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H F Lu

Publications and source records attributed to H F Lu.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of caffeic acid, chlorogenic acid and ferulic acid on growth and arylamine N-acetyltransferase activity in Shigella sonnei (group D).

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) as substrates were determined in Shigella sonnei (group D) collected from patients with diarrhoeal disease. The NAT activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. Inhibition of growth studies from S. sonnei (group D) demonstrated that caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) elicited a dose-dependent bactericidal effect in S. sonnei (group D) cultures, i.e. the greater the concentration of CA, CGA and FA, the greater the inhibition of growth of S. sonnei (group D). Cytosols or suspensions of S. sonnei (group D) with and without selected concentrations of CA, CGA and FA co-treatment showed different percentages of 2-AF acetylation. The data indicated that there was reduced NAT activity associated with increased CA, CGA and FA in Shigella dysenteriae (group D) cytosols and intact cells. For the cytosol and intact bacteria examinations, the apparent values of K(m) and Vmax decreased after being co-treated with 400 microM CA, CGA and FA. This report is the first demonstration of plant phenolic inhibition (CA, CGA and FA) of arylamine NAT activity and growth in the bacterium S. sonnei (group D).

Arylamine N-Acetyltransferase↗
PubMed2000

Transrectal ultrasonographic findings in patients with hemospermia.

BACKGROUND: Hemospermia causes anxiety in many men. Its etiology includes inflammatory disorders, vascular lesions, malignancies and congenital anomalies. With the advent of modern transrectal ultrasonography (TRUS) and magnetic resonance imaging technology, we can now visualize the internal structures of the seminal vesicles and prostate, and identify a more accurate etiology for hemospermia. This study was performed to assess the efficacy of TRUS in the evaluation of hemospermia. METHODS: TRUS was used to evaluate 40 patients with hemospermia. Patients' ages ranged from 20 to 72 years, with an average age of 41 years. The duration of symptoms was 3 days to 10 years, (average age 12.4 months). Apart from three patients with hepatitis, there was no major illness found in the medical histories. TRUS was performed with a B & K, type 3535 transrectal transducer device, with an 1850 multiplane probe. RESULTS: TRUS revealed abnormalities in 33 (83%) of 40 patients, which included ejaculatory duct cysts, müllerian duct cysts, asymmetric seminal vesicle dilatation, preprostatic vein engorgement and prostatic enlargement. There were stones in the ejaculatory ducts, ejaculatory duct cysts, müllerian duct cysts or prostate. Nine patients with prostatic stones were under 40 years-of-age. Other than the development of stones, there were no significant differences between patients less than 40 years-of-age and those more than 40 years old. CONCLUSIONS: The results suggest that TRUS is an effective method for examining the seminal vesicles, ejaculatory duct and prostate complex. Sonoguided aspiration, injection of contrast medium or biopsy can further help to differentiate the nature of hemospermia.

Adult↗
PubMed1999

Study of prognostic predictors for non-small cell lung cancer.

BACKGROUND: The outcome of treatment in non-small cell lung cancer (NSCLC) remains poor. One of the reasons is that in many patients its biological behavior does not follow a definite pattern, and can not be accurately predicted prior to treatment. In the present study we have examined the significant prognostic predictors. METHODS: One hundred and fifty-eight patients with NSCLC entered this study. They received surgery alone (95 cases) or combined therapy with postoperative irradiation (63 cases). Three types of data have been collected: (1) clinical characteristics: age, sex, Karnofsky performance status, weight loss, T stage, and N stage; (2) histopathology studies: histological types, tumor differentiation, status of vascular and lymphatic vessel invasions; (3) laboratory measurements by immunohistochemistry assay: oncoprotein overexpression, including pan-ras, c-myc, neu, epidermal growth factor receptor (EGFR) and p53, and tumor cell proliferation by proliferating cell nuclear antigen (PCNA). RESULTS: For the entire group, 5-year actuarial survival, local control and distant metastasis rates were 44, 63 and 40%, respectively. In the univariate analyses, T stage, N stage and lymphatic vessel invasion correlated to survival; T stage and N stage to local control; N stage, lymphatic vessel invasion and pan-ras protein positive stain to distant metastasis. When the index of oncoprotein positive stains was used, the higher index was associated with a higher distant metastasis rate. In the multivariate analyses, T stage, N stage and lymphatic vessel invasion could be independent predictors for survival; T stage for local control; N stage, lymphatic vessel invasion and index of positive oncoprotein stains for distant metastasis. CONCLUSIONS: Late T and N stages, lymphatic vessel invasion and multi-oncoprotein positive stains would predict poor prognoses for NSCLC.

Actuarial Analysis↗
PubMed1999

Effects of berberine on arylamine N-acetyltransferase activity in human bladder tumour cells.

Berberine was used to determine inhibition of arylamine N-acetyltransferase (NAT) activity in human bladder tumour cells. The NAT activity was measured by HPLC assaying for the amounts of N-acetyl-2-aminofluorene (AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and remaining 2-aminofluorene (AF) and p-aminobenzoic acid (PABA). Two assay systems were performed, one with cellular cytosols, the other with intact bladder tumour cell suspensions. The NAT activity in human bladder tumour cells was inhibited by berberine in a dose-dependent manner, that is, the higher the concentration of berberine, the higher the inhibition of NAT activity. The values of apparent Km and Vmax calculated from cytosol NAT and intact cells were also decreased by berberine. This report is the first demonstration to show berberine did affect human bladder tumour cell NAT activity.

4-Aminobenzoic Acid↗
PubMed1999

Effects of the butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the arylamines N-acetyltransferase activity in rat white blood cells.

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used to determine any effects on the N-acetyltransferase (NAT) activity in rat whole blood and white blood cells as measured by high performance liquid chromatography assay for the amounts of N-acetyl-2-aminofluorene (AAF) and 2-aminofluorene (AF). Two assay systems were performed, one with cellular cytosols, the other with intact white blood cells. The NAT activity in the whole blood and white blood cell cytosols was suppressed by BHA and BHT in a dose-dependent manner, i.e. the higher the concentrations of BHA and BHT, the higher the inhibition of NAT activity. Time-course experiments showed that NAT activity measured from the intact white blood cells was inhibited by BHA and BHT up to 24 h. The results suggest that BHA and BHT suppressed AF acetylation in rat blood with intact white blood cells.

Acetylation↗
PubMed1998

Synthesis, biological activity, and conformational analysis of peptidomimetic analogues of the Saccharomyces cerevisiae alpha-factor tridecapeptide.

Biochemical and biophysical investigations on the Saccharomyces cerevisiae alpha-factor indicate that this tridecapeptide mating pheromone (WHWLQLKPGQPMY) might adopt a type II beta-turn in the center of the peptide when it binds to its G protein-coupled receptor. To test this hypothesis we synthesized analogues of alpha-factor incorporating a (R or S)-gamma-lactam conformational constraint [3-(R or S)-amino-2-oxo-1-pyrrolidineacetamido] in place of the Pro-Gly at residues 8 and 9 of the peptide and tested their biological activities and receptor binding. Analogues were purified to >99% homogeneity as evidenced by high-performance liquid chromatography and capillary electrophoresis and characterized by amino acid analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The restricted alpha-factor analogue WHWLQLK[(R)-gamma-lactam]QP[Nle]Y was more active than its lactam-containing diastereomeric homologue WHWLQLK[(S)-gamma-lactam]QP[Nle]Y and about equally active with the [Nle12]-alpha-factor in growth arrest and FUS1-lacZ gene induction assays. Both lactam analogues competed with tritiated [Nle12]-alpha-factor for binding to the alpha-factor receptor (Ste2p) with the (R)-gamma-lactam-containing peptide having 7-fold higher affinity than the (S)-gamma-lactam-containing homologue. Two-dimensional NMR spectroscopy and modeling analysis gave evidence that the (R)-gamma-analogue is a flexible peptide that assumes a transient gamma-turn structure around the lactam moiety. The results represent the first example of an alpha-factor analogue containing a peptidomimetic constraint that is as active as the native pheromone. The correlation between activity and structure provides further evidence that the biologically active conformation of the molecule contains a turn in the middle of the pheromone. This study provides new insights into the structural basis of alpha-factor activity and adds to the repertoire of conformationally biasing constraints that can be used to maintain and even enhance biological activity in peptide hormones.

Amides↗
PubMed1998

A HU-like protein binds to specific sites within nod promoters of Rhizobium leguminosarum.

Nodulation genes (nod) of rhizobia are essential for establishment of its symbiosis with specific legume hosts and are usually located on the Sym(biosis) megaplasmid. In this work we identified a new Sym plasmid independent protein in Rhizobium leguminosarum, Px, by its ability to bind to nod promoters and induce DNA bending. Depending upon its concentrations relative to DNA templates, Px could either stimulate or inhibit in vitro transcription of the major regulatory nodulation gene nodD. This may result from its property to bind to specific sites within nod promoters at lower concentration or in the presence of competitor calf thymus DNA but nonspecifically associate with DNA at higher levels or in the absence of competitors. Its binding sites within nodD and nodF promoters were determined by DNase I footprinting but showed no sequence consensus. N-terminal sequencing and Western blot revealed that Px belongs to the HU class of prokaryotic histone-like proteins. Its binding feature and functioning mechanism were discussed in the light of this discovery.

Bacterial Proteins↗
PubMed1998

Structure-activity studies of fluoroalkyl-substituted gamma-butyrolactone and gamma-thiobutyrolactone modulators of GABA(A) receptor function.

Dihydro-2(3H)-furanones (gamma-butyrolactones) and dihydro-2(3H)-thiophenones (gamma-thiobutyrolactones) containing fluoroalkyl groups at positions C-3, C-4, and C-5 of the heterocyclic rings were prepared. The anticonvulsant/convulsant activities of the compounds were evaluated in mice. Brain concentrations of the compounds were determined and the effects of the compounds on [35S]-tert-butylbicyclophosphorothionate ([35S]TBPS) binding to the picrotoxin site on GABAA receptors were investigated. The effects of the compounds on GABAA receptor function were studied using electrophysiological methods and cultured rat hippocampal neurons. Fluorination at C-3 results in either subtle or pronounced effects on the pharmacological activity of the compounds. When hydrogens are replaced with fluorines at the methylene carbon of an ethyl group, as in 3-(1,1-difluoroethyl)dihydro-3-methyl-2(3H)-furanone (1), the anticonvulsant actions of the compound are not much changed from those found for the corresponding alkyl-substituted analogue. In marked contrast, fluorination at the methyl carbon of the ethyl group, as in dihydro-3-methyl-3-(2,2,2-trifluoroethyl)-2(3H)-furanone (3), produces a compound having convulsant activity. This convulsant activity seems to be due to an increased affinity of the compound for the picrotoxin site on GABAA receptors caused by an interaction that involves the trifluoromethyl group. Results obtained with gamma-butyrolactones containing either a 3-(1-trifluoromethyl)ethyl or a 3-(1-methyl-1-trifluoromethyl)ethyl substituent indicate that the interactions of the trifluoromethyl group with the picrotoxin binding site are subject to both stereochemical and steric constraints. Sulfur for oxygen heteroatom substitution, as in the corresponding gamma-thiobutyrolactones, affects the type (competitive, non-competitive, etc.) of binding interactions that these compounds have with the picrotoxin site in a complex manner. Fluorination of alkyl groups at the C-4 and C-5 positions of gamma-butyrolactones having convulsant activity increases convulsant potency.

4-Butyrolactone↗
PubMed1998

Structure-function analysis of the Saccharomyces cerevisiae tridecapeptide pheromone using alanine-scanned analogs.

Twenty-six peptide analogs of the Saccharomyces cerevisiae alpha-factor, a tridecapeptide mating pheromone (W1H2W3L4Q5L6K7p8G9Ql0P11M12Y13) with either L- or D-alanine replacement of each amino acid residue (Ala-scanned) and with the isosteric replacement of methionine at position 12 by norleucine, were synthesized, purified to homogeneity and assayed for biological activity and receptor binding. Two new and effective antagonists, [D-Ala3,Nle12]alpha-factor and [D-Ala4,Nle12]alpha-factor, were found among the series, and the [D-Ala10,Nle12]alpha-factor demonstrated a marked ability to increase the biological activity of [Nle12]alpha-factor without having any effect by itself. One analog, the [L-Ala1 alpha-factor, showed a 3-fold increase in bioactivity over the [Nle12]alpha-factor, although its binding to the alpha-factor receptor was about 70-fold less than [Nle12]alpha-factor. Residues near the carboxyl terminus contributed more strongly to receptor binding than other residues, whereas those near the amine terminus of the alpha-factor played an important role in signal transduction. The effect of insertion of D-Ala residues at positions 7, 8, 9 and 10 on bioactivity and receptor binding of the peptide suggested a specific positioning role of the central loop in establishing optimal contacts between the receptor and the ends of the pheromone. We conclude that the alpha-factor may be divided into segments with dominant roles in forming the biologically active pheromone conformation, in receptor binding and in initiating signal transduction. The discovery of such relationships was made possible by the systematic variation of each residue in the peptide and by the testing of each analog in highly defined biological and binding assays.

Alanine↗
PubMed1997

Design and synthesis of novel imidazole-substituted dipeptide amides as potent and selective inhibitors of Candida albicans myristoylCoA:protein N-myristoyltransferase and identification of related tripeptide inhibitors with mechanism-based antifungal activity.

A new class of antifungal agents has been discovered which exert their activity by blockade of myristoylCoA: protein N-myristoyltransferase (NMT; EC 2.1.3.97). Genetic experiments have established that NMT is needed to maintain the viability of Candida albicans and Cryptococcus neoformans,the two principal causes of systemic fungal infections in immunocompromised humans. Beginning with a weak octapeptide inhibitor ALYASKLS-NH2 (2, Ki = 15.3 +/- 6.4 microM), a series of imidazole-substituted Ser-Lys dipeptide amides have been designed and synthesized as potent and selective inhibitors of Candida albicans NMT. The strategy that led to these inhibitors evolved from the identification of those functional groups in the high-affinity octapeptide substrate GLYASKLS-NH2 1a necessary for tight binding, truncation of the C-terminus, replacement of the four amino acids at the N-terminus by a spacer group, and substitution of the glycine amino group with an N-linked 2-methylimidazole moiety. Initial structure-activity studies led to the identification of 31 as a potent and selective peptidomimetic inhibitor with an IC50 of 56 nM and 250-fold selectivity versus human NMT. 2-Methylimidazole as the N-terminal amine replacement in combination with a 4-substituted phenacetyl moiety imparts remarkable potency and selectivity to this novel class of inhibitors. The (S,S) stereochemistry of serine and lysine residues is critical for the inhibitory activity, since the (R,R) enantiomer 40 is 10(3)-fold less active than the (S,S) isomer 31. The inhibitory profile exhibited by this new class of NMT ligands is a function of the pKa of the imidazole substituent as illustrated by the benzimidazole analog 35 which is about 10-fold less potent than 31. The measured pKa (7.1 +/- 0.5) of 2-methylimidazole in 31 is comparable with the estimated pKa (approximately 8.0) of the glycyl residue in the high-affinity substrate 1a. Groups bulkier than methyl, such as ethyl, isopropyl, or iodo, at the imidazole 2-position have a detrimental effect on potency. Further refinement of 31 by grafting an alpha-methyl group at the benzylic position adjacent to the serine residue led to 61 with an IC50 of 40 nM. Subsequent chiral chromatography of 61 culminated in the discovery of the most potent Candida NMT inhibitor 61a reported to date with an IC50 of 20 nM and 400-fold selectivity versus the human enzyme. Both 31 and 61a are competitive inhibitors of Candida NMT with respect to the octapeptide substrate GNAASARR-NH2 with Ki(app) = 30 and 27 nM, respectively. The potency and selectivity displayed by these inhibitors are dependent upon the size and orientation of the alpha-substituent. An alpha-methyl group with the R configuration corresponding to the (S)-methyl-4-alanine in 2 confers maximum potency and selectivity. Structural modification of 31 and 61 by appending an (S)-carboxyl group beta to the cyclohexyl moiety provided the less potent tripeptide inhibitors 73a and 73b with an IC50 of 1.45 +/- 0.08 and 0.38 +/- 0.03 microM, respectively. However, these tripeptides (73a and 73b) exhibited a pronounced selectivity of 560- and 2200-fold versus the human NMT. More importantly 73a displayed fungistatic activity against C albicans with an EC50 of 51 +/- 17 microM in cell culture. Compound 73b also exhibited a similar antifungal activity. An Arf protein gel mobility shift assay for monitoring intracellular myristoylation revealed that a single dose of 200 microM of 73a or 73b produced < 50% reduction in Arf N-myristoylation, after 24 and 48 h, consistent with their fungistatic rather than fungicidal activity. In contrast, the enantiomer 73d which had an IC50 > 1000 microM against C. albicans NMT did not exhibit antifungal activity and produced no detectable reduction in Arf N-myristoylation in cultures of C. albicans. These studies confirm that the observed antifungal activity of 73a and 73b is due to the attenuation of NMT activity and that NMT represents an attractive tar

Acyltransferases↗
PubMed1997

Conformationally constrained [p-(omega-aminoalkyl)phenacetyl]-L-seryl-L-lysyl dipeptide amides as potent peptidomimetic inhibitors of Candida albicans and human myristoyl-CoA:protein N-myristoyl transferase.

MyristoylCoA:protein N-myristoyltransferase (NMT) covalently attaches the 14-carbon saturated fatty acid myristate, via an amide bond, to the N-terminal glycine residues of a variety of cellular proteins. Genetic studies have shown that NMT is essential for the viability of the principal fungal pathogens which cause systemic infection in immunosuppressed humans and hence is a target for development of fungicidal drugs. We have generated a class of potent peptidomimetic inhibitors of the NMT from one such fungal pathogen, Candida albicans. The N-terminal tetrapeptide from a substrate analog inhibitor, ALYASKL-NH2, was replaced with an omega-aminoalkanoyl moiety having an optimal 11-carbon chain for inhibition (11-aminoundecanoyl-SKL-NH2, 3a, IC50 = 1.2 +/- 0.14 microM). A series of replacements for the C-terminal Leu established that residues containing a lipophilic side chain were most effective, with cyclohexylalanine having the greatest potency (3g, IC50 = 0.36 +/- 0.06 microM). Removal of the carboxamide moiety led to a metabolically stable dipeptide inhibitor containing an N-(cyclohexylethyl)lysinamide (17e, IC50 = 0.11 +/- 0.03 microM). Partial rigidification of the flexible aminoundecanoyl chain produced the dipeptide p-(omega-aminohexyl)phenacetyl-L-seryl-L-lysyl-N-(cyclohexyleth yl)amide (26b, IC50 = 0.11 +/- 0.04 microM). Subsequent incorporation of an alpha-methyl substituent into 26b provided the dipeptide analog [2-[p-(omega-aminohexyl)phenyl]propionyl]-L-seryl-L-lysyl-N-(cyclohex ylethyl)amide, a very potent inhibitor (48, IC50 = 0.043 +/- 0.006 microM), which retained the three essential elements required for recognition by the acyl transferase's peptide binding site.

Acyltransferases↗
PubMed1997

Selective peptidic and peptidomimetic inhibitors of Candida albicans myristoylCoA: protein N-myristoyltransferase: a new approach to antifungal therapy.

MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins. Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.

Acyltransferases↗
PubMed1997

Expression of the integrin alpha5 subunit and its mediated cell adhesion in hepatocellular carcinoma.

Tumor invasion and metastasis are complex processes, requiring the ability of tumor cells to interact with proteins of the extracellular matrix through cell-adhesion molecules on the cell surface. Integrins are heterodimeric membrane glycoproteins, consisting of alpha and beta subunits, which enable cells to recognize adhesive substrates in the extracellular matrix. The roles of the integrin alpha5beta1 in tumor invasion are highlighted by finding that some tumor cells have lost or reduced alpha5beta1 expression. It therefore functions as a negative signaling regulator. Expression of alpha5beta1 and its mediation of cell adhesion in hepatocellular carcinoma (HCC) have not been elucidated. In surgical specimens of HCC we found, by immunohistochemistry and Northern blot analysis, that the alpha5-positive rates in cancerous tissues were lower than the corresponding rates in non-cancerous tissues. Reduced expression of the integrin alpha5 occurred more frequently in HCC with more malignant phenotypes, such as poor differentiation, large size (more than 10-cm in diameter), absence of capsule and high invasion. Reverse transcription/polymerase chain reaction, a more sensitive assay, was used to detect the alpha5 mRNA level in LCID20, a highly metastatic model of human HCC, and LCID35, a low-metastasis model. The results showed that integrin alpha5 was negative in the former and positive in the latter. Cell adhesion assays showed the maximal percentage inhibition of anti-alpha5 mAb on SMMC 7721 cell adhesion to fibronectin to be 68.9 +/- 4.9% at the saturation concentrations of each antibody (200 microg/ml). If anti-alpha5 mAb was combined with anti-beta1 mAb, the inhibition was 74.1 +/- 11.1%. It is concluded that reduced expression of the integrin alpha5 subunit is correlated with more malignant phenotypes of human HCC. Any change in the adhesion of hepatocellular carcinoma cells to fibronectin is mainly dependent upon the function of the integrin alpha5beta1.

Antigens, CD↗
PubMed1997

Position one analogs of the Saccharomyces cerevisiae tridecapeptide pheromone.

Analogs of the Saccharomyces cerevisiae alpha-mating factor [WHWLQLKPGQPMY], in which a variety of residues replaced Trp1 were synthesized and assayed for biological activity and receptor affinity. Analogs containing Gly or Leu or many different aromatic residues in position 1 of the peptide exhibited bioactivity in a growth arrest assay slightly greater than, or equal to, that of the parent pheromone, whereas the Glu1 and Lys1 analogs exhibited significantly lower bioactivity. Analogs with an aromatic replacement at position 1 had 3- to 6-fold lower receptor affinity than the parent peptide, whereas analogs with a hydrophilic residue at the N-terminus exhibited large reductions in receptor affinity with the peptide with Glu in position 1 showing a 120-fold reduction. N alpha-Acetylation had little effect on bioactivity but lowered receptor affinity by 20- to 40-fold. Amidation of the carboxyl terminus resulted in a 10-fold decrease in activity and a 160-fold decrease in receptor affinity. These results indicate that the alpha-factor receptor has a large hydrophobic binding pocket, possibly containing a negatively charged side-chain, which interacts with the N-terminus of alpha-factor. The lack of correlation between activity and binding and several analogs suggests that small residues near the N-terminus of alpha-factor may be very efficient in triggering isomerization of the receptor to its activated state in the first step of the signal transduction pathway.

Gene Expression↗
PubMed1996

Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs.

Analogs of the Saccharomyces cerevisiae alpha-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys7 and Gln10 were replaced with Cys, Cys(CH3), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo7,10[Cys7,X9,Cys10,Nle12]alpha-factor , where X=D-Val, D-Ala, L-Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20-30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys7 and Gln10 with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5-10-fold more active than their sulfhydryl-containing homologs, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH3) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the alpha-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form beta-turns spanning residues 7-10 than their less active linear counterparts. The results provide strong evidence that this beta-turn is important for optimal signal transduction by alpha-factor.

Amino Acid Sequence↗
PubMed1995

Cloning and comparative sequence analysis of the gene encoding canine intercellular adhesion molecule-1 (ICAM-1).

Canine intercellular adhesion molecule-1 (ICAM-1) plays a primary role in the adherence of canine neutrophils to endothelial cells and in the cytotoxicity of canine neutrophils for adult cardiac myocytes. We have cloned the canine ICAM-1 gene and have analyzed the conservation of ICAM-1 amino acid (aa) sequences in man, chimpanzee, mouse, rat and dog. Canine ICAM-1 displays 61% identity with human ICAM-1. Cys residues critical to the immunoglobulin (Ig) fold structure and four sites of N-linked glycosylation are absolutely conserved in ICAM-1 from all species. Residues in the cytoplasmic tail associated with cytoskeletal alpha-actinin binding are highly conserved, supporting the hypothesis that intracellular attachment is indeed important for ICAM-1 function. Residues critical for human ICAM-1 binding to the beta 2-integrin leukocyte-function-associated antigen 1 (LFA-1) are highly conserved between all species, whereas those residues demonstrated to play an important role in interaction of human ICAM-1 with macrophage activation complex 1 (Mac-1) are not highly conserved. Residues critical for ICAM-1 binding to rhinovirus and malaria-infected red blood cells (IRBC) are not highly conserved.

Amino Acid Sequence↗