Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

p73 is a recently cloned tumor suppressor gene that is highly homologous to p53, and the products of both possess similar functions in inhibiting cell growth and inducing apoptosis. Interestingly, the COOH-terminal region of p53 displays no significant homology with that of p73. Moreover, p73 has an additional segment at its COOH terminus. Recently, we have found two mutations of p73 with amino acid substitution (P405R and P425L) in primary neuroblastomas. Because the region (amino acid residues 382-491) contains a glutamine- and proline-rich domain, we hypothesized that it has a transactivation function, and the mutations found in tumors result in loss of function. To test it, we used the yeast GAL4 DNA-binding fusion system. Yeast transformants expressing a GAL4-p73(1-112) or a GAL4-p73alpha(380-513) fusion protein were grown in SD medium lacking histidine and tryptophan and exhibited a significant induction of beta-galactosidase activity. Transient transfection experiments revealed that both of fusion proteins could induce the chloramphenicol acetyltransferase activity in mammalian cells, indicating that the COOH-terminal as well as NH2-terminal regions of p73 had significantly high levels of transactivation activity. Furthermore, the former activity was severely impaired in two naturally occurring mutant forms found in neuroblastomas. These suggest that, unlike p53, p73 has two domains with transactivation function, one in the NH2-terminal region and the other in the COOH-terminal region. Loss of function mutation in the latter might be involved in tumorigenesis and/or tumor progression.
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PMID:Identification of a transactivation activity in the COOH-terminal region of p73 which is impaired in the naturally occurring mutants found in human neuroblastomas. 1038 37

To investigate the influence of proline residues on the activity of alpha-helical peptides, variants were synthesized with insertions of proline residues to create peptides without proline, or with one or two prolines. The influence of the proline-induced bends was assessed by circular dichroism in the presence of liposomes, and the ability of the peptides to kill microorganisms, to permeabilize the outer and cytoplasmic membranes of Escherichia coli, to bind to liposomes, to form channels in planar lipid bilayers, and to synergize with conventional antibiotics. Representative peptides adopted alpha-helical conformations in phosphatidylcholine/phosphatidylglycerol (POPC/POPG, 7:3) liposomes as well as in 60% trifluoroethanol solution, as revealed by circular dichroism (CD) spectroscopy. However, the percent of helicity decreased as the number of proline residues increased. Tryptophan fluorescence spectroscopy showed that all of these peptides inserted into the membranes of liposomes as indicated by a blue shift in the emission maximum and an increase in the fluorescence intensity of the single tryptophan at residue 2. Quenching experiments further prove that the tryptophan residue was no longer accessible to the aqueous quencher KI. The peptide that lacked proline exhibited the highest activity [minimal inhibitory concentrations (MICs) of 0.5-4 microg/mL] against all tested Gram-negative and Gram-positive bacteria, but was hemolytic at 8 microg/mL. The single-proline peptides exhibited intermediate antibacterial activity. Peptides with two proline residues were even less active with moderate MICs only against E. coli. With only one exception from each group, the peptides were nonhemolytic. The ability of the peptides to demonstrate synergy in combination with conventional antibiotics increased as the antibacterial effectiveness decreased. All peptides bound to bacterial lipopolysaccharide and permeabilized the outer membrane of E. coli to similar extents. However, their ability to permeabilize the cytoplasmic membrane of E. coli as assessed by the unmasking of cytoplasmic beta-galactosidase decreased substantially as the number of proline residues increased. Correspondingly, increasing the number of proline residues caused a decreased ability to form channels in planar lipid bilayers, and the hemolytic, proline-free peptide tended to cause rapid breakage of planar membranes. Thus, the number of bends created by insertion of proline residues is an important determinant of antimicrobial, hemolytic, and synergistic activity.
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PMID:Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides. 1038 56

Analogues based on the insect cecropin-bee melittin hybrid peptide (CEME) were studied and analyzed for activity and salt resistance. The new variants were designed to have an increase in amphipathic alpha-helical content (CP29 and CP26) and in overall positive charge (CP26). The alpha-helicity of these peptides was demonstrated by circular dichroism spectroscopy in the presence of liposomes. CP29 was shown to have activity against gram-negative bacteria that was similar to or better than those of the parent peptides, and CP26 had similar activity. CP29 had cytoplasmic membrane permeabilization activity, as assessed by the unmasking of cytoplasmic beta-galactosidase, similar to that of CEME and its more positively charged derivative named CEMA, whereas CP26 was substantially less effective. The activity of the peptides was not greatly attenuated by an uncoupler of membrane potential, carbonyl cyanide-m-chlorophenylhydrazone. The tryptophan residue in position 2 was shown to be necessary for interaction with cell membranes, as demonstrated by a complete lack of activity in the peptide CP208. Peptides CP29, CEME, and CEMA were resistant to antagonism by 0.1 to 0.3 M NaCl; however, CP26 was resistant to antagonism only by up to 160 mM NaCl. The peptides were generally more antagonized by 3 and 5 mM Mg2+ and by the polyanion alginate. It appeared that the positively charged C terminus in CP26 altered its ability to permeabilize the cytoplasmic membrane of Escherichia coli, although CP26 maintained its ability to kill gram-negative bacteria. These peptides are potential candidates for future therapeutic drugs.
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PMID:Salt-resistant alpha-helical cationic antimicrobial peptides. 1039 Feb

HIV-2 GH-1 is a molecular clone derived from an AIDS patient from Ghana. In contrast to the prototypic molecular clone ROD, GH-1 exhibits a narrow range of target cell specificity. By an infectious assay using HeLa-CD4 cells stably transfected with an HIV-1 LTR-beta-galactosidase reporter gene and transiently expressing various cloned chemokine receptors, we have examined the coreceptor usage of GH-1. In contrast to ROD, which uses principally CXCR4, GH-1 was found to use mainly if not exclusively CCR5 but not CXCR4. The distinct coreceptor usage of these two molecular clones allowed us to further map the region of gp120 that is important for the coreceptor specificity. By constructing a series of chimeric viruses between GH-1 and ROD, we have demonstrated that the C-terminal half of the V3 loop region of gp120 determines the differential coreceptor usage between GH-1 and ROD, and only a few amino acid differences in this region appear to be able to shift the specificity between CCR5 and CXCR4. Notably, the shift in the coreceptor usage from CCR5 to CXCR4 is associated with an increase in the net positive charge in the V3 region.
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PMID:Small amino acid changes in the V3 loop of human immunodeficiency virus type 2 determines the coreceptor usage for CXCR4 and CCR5. 1054 50

A highly desirable approach to prevention of human immunodeficiency virus type 1 (HIV-1) transmission during sexual intercourse is the development of nontoxic, topical, broad spectrum microbicides effective against transmission of cell-associated and cell-free virus. Toward this end, the HIV-1 inactivation potential of surface active agents C31G and an alkyl sulfate, sodium dodecyl sulfate (SDS) was assessed. Because of its extensive use as a microbicidal agent, nonoxynol-9 (N-9) was used as a reference against which C31G and SDS were compared. Viral inactivation was measured using HIV-1 LTR-beta-galactosidase indicator cells (expressing CD4 or CD4/CCR5) derived from HeLa cells, a cell line of human cervical adenocarcinoma origin. In experiments which examined inactivation of cell-free HIV-1, C31G was generally more effective than N-9. Viral inactivation by SDS occurred at twice the concentration necessary to achieve similar levels of inactivation using either N-9 or C31G. Using HeLa and HeLa-derived cells in cytotoxicity studies, it was demonstrated that SDS is as much as 11 and five times less cytotoxic than N-9 or C31G, respectively, during 48 h of continuous exposure. SDS (unlike C31G and N-9) can inactivate non-enveloped viruses such as human papillomavirus (HPV) [Howett, M.K., Neely, E.B., Christensen, N.D., Wigdahl, B., Krebs, F.C., Malamud, D., Patrick, S.D., Pickel, M.D., Welsh, P.A., Reed, C.A., Ward, M.G., Budgeon, L.R., Kreider, J.W., 1999. A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrob. Agents Chemother. 43(2), 314-321]. Since addition of SDS to C31G or N-9 may make the resulting microbicidal mixtures broadly effective against both enveloped and non-enveloped viruses, several surface active agent combinations were evaluated for their abilities to inactivate HIV-1. Addition of SDS to either C31G or N-9 resulted in mixtures that were only slightly less effective than equivalent concentrations of C31G or N-9 alone. To investigate inactivation of cell-associated infectivity, HIV-1 IIIB-infected SupT1 cells were treated with N-9, C31G, or SDS. Inactivation of cell-associated infectivity required higher microbicide concentrations than were needed for inactivation of cell-free virus. However, the relative activities of N-9, C31G, or SDS were similar to those seen in assays of inactivation using cell-free virus. These studies suggest that C31G and SDS may be attractive candidates for human trials as topical microbicides effective against HIV-1 transmission since both function at concentrations that provide effective viral inactivation with low levels of cytotoxicity. SDS microbicides (used alone or with other microbicides) may provide the added advantage of protection from HPV infection.
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PMID:Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate. 1055 74

An extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp. has been purified to homogeneity by successive DEAE cellulose chromatography followed by gel filtration on Sephacryl S-300. The native molecular mass of the enzyme is 250,000 and it is composed of two identical subunits with molecular mass of 120,000. It is an acidic protein with a pI of 4.2. Purified beta-galactosidase is a glycoprotein and contains 8% neutral sugar. The optimum pH and temperature for enzyme activity are 4.5 and 60 degrees C, respectively. The enzyme is stable at 60 degrees C for 4 h, and has a t(1/2) of 150 min(-1) at 70 degrees C which is one of the highest reported for fungal beta-galactosidases. Substrate specificity studies indicated that the enzyme is specific for beta-linked galactose residues with a preference for p-nitrophenyl-beta-D-galactopyranoside (pNPG). The Km and Vmax values for the synthetic substrates pNPG and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 0.66 mM and 1.32 mM; and 22.4 mmol min(-1) mg(-1) and 4.45 mmol min(-1) mg(-1), respectively, while that for the natural substrate, lactose, was 50.0 mM and 12 mmol min(-1) mg(-1). The end product galactose and the substrate analogue isopropyl thiogalactopyranoside (ITPG) inhibited the enzyme with Ki of 2.6 mM and 12.0 mM, respectively. The energy of activation for the enzyme using pNPG and oNPG were 27.04 kCal and 9.04 kCal, respectively. The active site characterization studies using group-specific reagents revealed that a tryptophan and lysine residue play an important role in the catalytic activity of the enzyme.
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PMID:Characterization of a thermostable extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp. 1057 53

Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle-functional interaction between viral glycoproteins and the cell during attachment and fusion-and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-betagal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the beta-galactosidase (betagal) produced as a result of this fusion. The analog alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) interfered with fusion, decreasing betagal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN-receptor interaction; 10 mM DANA completely blocked fusion and betagal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog alpha-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN-receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo.
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PMID:The use of a quantitative fusion assay to evaluate HN-receptor interaction for human parainfluenza virus type 3. 1060 17

Submandibular glands obtained post-mortem from mature ferrets of both sexes were examined with the use of light microscopical histochemical methods for proteins, mucosubstances and enzymes associated with cell functions or organelles. Demilunar cells showed carboxylated mucosubstances that were mainly non-sulphated, and diffuse activity for peroxidase, E600-sensitive esterase and acid phosphatase. Thiol groups were also detected in these cells. Central acinar cells showed sulphated mucosubstances, disulphides and reticular staining for thiamine pyrophosphatase. Intercalary ducts showed diffuse activity for NADH and NAD(P)H dehydrogenases. Striated ducts contained protein, tryptophan, disulphides, neutral mucosubstances and E600-sensitive esterase periluminally. Basally, the striated ductal cells showed variable activity for peroxidase, cytochrome oxidase, succinate dehydrogenase and acid phosphatase. Basolateral plasma membranes of these cells exhibited ouabain-sensitive Na,K-ATPase activity. The collecting ducts were characterized by variable periluminal staining for acid phosphatase, beta-glucuronidase, acid beta-galactosidase and E600-resistant esterase. The results suggest that the histological appearances of the acini of the submandibular gland of the ferret are dependent on the synthesis of secretory acid glycoproteins, that the striated ducts are involved with the secretion of tryptophan-rich product comprising neutral glycoproteins and showing esterase activity and with marked transport of ions and that the collecting ducts are involved with absorption.
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PMID:Morphological phenotypes and functional capabilities of submandibular parenchymal cells of the ferret investigated by protein, mucosubstance and enzyme histochemistry. 1066 22

Tumour growth is dependent upon a blood supply and is associated with the switch to the angiogenic phenotype. We are developing strategies for targeting gene expression to endothelial cells in the tumour vasculature. Recombinant retroviruses have been generated that incorporate regulatory sequences of the prepro-endothelin-1 (ppET1) promoter. Following reverse transcription and integration these modifications are duplicated in the proviral 5' LTR for transcription of the internal beta-galactosidase reporter gene. The titres and endothelial specificity of retroviral vectors harbouring different modifications have been analysed. In the optimal strategy, replacing the MLV enhancer with ppET1 promoter sequences containing the GATA and AP1 elements whilst maintaining sequences from the viral promoter resulted in endothelial cell-specific expression of the reporter gene, and viral titres comparable to those of the unmodified vector. A panel of endothelial and non-endothelial cells infected with the modified virus from a high titre producer clone showed a pattern of expression consistent with the activity of the endogenous ppET1 promoter. The modified LTR retained specificity in vivo, in subcutaneous tumours arising from the co-injection of tumour cells and irradiated virus producer cells. This simple model achieves high efficiency of transduction and can be used routinely for the screening of targeted retroviral vectors. Gene Therapy (2000) 7, 368-376.
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PMID:Generation of a high titre retroviral vector for endothelial cell-specific gene expression in vivo. 1069 18

Phenotypic drug susceptibility assays of human immunodeficiency virus type 1 (HIV-1) isolates generally use time-consuming, expensive assays with peripheral blood mononuclear cells. A new HIV-1 indicator cell line, MAGI-CCR5, has been developed and applied for this purpose. This cell line expresses human CD4, the two major HIV-1 coreceptors, CCR5 and CXCR4, the reporter gene beta-galactosidase driven by the HIV-1 LTR, and quantitates infection within 48 h. A panel of reference strains and primary HIV-1 isolates were all found to infect this cell line. Susceptibility assays with a nucleoside (zidovudine, ZDV) and a non-nucleoside reverse transcriptase inhibitor (nevirapine, NVP) were performed with reference and primary isolates. The assay was modified into two steps for protease inhibitor (indivinavir, IDV and ritonavir, RTV) susceptibility assays. Primary isolates derived from drug naive patients displayed mean baseline 50% effective concentrations (EC50) of 0.14 microM for ZDV, 0.33 microM for NVP, and 0.02 microM for IDV. Isolates derived from patients under treatment displayed increased EC50 concentrations. The MAGI-CCR5 cell line offers a rapid, efficient, and reproducible method of testing a wide range of HIV-1 isolates for drug susceptibility.
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PMID:Rapid phenotypic drug susceptibility assay for HIV-1 with a CCR5 expressing indicator cell line. 1071 48


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