/VAMPs in vitro . SNAP-23 is 59% identical with SNAP-25 . Unlike SNAP-25, SNAP-23 was expressed in all tissues examined . These findings suggest that SNAP-23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion in all mammalian cells. J Biol Chem, 1996 Jun 7, 271(23), 13304 - 7 Mouse p170 is a novel phosphatidylinositol 3-kinase containing a C2 domain; Virbasius JV et al.; Phosphatidylinositol (PI) 3-kinases catalyze the formation of 3'-phosphoinositides, which appear to promote cellular responses to growth factors and such membrane trafficking events as insulin-stimulated translocation of intracellular glucose transporters . We report here the cloning of a novel PI 3-kinase, p170, from cDNA of insulin-sensitive mouse 3T3-L1 adipocytes . Mouse p170 utilizes PI and to a limited extent PI 4-P as substrates, in contrast to the PI-specific yeast VPS34 homolog PtdIns 3-kinase and the p110 PI 3-kinases, which phosphorylate PI, PI 4-P, and PI 4,5-P2 . Mouse p170 is also distinct from PtdIns 3-kinase or the p110 PI 3-kinases in exhibiting a 10-fold lower sensitivity to wortmannin . Unique structural elements of p170 include C-terminal sequences strikingly similar to the phosphoinositide-binding C2 domain of protein kinase C isoforms, synaptotagmins, and other proteins . These features of mouse p170 are shared with a recently cloned Drosophila PI 3-kinase, DmPI3K_68D . Together, these proteins define a new class of PI 3-kinase likely influenced by cellular regulators distinct from those acting upon p110- or VPS34-like PI 3-kinases. J Biol Chem, 1996 Jun 7, 271(23), 13448 - 53 Differential responsiveness of a splice variant of the human type I interferon receptor to interferons; Cook JR et al.; Chinese hamster ovary cells containing the yeast artificial chromosome F136C5 (alphaYAC) respond to all type I human interferons including IFN-alphaA, IFN-beta, and IFN-omega . The alphaYAC contains at least two genes encoding interferon-alpha receptor (IFN-alphaR) chains that are required for response to type I human interferons: Hu-IFN-alphaR1 and Hu-IFN-alphaR2 . We previously isolated a splice variant of the Hu-IFN-alphaR1 chain designated Hu-IFN-alphaR1s . Chinese hamster ovary cells containing a disrupted alphaYAC, which contains a deletion in the human IFNAR1 gene, were transfected with expression vectors for the Hu-IFN-alphaR1 and Hu-IFN-alphaR1s chains . With these cells, two type I interferons have been identified which can interact with the splice variant (Hu-IFN-alphaR1s) and with the Hu-IFN-alphaR1 chains: Hu-IFN-alphaA and IFN-omega . Two other type I interferons, Hu-IFN-alphaB2 and Hu-IFN-alphaF, are capable of signaling through the Hu-IFN-alphaR1 chain only and cannot utilize the splice variant Hu-IFN-alphaR1s . Hu-IFN-alphaR1 and Hu-IFN-alphaR1s differ in that the latter is missing a single subdomain of the receptor extracellular domain encoded by exons 4 and 5 of the IFNAR1 gene . These results therefore indicate that different type I interferons require different subdomains of the Hu-IFN-alphaR1 receptor chain, and that the splice variant chain (Hu-IFN-alphaR1s) is functional. Biochim Biophys Acta, 1996 Jun 3, 1307(1), 31 - 4 Cloning of a cDNA encoding a human homologue of CDC47, a member of the MCM family; Kiyono T et al.; A complementary DNA (cDNA) clone encoding a 719-amino acid (aa) protein was isolated, which has a 49% aa identity with budding yeast CDC47, a member of the MCM family . Antiserum raised against a C-terminal polypeptide bacterially produced from the cDNA clone detected a cellular protein about 80 kDa, which coincides with the size of the in vitro transcription/translation product of the cDNA . These results indicate that the cDNA covers the entire coding region of a human homologue of CDC47. J Ind Microbiol, 1996 Jun, 16(6), 348 - 50 Effect of inoculum level of xylitol production from rice straw hemicellulose hydrolysate by Candida guilliermondii; Roberto IC et al.; The effect of inoculum level on xylitol production by Candida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate . High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L-1 to 2.41 g L-1 decreased both the rate of xylose utilization and xylitol accumulation . The maximum xylitol yield (0.71 g g-1) and volumetric productivity (0.56 g L-1 h-1) were reached with an inoculum level of 0.9 g L-1 . These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeast C . guilliermondii with efficiency values as high as 77% of the theoretical maximum. Pharmacol Res, 1996 Jun, 33(6), 367 - 73 Oxaceprol, an atypical inhibitor of inflammation and joint damage; Ionac M et al.; Oxaceprol, an established therapeutic agent for osteoarthritis, had no effect on macrophage prostaglandin E2 release in vitro and inhibited carrageenan paw oedema at high doses (18-150 mg/kg p.o.) . In the same dose range, oxaceprol was comparable to indomethacin (3 mg/kg p.o.) as an inhibitor of yeast hyperalgaesia and at 6-50 mg/kg/day p.o . had a mild, variable inhibitory effect on cotton pellet granuloma formation . In adjuvant arthritic rats, oxaceprol (6-54 mg/kg/day p.o.) given therapeutically had no effect on the primary paw oedema response, but inhibited secondary lesions in the ears and tail . Histologically, oxaceprol markedly inhibited inflammatory cell infiltration and bone damage in the adjuvant-injected paw . In contrast to indomethacin, oxaceprol was more effective at inhibiting periarticular soft tissue inflammation but did not affect cartilage breakdown in this model . Oxaceprol has a clearly different spectrum of action to NSAIDs such as indomethacin and may act by inhibiting leucocyte infiltration and late connective tissue changes during inflammatory joint disease. Comp Biochem Physiol A Physiol, 1996 Jun, 114(2), 175 - 87 Mechanical responses of chromium-deficient developing rat heart; Penefsky ZJ et al.; Mechanical responses were compared between controls, developing Sprague-Dawley rat papillary muscle and age-matched weanlings fed with Torula yeast, a food source deficient in chromium . At 8 weeks postnatal, deficient rats differed in significant ways from their normal counterparts . Deficient rats in contrast to controls weighed less, their interval-force (I-F) relationship was more negative and their inotropic response to high calcium concentrations was greater . At this time, however, deficient and control rats responded equally to alpha (phenylephrine) and beta (isoproterenol) agonists . At 10 weeks of age, the controls exhibited a less negative I-F and a negative inotropic response to high calcium concentrations while the response to alpha and beta agonists was unchanged . In contrast, at 10 weeks of age, the chromium-deficient rats exhibited a highly negative I-F response and significant inotropic response to high calcium concentrations . The response of the deficient hearts to beta-agonists diminished . At 13 weeks postnatal, control hearts showed only a 10-15% negative I-F response, a persistent response to catecholamines and negative inotropic responses to high calcium concentrations . In deficient hearts, the negative I-F response was reduced and the response to beta-agonists was further diminished but a positive inotropic response to phenylephrine and high calcium concentrations persisted . These observations in deficient animals are explained in terms of a retarded development of the calcium handling elements in the heart and a lack of an insulin-like growth factor. Protein Eng, 1996 Jun, 9(6), 499 - 505 Effect of replacing helical glycine residues with alanines on reversible and irreversible stability and production of Aspergillus awamori glucoamylase; Chen HM et al.; To decrease irreversible thermoinactivation of Aspergillus awamori glucoamylase, five Gly residues causing helix flexibility were replaced with Ala residues . Mutation of Gly57 did not affect thermostability . Mutation of Gly137 doubled it at pHs 3.5 and 4.5 but barely changed it at pH 5.5 . The Gly139-->Ala mutation did not change thermostability at pH 3.5, improved it at pH 4.5 and worsened it at pH 5.5 . The Gly 137/Gly139-->Ala/Ala mutation gave 1.5-2-fold increased thermostabilities at pHs 3.5-5.5 . Mutations of Gly251 and Gly383 decreased it at all pHs . Gly137-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases are the most stable yet produced by mutation . Guanidine treatment at pH 4.5 decreased the reversible stabilities of Gly137-->Ala, Gly139-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases at infinite dilution while not changing those of Gly251-->Ala and Gly383-->Ala glucoamylases, which is, in general, opposite to what occurred with thermoinactivation . Mutation of Gly57 greatly improved the extracellular glucoamylase production by yeast, that of Gly137 barely affected it and those of Gly139 and of both Gly137 and Gly139 strongly impeded it . These observations suggest that alpha-helix rigidity can affect reversible and irreversible glucoamylase stability differently, that the effects of multiple mutations within one alpha-helix to improve stability are not always additive and that even single mutations can strongly affect extracellular enzyme production. Immunopharmacology, 1996 Jun, 33(1-3), 217 - 21 Removal and restoration of epithelial chloride secretory activity of kinins by gene manipulation; Cuthbert A et al.; Kinins are known to stimulate electrogenic chloride secretion in many mammalian epithelia, including those of the airways and the alimentary tract . In this study the chloride secretory activity of lysylbradykinin (LBK) on murine colonic epithelium has been examined, specifically to discover the primary and final effector mechanisms in this process, i.e., which kinin receptors are involved and which chloride channels are responsible for chloride secretion . The approach used was to modify the mice genetically and assess the effects on kinin mediated chloride secretion using voltage clamping at zero potential . Briefly, LBK increased SCC in mouse colon by approximately 150 microA cm-2 with an EC50 of approximately 5 nM . In null CF mice LBK, 1 microM had no effect on chloride secretion, but reduced SCC due to K+ secretion . This effect is normally masked in wild-type tissues by dominant chloride secretion, but can be shown to occur to the same extent by measuring K+ secretion with radioisotopes . Null CF mice produce no cftr, but CFTR was introduced into CF mice by injecting a YAC containing the human CF gene into the pronucleus of CF zygotes . Colonic epithelia from mice with the incorporated YAC showed the same sensitivity to LBK as wild-type tissues and achieved the same maximal chloride secretory response . Colonic epithelia from mice in which the B2r gene had been disrupted showed no response to LBK at normally supramaximally effective concentrations, although responses to other secretagogues were normal . Similarly des-Arg-BK caused no acute chloride secretory response in colonic epithelia from B2 knockout mice, however small responses appeared if tissues were incubated in vitro for 3-6 h . It is concluded that cftr chloride channels and B2rs are required for electrogenic chloride secretion . Further CFTR can replace cftr with no effect on either the sensitivity or extent of chloride secretion . In vitro, colonic epithelia may generate B1rs which, upon activation, have a minor effect on chloride secretory activity. Genome Res, 1996 Jun, 6(6), 504 - 14 Construction of a YAC contig encompassing the Usher syndrome type 1C and familial hyperinsulinism loci on chromosome 11p14-15.1; Ayyagari R et al.; The Usher syndrome type 1C (USH1C) and familial hyperinsulinism (HI) loci have been assigned to chromosome 11p14-15.1, within the interval D11S419-D11S1310 . We have constructed a yeast artificial chromosome (YAC) contig, extending from D11S926 to D11S899, which encompasses the critical regions for both USH1C and HI and spans an estimated genetic distance of approximately 4 cM . A minimal set of six YAC clones constitute the contig, with another 22 YACs confirming the order of sequence-tagged sites (STSs) and position of YACs on the contig . A total of 40 STSs, including 10 new STSs generated from YAC insert-end sequences and inter-Alu PCR products, were used to order the clones within the contig . This physical map provides a resource for identification of gene transcripts associated with USH1C, HI, and other genetic disorders that map to the D11S926-D11S899 interval. Genome Res, 1996 Jun, 6(6), 478 - 91 Transcription mapping in a 700-kb region around the DXS52 locus in Xq28: isolation of six novel transcripts and a novel ATPase isoform (hPMCA5); Heiss NS et al.; The chromosomal band Xq28 has been a focus of interest in human genetics because > 20 hereditary diseases have been mapped to this region . However, about two-thirds of the disease genes remain uncloned . The region around the polymorphic DXS52 locus (ST14) within Xq28 lies in the candidate regions for several as-yet-uncloned disease genes . So far, only four melanoma antigen genes (MAGE) and the human biglycan (BGN) gene, have been mapped within the 700-kb stretch around DXS52, suggesting that more genes may reside in this region . By combining exon trapping and direct cDNA selection methods, we sought to identify novel transcripts around the DXS52 locus . In addition to recovering the MAGE and BGN genes, we isolated and mapped six putative novel genes (XAP103-XAP108), the caltractin gene, and a gene encoding a novel Ca(2+)-transporting ATPase isoform (hPMCA5) . The newly isolated sequences were considered as representing parts of putative genes if they contained at least one unique exon-trap product and/or at least one expressed sequence tag (EST) from sequence data bases and if, in addition, they showed evidence of expressed RT-OCT and/or Northern blot analysis . Our data facilitated the integration of the transcription map with the physical map around the DXS52 locus . Future analysis of the novel genes as candidates for Barth syndrome (BTHS) and chondrodysplasia punctata (CDPX2) is in progress. Genes Chromosomes Cancer, 1996 Jun, 16(2), 94 - 105 Narrowing the critical region for a rhabdoid tumor locus in 22q11; Biegel JA et al.; Rhabdoid tumor is a rare malignant neoplasm of childhood that may occur in various locations, including the central nervous system and the kidney . Previous cytogenetic studies of primary rhabdoid tumors have demonstrated monosomy or deletion of chromosome 22 and have implicated the presence of a rhabdoid tumor suppressor gene that maps to 22q . We have employed fluorescence in situ hybridization to narrow the region for this locus in four rhabdoid tumor cell lines with translocations or deletions involving chromosome segment 22q11 . The completion of a cosmid and yeast artificial chromosome contig spanning the immunoglobulin lambda gene locus to BCR has allowed us to map a critical region for a rhabdoid tumor gene to a 500 kb span of chromosome segment 22q11. Chem Biol, 1996 Jun, 3(6), 491 - 7 Cytochrome c folding triggered by electron transfer; Mines GA et al.; BACKGROUND: Experimental and theoretical studies of protein folding suggest that the free-energy change associated with the folding process is a primary factor in determining folding rates . We have recently developed a photochemical electron-transfer-triggering method to study protein-folding kinetics over a wide range of folding free energies . Here, we have used this technique to investigate the relationship between folding rate and free-energy change using cytochromes c from horse (h-cyt c) and yeast (y-cyt c), which have similar backbone folds but different amino-acid sequences and, consequently, distinct folding energies . RESULTS: The folding free energies for oxidized and reduced h-cyt c and y-cyt c are linear functions of the denaturant (guanidine hydrochloride) concentration, but the concentration required to unfold half of the protein is 1.5 M lower for y-cyt c . We measured the folding rates of reduced h-cyt c and y-cyt c over a range of guanidine hydrochloride concentrations at two temperatures . When driving forces are matched at the appropriate denaturant concentrations, the two homologs have comparable folding rates . The activation free energies for folding h-cyt c and y-cyt c are linearly dependent on the folding free energies . The slopes of these lines are similar (approximately 0.4) for the two proteins, suggesting an early transition state along the folding reaction coordinate . CONCLUSIONS: The free-energy relationships found for h-cyt c and y-cyt c folding kinetics imply that the height of the barrier to folding depends upon the relative stabilities of the unfolded and folded states . The striking correspondence in rate/free-energy profiles for h-cyt c and y-cyt c suggests that, despite low sequence homology, they follow similar folding pathways. Curr Opin Struct Biol, 1996 Jun, 6(3), 317 - 21 Hammering away at RNA global structure; Hagerman PJ et al.; A major goal of the study of RNA tertiary structure is an understanding of the rules relating sequence and global conformation . This goal has been furthered during the past year for two important structural elements: yeast tRNAPhe and the self-cleaving hammerhead RNA . In both cases, a combination of solution and crystallographic studies has yielded strongly concordant views of their global conformations. J Med Vet Mycol, 1996 Jun-Jul, 34(3), 175 - 80 Differential expression of the 45 kDa protein (actin) during the dimorphic transition of Sporothrix schenckii; Han-Yaku H et al.; We investigated the gene expression of a protein during the dimorphic transition from yeast to mycelial form of Sporothrix schenckii . Yeast cells were converted to mycelial cells in Sabouraud glucose broth at 25 degrees C . After 1, 3 and 5 days of culture, the intermediate form of cells between yeast and mycelium was obtained, and after 7 days these cells were morphologically similar to the mycelial cells . Proteins having the molecular weight of 45 kDa were found to by synthesized preferentially by intermediate form of day 7 and mycelial cells . On the other hand, the 45 kDa proteins were predominantly translated by the RNA isolated from the intermediate of day 7 and mycelial cells using in vitro cell-free translation assay . The 45 kDa proteins synthesized by mycelial cells were found to be identical with the 45 kDa translation products directed by the mRNA isolated from the intermediate and mycelial cells by V8 protease peptide mapping . The 45 kDa protein was considered to be actin by Western blot analysis using an anti-actin monoclonal antibody . These results suggest that the intermediate form of day 7 has the same phenotypes in the morphology and biosynthesis of actin as those of mycelial cells . The expression of the actin gene may be involved in the dimorphic transition of S . schenckii. Trends Microbiol, 1996 Jun, 4(6), 246 - 51 Role of cell-surface molecules of Blastomyces dermatitidis in host-pathogen interactions; Klein BS et al.; The fungal pathogen Blastomyces dermatitidis produces an adhesin (WI-1) in yeast stages, which contains repetitive regions that bind host-cell receptors . Adhesin and glucan may modulate fungal interactions with macrophages; their level of expression is altered in hypovirulent mutants . Adhesin is also involved in immune responses, and may be important in eliciting the clearance of the fungus. Curr Opin Immunol, 1996 Jun, 8(3), 412 - 8 Phosphatidylinositol 3-kinase related kinases; Abraham RT; Studies in yeast, files and mammalian cells have uncovered a novel family of signal-transducing kinases which bear an evolutionary relationship to phosphatidylinositol 3-kinase . These phosphatidylinositol 3-kinase related enzymes play critical roles in DNA repair, V(D)J recombination and cell-cycle checkpoints, and their dysfunction leads to clinical manifestations ranging from immunodeficiency to cancer. Curr Biol, 1996 Jun 1, 6(6), 651 - 4 Cell division: why daughters cannot be like their mothers; Chang F et al.; A cell-fate determinant that segregates asymmetrically at cell division has been identified in budding yeast . Possible mechanisms for this asymmetric segregation are suggested by the identification of mutants in genes encoding cortically localized proteins. Mol Endocrinol, 1996 Jun, 10(6), 631 - 41 Evidence for the direct interaction of the insulin-like growth factor I receptor with IRS-1, Shc, and Grb10; Dey BR et al.; We have used the yeast two-hybrid system to study the interaction between the IGF-I receptor and two putative substrates, IRS-1 and Shc . In addition, we have identified Grb10 as a protein that binds to the insulin-like growth factor I (IGF-I) receptor . This two-hybrid system (the interaction trap) utilizes a hybrid protein containing the LexA DNA-binding domain fused to the intracellular portion of the IGF-I receptor (LexA-IGFIR beta) and hybrids containing an activation domain fused to either IRS-1 (Ad-IRS-1), Shc (Ad-Shc), or a cDNA library . A positive interaction of LexA-IGFIR beta with the activation domain hybrid results in activation of reporter genes, LacZ and LEU2, in the yeast . Western blotting of extracts from transformed yeast demonstrated that the LexA-IGFIR beta fusion protein was expressed and phosphorylated on tyrosine residues . Coexpression of LexA-IGFIR beta with Ad-IRS-1 resulted in strong activation of both reporter genes; activation did not occur with a kinase-negative receptor mutant . IRS-1 residues 160-516 were sufficient for this strong interaction . Coexpression of LexA-IGFIR beta with Ad-Shc also resulted in strong activation of both LacZ and LEU2 reporter genes . This interaction was also dependent upon a tyrosine kinase-active receptor and required tyrosine 950 in the juxtamembrane region of the receptor . An N-terminal fragment of Shc (amino acids 1-232) interacted almost as strongly as full-length Shc whereas the Shc SH2 domain only activated the more sensitive LEU2 reporter . Full-length Shc was phosphorylated on tyrosine when coexpressed with IGFIR beta but not when coexpressed with the kinase-negative receptor mutant . To identify additional proteins that interact with the IGFIRs, a human fetal brain cDNA library was screened using the interaction trap system . This analysis identified partial cDNAs for Grb10 . Coexpression of LexA-IGFIR beta with Ad-Grb10 resulted in strong activation of both LacZ and LEU2 reporter genes; this interaction was dependent upon a tyrosine kinase-active receptor but did not require tyrosine 950. Hum Mol Genet, 1996 Jun, 5(6), 827 - 33 Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP); Roepman R et al.; The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene . In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification . In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls . A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries . Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region . So far, no point mutations in these putative exon sequences have been identified. Recenti Prog Med, 1996 Jun, 87(6), 271 - 4 Vaccination against hepatitis B virus in children and adolescents in a pediatric hospital; Catania G et al.; Vaccination against hepatitis B has become compulsory in Italy and is routinely administered in outpatient clinics of Local Health Service . In the setting of a pediatric hospital 205 children (less than 10-year-old) and 144 adolescents (10-19-year-old) have been vaccinated with anti-HBV recombinant yeast-derived vaccine (Engerix B 10 U.I . at 0.1 and 6 months in children and 20 U.I . in adolescents) . Anti-HBs titers were evaluated 1 month after least dose . Geometric mean titer (GMT) was 9500 mU/I in children and 18,000 in adolescents; 99.85% of subjects had a protective anti-HBs titer (10 mU/I or more) at one month after the third dose and 89.1% had titers of 1000 mU/I or more . Only 9.7% of subjects had adverse events, mainly (75%) at the injection site, anyway of trivial importance, without any difference of sex and age . Anti-hepatitis B yeast-derived vaccine is highly immunogenic, without relevant adverse events; the hospital setting is appropriate to participate to the program of vaccination in pediatric age. Am J Physiol, 1996 Jun, 270(6 Pt 1), C1647 - 55 Is gamma-actin a regulator of amino acid transport? Lin G, McCormick JI, Johnstone RM. A mutated yeast cell line incapable of growth in minimal medium with proline as the sole nitrogen source was restored to normal growth by transfection with a cDNA from mouse Ehrlich cells . The cloned cDNA (E51) was found to be 90% homologous to gamma-actin . Immediately after transfection with E51 cDNA, both alpha-aminoisobutyric acid (AIB) and proline uptake in the mutated yeast were increased, particularly at pH 5 . The expression of the same E51 cDNA also enhanced amino acid uptake in Xenopus laevis oocytes after injection into the Xenopus nuclei . A mutated mammalian lymphocyte cell line (GF-17), deficient in system A transport, also showed increased Na(+)-dependent transport after transfection with E51 cDNA . Whereas the mock transfected GF-17 cells failed to grow in the selection medium, the transfectants with E51 cDNA grew better than the untransfected cells . The data are consistent with the conclusion that expression of E51 cDNA can modify inactive, endogenous amino acid transporters, permitting substantial amino acid uptake in cells deficient in amino acid transporter(s) and permitting rapid cell growth . The data suggest that the gamma-actin-like protein coded for by E51 cDNA may play a significant regulatory role in amino acid transport. J Cell Biochem, 1996 Jun 1, 61(3), 459 - 66 Mammalian CAP interacts with CAP, CAP2, and actin; Hubberstey A et al.; We previously identified human CAP, a homolog of the yeast adenylyl cyclase-associated protein . Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions . We have explored the interactions of human CAP with various proteins . First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP . These peptides include regions derived from CAP and BAT3, a protein with unknown function . We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST . Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves . Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts . Similar results demonstrate that human CAP can also interact with human CAP2 . We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts . This interaction requires the C-terminal domain of CAP, but not the N-terminal domain . Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin . We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts. Curr Opin Cell Biol, 1996 Jun, 8(3), 331 - 9 Biochemistry and regulation of pre-mRNA splicing; Adams MD et al.; During the past year, significant advances have been made in the field of pre-mRNA splicing . It is now clear that members of the serine-arginine-rich protein family are key players in exon definition and function at multiple steps in the spliceosome cycle . Novel findings have been made concerning the role of exon sequences, which function as both constitutive and regulated enhancers of splicing, in trans-splicing and as targets for tissue-specific control of splicing patterns . By combining biochemical approaches in human and yeast extracts with genetic analysis, much has been learned about the RNA-RNA and RNA-protein interactions that are necessary to assemble the various complexes that are found along the pathway to the catalytically active spliceosome. Biochem J, 1996 Jun 1, 316 ( Pt 2), 681 - 4 An H(+)-ATPase regulates cytoplasmic pH in Pneumocystis carinii trophozoites; Docampo R et al.; Pneumocystis carinii is an opportunistic fungus which causes interstitial pneumonia in patients with acquired immunodeficiency syndrome (AIDS) . Cytoplasmic pH (pHi) regulation in short-term-cultured P . carinii trophozoites was studied using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein . With an extracellular pH of 7.4, the mean baseline pHi of P . carinii trophozoites was 7.40 +/- 0.10 (n = 8) . This steady-state pHi was not significantly affected in the absence of extracellular Na+ or K+ . Moreover, steady-state pHi was maintained in the nominal absence of HCO3- and was not affected by the Cl-/HCO(3-)-exchanger inhibitor 4, 4'-di-isothiocyanato-dihydrostilbene-2, 2'-disulphonic acid (100 microM), or the Na+/H(+)-exchanger inhibitor N-ethyl-N-isopropylamiloride (100 microM) . In contrast, the general inhibitors of ATPases, N-ethylmaleimide (1 mM), and dicyclohexylcarbodi-imide (100 microM), and the inhibitor of yeast H(+)-ATPase, diethylstilbestrol (12.5-100 microM), decreased pHi, while the K+/H(+)-ATPase inhibitor omeprazole (50-400 microM), and the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (1-5 microM) only produced a dose-dependent acidification of the cells when used at high concentrations . In addition, steady-state pHi depended on the availability of cellular ATP, since it was decreased by the ATP synthase inhibitors oligomycin (1 microgram/ml) and sodium azide (1 mM), and by the uncoupler of oxidative phosphorylation carbonyl cyanide p-trifluorophenylhydrazone (1 microM), agents that were able to deplete significantly the intracellular ATP levels . Taken together, these results are consistent with an important role of an H(+)-ATPase similar to those found in other fungi in the regulation of pHi homoeostasis in P . carinii trophozoites. J Cell Biol, 1996 Jun, 133(6), 1293 - 305 Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila; Hopmann R et al.; Regulation of actin filament length and orientation is important in many actin-based cellular processes . This regulation is postulated to occur through the action of actin-binding proteins . Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant . Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or