Biomedical subjects

L J Ross

Publications and source records attributed to L J Ross.

At least 19 recordsLinked to original sources

PubMed2000

The two toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers.

Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I.HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.

Animals↗

PubMed2000

Crystallization of the Mycobacterium tuberculosis cell-division protein FtsZ.

Mycobacterium tuberculosis FtsZ (MtbFtsZ), an essential protein in bacterial cell division, has been crystallized in the presence of a new inhibitor of MtbFtsZ polymerization and GTPase activity, ethyl (6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1, 4]diazepin-8-yl)carbamate (SRI-7614). Crystals of the MtbFtsZ-SRI-7614 complex (form I, 30% polyethylene glycol 4000, 0.1 M sodium citrate pH 5.6, 0.2 M NH(4)OAc, 293 K) belong to space group P6(1) or P6(5), with unit-cell parameters a = 88.78, c = 178. 02 A, and diffract to 2.3 A resolution. A second crystal form, of the GDP complex, grows in the presence or absence of Mg(2+) from PEG 4000 at 277 K or from (NH(4))(2)SO(4) at 293 K, respectively (form II, space group P6(2)22 or P6(4)22, with unit-cell parameters a = 135.02, c = 328.97 A or a = 129.30, c = 327.97 A, respectively). Complete data sets to approximately 7 A resolution have been collected from both. Exceptional form II crystals diffract to at least 4.5 A resolution. Determination of the MtbFtsZ structure may advance the design of improved inhibitors of FtsZ polymerization.

Azepines↗

PubMed2000

Slow polymerization of Mycobacterium tuberculosis FtsZ.

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90 degrees light scattering. The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg(2+) and GTP. The minimum concentration of FtsZ needed for polymerization is 3 microM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

Bacterial Proteins↗

PubMed1999

Crystal structures of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase-GMP and -IMP complexes: comparison of purine binding interactions with the XMP complex.

The crystal structures of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT) have been determined at 1.65 and 1.90 A resolution. These complexes, which crystallize in space groups P2(1) (a = 65.45 A, b = 90.84 A, c = 80. 26 A, and beta = 92.53 degrees ) and P2(1)2(1)2(1) (a = 84.54 A, b = 102.44 A, and c = 108.83 A), each comprise a tetramer in the crystallographic asymmetric unit. All active sites in the tetramers are fully occupied by the nucleotide. Comparison of these structures with that of the xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex reported in the following article [Héroux, A., et al. (1999) Biochemistry 38, 14495-14506] shows how T. gondii HGPRT is able to recognize guanine, hypoxanthine, and xanthine as substrates, and suggests why the human enzyme cannot use xanthine efficiently. Comparison with the apoenzyme reveals the structural changes that occur upon binding of purines and ribose 5'-phosphate to HGPRT. Two structural features important to the HGPRT mechanism, a previously unrecognized active site loop (loop III', residues 180-184) and an active site peptide bond (Leu78-Lys79) that adopts both the cis and the trans configurations, are presented.

Amino Acid Sequence↗

PubMed1999

Crystal structure of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase with XMP, pyrophosphate, and two Mg(2+) ions bound: insights into the catalytic mechanism.

The crystal structure of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex has been determined at 1. 60 A resolution. This biproduct, post-transition state structure is of a T. gondii HGPRT mutant (Asp150Ala or D150A). The D150A mutant has reduced activity (k(cat) lower by 11-, 296-, and 8.6-fold for hypoxanthine, guanine, and xanthine, respectively) compared to wild-type T. gondii HGPRT. The Michaelis constants for purine bases are altered only slightly, whereas those for alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP) are lower by approximately 6.5-fold. The ternary complex crystallizes in space group C222(1) (a = 55.21 A, b = 112.25 A, and c = 144.28 A) with two subunits in the asymmetric unit; the HGPRT tetramer is completed by the application of 2-fold crystallographic symmetry. All active sites contain XMP ¿bound in a fashion similar to that of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes reported in the preceding article [Héroux, A., et al. (1999) Biochemistry 38, 14485-14494]¿, pyrophosphate, and two Mg(2+) ions. Each Mg(2+) ion is octahedrally coordinated by two terminal pyrophosphate oxygen atoms and several ordered water molecules. This structure shows how HGPRT uses two Mg(2+) ions to orient and activate the pyrophosphate moiety of PRPP for attack by a purine base, and why mutation in humans of the residue corresponding to Asp206, the only HGPRT amino acid that directly contacts the Mg(2+) ions, causes Lesch-Nyhan syndrome (HGPRT(Kinston), D193N). The Leu78-Lys79 peptide bond in the active site adopts the cis configuration, which it must to bind PRPP or pyrophosphate. The contribution of cis-trans isomerization of this peptide bond to the energetics of substrate binding and product release is discussed. A comprehensive description of the HGPRT reaction mechanism is also proposed.

Animals↗

PubMed1999

Michellamine alkaloids inhibit protein kinase C.

Michellamines A, B, and C have shown antiviral activity against HIV-1 and HIV-2 in cell culture. They act in a complex manner by at least two reported antiviral mechanisms, inhibition of HIV reverse transcriptase and inhibition of HIV-induced cellular fusion. On the basis of their structural similarity to other protein kinase C (PKC) inhibitors, we have investigated another possible mechanism-inhibition of PKC. The michellamines were found to inhibit rat brain PKC with IC50 values in the 15-35 microM range. Michellamine B was a noncompetitive PKC inhibitor with respect to ATP with a Ki value of 4-6 microM, whereas mixed-type inhibition was observed when the peptide concentration was varied. Michellamine B inhibited the kinase domain of PKC similarly. These results indicate that the michellamines bind to the PKC kinase domain and not its regulatory domain. Molecular modeling showed that all three michellamines can bind in the active site cleft of the PKC kinase domain, to block both the ATP and the peptide substrate subsites.

Adenosine Triphosphate↗

PubMed1999

PMTI, a broadly active unusual single-stranded polyribonucleotide, inhibits human immunodeficiency virus replication by multiple mechanisms.

Poly(1-methyl-6-thioinosinic acid), or PMTI, is a single-stranded polyribonucleotide and is the first homopolyribonucleotide devoid of Watson-Crick hydrogen bonding sites to show potent human immunodeficiency virus (HIV) inhibition. PMTI was found to be active when evaluated against a variety of low passage clinical HIV isolates in fresh human peripheral blood cells, including T cell-tropic and monocyte-macrophage-tropic viruses, syncytium-inducing and non-syncytium-inducing viruses and viruses representative of the various HIV-1 clades (A through F). The compound was active against HIV-2, all nucleoside and non-nucleoside reverse transcriptase (RT) inhibitor drug-resistant virus isolates tested and interacted with AZT or ddl to synergistically inhibit HIV infection. In biochemical inhibition assays, PMTI was determined to be a potent inhibitor of HIV-1 and HIV-2 RT, including RTs with mutations that engender resistance to nucleoside and non-nucleoside RT inhibitors. PMTI inhibited both the polymerase and RNase H activities of HIV RT. PMTI did not inhibit HIV-1 protease or integrase. Cell-based mechanism of action assays indicated that PMTI also interfered with early events in the entry of HIV into target cells. Furthermore, PMTI inhibited the fusion of gp120-expressing and CD4-expressing cells, but at concentrations approximately 1 log10 greater than those that inhibited virus entry. These results suggest that the homopolyribonucleotide PMTI blocks HIV replication in human cells at its earliest stages by multiple mechanisms, inhibition of virus entry and inhibition of RT.

Anti-HIV Agents↗

PubMed1998

Differential susceptibility to Marek's disease is associated with differences in number, but not phenotype or location, of pp38+ lymphocytes.

Flow cytometric and immunocytochemical techniques were used to quantify, identify and locate Marek's disease herpesvirus (MDV)-infected lymphocytes in lymphoid organs of infected chickens, by expression of the virus antigen pp38. Two closely related lines of chicken, one susceptible to Marek's disease (line 7(2)) and another resistant (line 6(1)), were infected at 2 weeks of age and compared at 10 sampling times between 0 and 50 days post-infection. In both lines 6(1) and 7(2), pp38+ lymphocytes were detected at 4-6 days in the spleen, thymus and bursa. pp38+ cells could not be detected from day 8 onwards. In both lines, pp38+ lymphocytes were located in the peri-ellipsoidal area of the spleen, the medulla of the thymic lobes and the medulla of the bursal follicles. In both lines, pp38+ cells were predominantly B lymphocytes, but CD4+ and CD8+ TCR alphabeta+ T lymphocytes were also detected in the thymus and spleen. For each organ, the mean number of pp38+ lymphocytes was greater in line 7(2) than in line 6(1). pp38+ lymphocytes were not detected in the peripheral blood at any time. The data show that the differential susceptibility of lines 6(1) and 7(2) to the development of Marek's disease lymphoma is not attributable to differences in phenotype or location of pp38+ lymphocytes, or the time of expression of pp38. However, susceptibility is associated with greater numbers of pp38+ lymphocytes.

Animals↗

PubMed1996

Dermatoglyphics in offspring of women given gamma globulin prophylaxis during pregnancy.

The fingertip dermatoglyphic patterns were studied in 93 infants whose mothers had been given gamma globulin (GG) prophylaxis against hepatitis on known gestational dates. The prevalence of infants with whorl patterns on 0-3 fingertips and that of infants with ulnar loops on 9 or 10 fingertips were significantly higher (P < 0.02 and P = 0.01, respectively) in offspring of women given prophylaxis during the first 162 days of pregnancy than in those whose mothers received GG at a later stage of gestation. These findings were particularly prominent in infants who had been exposed to > or = 5 ml of GG in the first 162 days of gestation. In light of these observations, and of previously noted dermatoglyphic changes in offspring of women who had gestational prophylaxis against rubella, it appears that exogenous GG can influence the prenatal development of fingertip skin ridge patterns, and that this influence is responsive to the gestational timing and dosage of GG administered.

Case-Control Studies↗

PubMed1995

Gene organization in herpesvirus of turkeys: identification of a novel open reading frame in the long unique region and a truncated homologue of pp38 in the internal repeat.

The DNA sequence of a 4.792-kb fragment comprising 3.176 kb of the long unique region (UL) and 1.605 kb of the internal repeat (IRL) flanking UL of herpesvirus of turkeys (HVT) was determined. Three potential open reading frames (ORFs) and an origin of replication have been identified. ORF 1, which maps entirely within UL, has the capacity to code for an 82K protein, 731 amino acids long, which has a counterpart in Marek's disease virus (MDV) but not in other herpesviruses. ORF 2 has the potential to encode a protein consisting of 129 amino acids with a predicted M(r) of 13.5K which appears to be unique to HVT. ORF 3 is encoded entirely within IRL and codes for an 84 amino acids long protein with a predicted M(r) of 8.5K. ORF 3 shows significant homology with the C-terminal region of the MDV-1-specific phosphoprotein pp38 and its recently identified homologue in MDV-2. Northern blot analysis of RNA extracted from HVT-infected chick embryo fibroblasts identified a 5.6-kb RNA transcribed in a leftward direction toward UL scanning ORF 1, ORF 2, and ORF 3 and a 2.8-kb also transcribed leftward toward UL which spanned only ORFs 2 and 3. In addition, a 2.3- to 2.8-kb RNA family was transcribed rightwards through the origin of replication. In vitro transcription and translation of ORF 1 and ORF 3 resulted in the synthesis of polypeptides consistent with their expected M(r), but ORF 2 failed to produce any translation product.

Amino Acid Sequence↗

PubMed1994

Identification of novel transcripts complementary to the Marek's disease virus homologue of the ICP4 gene of herpes simplex virus.

Libraries of cDNA were generated from polyadenylated RNAs derived from Marek's disease virus (MDV)-transformed cell lines by directional cloning of oligo-(dT)-primed cDNAs in lambda gt22A. Analysis of the libraries for viral sequences showed that a number of cDNA clones originated from transcripts mapping in the BamHI A region of the MDV genome. Sequencing and fine mapping of these cDNAs suggested that the RNA transcripts expressed from this region were either in the sense or antisense direction with respect to the MDV homologue of the ICP4 gene of herpes simplex virus. The longest cDNA clone from antisense transcripts was 2756 bp long and partially overlapped the 5' end of the coding region of the ICP4 gene. The cDNA clone contained at least four introns, shown by comparison of its sequence with the sequence of the ICP4 gene. The presence of introns was confirmed by PCR analysis. All the introns have the consensus splice donor and acceptor signals at their 5' and 3' ends respectively. Northern blot analysis showed that the ICP4 gene homologue of MDV was abundantly transcribed only in lytically infected fibroblasts, whereas transcripts complementary to the ICP4 gene were the major transcripts in MDV-transformed cell lines and lymphomas. The transcripts complementary to ICP4 consist of two major RNA species approximately 15 kb and 1.32 kb long. The results suggest that there might be an inverse relationship between the abundance of ICP4 transcripts and their complementary transcripts in MDV-infected and transformed cells.

Amino Acid Sequence↗

PubMed1993

Thiazolobenzimidazole: biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor.

Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

Antiviral Agents↗

PubMed1993

Lack of synergy in the inhibition of HIV-1 reverse transcriptase by combinations of the 5'-triphosphates of various anti-HIV nucleoside analogs.

3'-Deoxy-3'-azidothymidine (AZT) has been shown to synergistically inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in cell culture when combined with several other 2',3'-dideoxynucleoside analogs. In an effort to understand the biochemical mechanism of this synergy, we have examined the effect of combinations of the 5'-triphosphate of AZT (AZT-TP) with either ddCTP, ddATP, or the 5'-triphosphate of the carbocyclic analog of 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir) on both the RNA-directed and DNA-directed DNA polymerase activity of HIV-1 reverse transcriptase. Kinetic studies, which evaluated the ability of these combinations to competitively inhibit the enzyme, showed that AZT-TP could not bind to the enzyme with either the RNA or DNA template at the same time as either of the other three inhibitors. None of these analogs could affect the incorporation of another analog into the DNA chain by the HIV-1 reverse transcriptase. These results indicated that synergistic inhibition of the HIV-1 reverse transcriptase is not responsible for the synergistic antiviral activity seen in cell culture with combinations of these nucleoside analogs.

Antiviral Agents↗

PubMed1993

The complete sequence and gene organization of the short unique region of herpesvirus of turkeys.

The DNA sequence of the whole of the short unique region (U(s)) and that of part of the short terminal repeat (TRs) of herpesvirus of turkeys (HVT) were determined. HVT U(s) is 8.6 kbp long and contains eight potential open reading frames (ORFs). Seven of these have counterparts in the U(s) of herpes simplex virus type 1 (HSV-1). The homologous proteins include US1, US2, US10, protein kinase (US3) and the glycoproteins gD, gI and gE. In addition, HVT contains one ORF which has a counterpart in the U(s) of Marek's disease virus (MDV) but is not homologous to any other known herpesvirus gene. Although HVT and MDV proteins encoded by U(s) genes have evident similarities with proteins encoded by alphaherpesviruses, multiple alignment analysis of predicted amino acid sequences show that HVT proteins are more closely related to MDV proteins than to homologous proteins of mammalian alphaherpesviruses. The percentage amino acid identity between HVT and MDV U(s)-encoded proteins ranges from 35 to 65, the most conserved protein being encoded by the homologues of the HSV-1 US2 gene. Most of the genes are colinear with those of HSV-1 except US10 which is transposed in HVT and MDV. A characteristic feature of HVT is the fact that approximately two-thirds of the gE gene is located in the inverted repeats flanking U(s).

Amino Acid Sequence↗

PubMed1993

Construction and properties of a turkey herpesvirus recombinant expressing the Marek's disease virus homologue of glycoprotein B of herpes simplex virus.

A herpesvirus of turkeys (HVT) recombinant containing a 3.9 kbp fragment of Marek's disease virus (MDV) DNA encoding MDV glycoprotein B (gB), stably integrated into the thymidine kinase (TK) gene of HVT, has been constructed. The replication of the recombinant in chick embryo fibroblasts (CEF) was comparable to that of wild-type HVT. The recombinant expressed authentic MDV gB and its processed forms (110K, 65K and 48K) in CEF as shown by immunoblotting using an MDV-specific anti-peptide serum. Northern blot analysis showed that MDV gB mRNA was transcribed from MDV promoter sequences flanking the MDV gB open reading frame and also from the HVT TK promoter. However, the level of replication of the recombinant in vivo appeared to be lower than wild-type HVT as shown by the titres of HVT antibodies, determined by ELISA. Pathogenicity tests showed that the recombinant was safe and did not cause microscopic or gross Marek's disease lesions or other abnormalities. The results suggest that HVT has potential as a vector for recombinant vaccines.

Animals↗

PubMed1992

Interference with HIV-1 reverse transcriptase-catalyzed DNA chain elongation by the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine.

In an effort to better understand features in nucleotide analogs that result in the inhibition of HIV-1 reverse transcriptase, we have evaluated this enzyme with the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine (CdG-TP). CdG-TP was a reasonably potent competitive inhibitor of the incorporation of dGTP into DNA by HIV-1 reverse transcriptase using either a RNA or DNA template (Ki, 1 microM). CdG-TP was a good substrate for HIV-1 reverse transcriptase on both templates, but the DNA chain was poorly extended beyond the incorporation of CdG. These results indicate that substitution of ribose with a cyclopentane ring in nucleotides is not well tolerated by HIV-1 reverse transcriptase.

Antiviral Agents↗

PubMed1992

African-American women and abortion: a neglected history.

The history of African-American women's efforts to control their fertility is largely unknown. From slavery to the present, the growth rate of the African-American population has been cut in half. Demographers and historians frequently attribute this change to external factors such as poverty, disease, and coerced birth control, rather than the deliberate agency of African-American women. This essay assembles a brief historical record of the ways African-American women have sought to control their fertility through the use of abortion and birth control. It also examines the activism of African-American women in the establishment of family planning clinics and in defense of abortion rights.

Abortion, Illegal↗

PubMed1991

Mechanism of inhibition of human immunodeficiency virus type 1 reverse transcriptase and human DNA polymerases alpha, beta, and gamma by the 5'-triphosphates of carbovir, 3'-azido-3'-deoxythymidine, 2',3'-dideoxyguanosine and 3'-deoxythymidine. A novel RNA template for the evaluation of antiretroviral drugs.

Carbovir (the carbocyclic analog of 2'-3'-didehydro-2',3'-dideoxyguanosine) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. Assays were developed to assess the mechanism of inhibition by the 5'-triphosphate of carbovir of HIV-1 reverse transcriptase using either RNA or DNA templates that contain all four natural nucleotides. Carbovir-TP was a potent inhibitor of HIV-1 reverse transcriptase using either template with Ki values similar to that observed by AZT-TP, ddGTP, and ddTTP. The kinetic constants for incorporation of these nucleotide analogs into DNA by HIV-1 reverse transcriptase using either template were similar to the values seen for their respective natural nucleotides. In addition, the incorporation of either carbovir-TP or AZT-TP in the presence of dGTP or dTTP, respectively, indicated that the mechanism of inhibition by these two nucleotide analogs was due to their incorporation into the DNA resulting in chain termination. Carbovir-TP was not a potent inhibitor of DNA polymerase alpha, beta, or gamma, or DNA primase. Given the potent activity of carbovir-TP against HIV-1 reverse transcriptase and its lack of activity against human DNA polymerases, we believe that further evaluation of this compound as a potential drug for the treatment of HIV-1 infection is warranted.

Antiviral Agents↗