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Biomedical subjects

T Lu

Publications and source records attributed to T Lu.

At least 127 records · Page 7Linked to original sources
PubMed1994

[Investigation of skull base in nasopharyngeal carcinoma].

100 patients with destruction of skull base in nasopharyngeal carcinoma, confirmed by pathology and CT scanning before radiotherapy, were analysed. It showed that the primary characteristics of skull base destruction are headache and cranial nerve involvement. Although the destruction of skull base showed evident manifestation in TNM classification, it can be inferred that the erosion development probably took place within 1-6 months. Comparing the CT scanning with X-ray submento-vertical view, the result indicated that the erosions of the various positions of the skull base and the invasion of paranasal sinus, cavernous sinus and orbit were clearly displayed by CT scanning but only 23% was discovered by X-ray. There was significant difference between CT scanning and X-ray, and CT scanning had more advantages. The relation between the erosion of the skull base and the invasion of parapharynx space and the metastatics of the cervical nodes were also discussed.

Adolescent↗
PubMed1993

Comparison of the acyl chain specificities of human myristoyl-CoA synthetase and human myristoyl-CoA:protein N-myristoyltransferase.

Human myristoyl-CoA synthetase and myristoyl-CoA:protein N-myristoyltransferase (hNmt) have been partially purified from an erythroleukemia cell line. Their substrate specificities were examined using two in vitro assays of enzyme activity together with a panel of C7-C17 saturated fatty acids plus 72 myristic acid analogs containing oxygen, sulfur, ketocarbonyl, ester, amide, cis and trans double bonds, triple bonds, and para-substituted phenyl groups. There is an inverse relationship between the polarity and the activity of C14 fatty acid substrates of myristoyl-CoA synthetase. Surveys of tetradecenoic and tetradecynoic acids suggest that myristate is bound to the synthetase in a bent conformation with a principal bend occurring in the vicinity of C5-C6. The synthetase can tolerate a somewhat wider range of physical chemical properties in acyl chains than can the monomeric hNmt. However, like myristoyl-CoA synthetase, there is an inverse relationship between acyl chain polarity and the activities of hNmt's acyl-CoA substrates. Moreover, the acyl chain of myristoyl-CoA appears to be bound to hNmt in a bent conformation with bends located in the vicinity of C5 and C8. The acyl chain specificities of both enzymes make them well suited to utilize efficiently any cellular pools of 5Z-tetradecenoic and 5Z,8Z-tetradecadienoic acids and their CoA derivatives. This feature may account for the recent observation that in some mammalian cell lineages, certain N-myristoyl-proteins are heterogeneously acylated with these C14 fatty acids. Finally, the acyl-CoA binding sites of human and Saccharomyces cerevisiae Nmts appear to have been highly conserved. Given their overlapping yet distinct peptide substrate specificities, development of species-specific inhibitors of Nmts should probably focus on structural features recognized in the enzymes' peptide substrates rather than in the acyl chain of their acyl-CoA substrates.

Acyl Coenzyme A↗
PubMed1993

Protein-protein interaction studies using immobilized oligohistidine fusion proteins.

Using a human transcription factor, upstream stimulatory factor, as a model system, we developed a method for the rapid and efficient purification of proteins that interact with a cloned polypeptide expressed as a fusion with an oligohistidine domain. The complex between the oligohistidine fusion protein and its interacting partner was loaded onto a column of chelating resin charged with Ni2+. The bound complex could be eluted as a whole with high concentrations of imidazole. Alternatively, the secondary protein could be released separately using appropriate elution conditions. This procedure is both simple and efficient, and it presents distinct advantages over other affinity purification methods. In addition, this same method can be used to study protein-protein interactions.

Animals↗
PubMed1993

Early diagnosis of typhoid fever using monoclonal antibody.

In our study, a reverse passive hemagglutination assay (RPHA) method was established using monoclonal antibody-IgM to detect the Salmonella typhi O9 antigen. This method has the advantages of sensitivity, specificity, rapidity, and simplicity, and helps make an early diagnosis of typhoid fever using only small amounts of blood.

Animals↗
PubMed1993

Changes of calmodulin contents in single vascular smooth muscle cells from the tail arteries of spontaneously hypertensive rats.

By combining immunohistochemistry and fluorocytometry techniques, total calmodulin (total CaM), Ca(2+)-bound calmodulin (Ca.CaM) and total protein contents in single vascular smooth muscle cells (VSMC) enzymatically dispersed from the tail arteries of young (5-7 weeks old, prehypertensive) and adult (20-24 weeks old, established hypertensive) stroke-prone spontaneously hypertensive rats (SHRsp) were studied and compared with those of age-matched Wistar-Kyoto rats (WKY). No significant difference was found in total CaM, Ca.CaM and protein contents between young SHRsp and WKY. Total CaM and protein contents in adult SHRsp were increased by similar degrees (30.7% and 27.5%, respectively) as in age-matched WKY, suggesting that increased total CaM content may be a consequence of increased synthesis of cellular protein during hypertension. However, Ca.CaM contents in adult SHRsp were significantly increased over those in age-matched WKY by a much higher degree (86.2%), reflecting an abnormal Ca2+ homeostasis in single VSMCs during hypertension.

Age Factors↗
PubMed1992

Analogs of palmitoyl-CoA that are substrates for myristoyl-CoA:protein N-myristoyltransferase.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p; EC 2.3.1.97) is an essential enzyme that is highly selective for myristoyl-CoA in vivo. It is unclear why myristate (C14:0), a rare cellular fatty acid, has been selected for this covalent protein modification over more abundant fatty acids such as palmitate (C16:0), nor is it obvious how the enzyme's acyl-CoA binding site is able to discriminate between these two fatty acids. Introduction of a cis double bond between C5 and C6 of palmitate [(Z)-5-hexadecenoic acid] or a triple bond between C4 and C5 or C6 and C7 (Y4- and Y6-hexadecenoic acids) yields compounds that, when converted to their CoA derivatives, approach the activity of myristoyl-CoA as Nmt1p substrates in vitro. Kinetic studies of 42 C12-C18 fatty acids containing triple bonds, para-phenylene, or a 2,5-furyl group, as well as cis and trans double bonds, suggest that the geometry of the enzyme's acyl-CoA binding site requires that the acyl chain of active substrates assume a bent conformation in the vicinity of C5. Moreover, the distance between C1 and the bend appears to be a critical determinant for optimal positioning of the acyl-CoA in this binding site so that peptide substrates can subsequently bind in the sequential ordered bi-bi reaction mechanism. Identification of active, conformationally restricted analogs of palmitate offers an opportunity to "convert" wild-type or mutant Nmts to palmitoyltransferases so that they can deliver these C16 fatty acids to critical N-myristoylproteins in vivo. nmt181p contains a Gly-451-->Asp mutation, which causes a marked reduction in the enzyme's affinity for myristoyl-CoA. Strains of S. cerevisiae containing nmt1-181 exhibit temperature-sensitive myristic acid auxotrophy: their complete growth arrest at 37 degrees C is relieved when the medium is supplemented with 500 microM C14:0 but not with C16:0. The CoA derivatives of (Z)-5-hexadecenoic and Y6-hexadecynoic acids are as active substrates for the mutant enzyme as myristoyl-CoA at 24 degrees C. However, unlike C16:0, they produce growth arrest of nmt181p-producing cells at this "permissive" temperature, suggesting that these C16 fatty acids do not allow expression of the biological functions of essential S. cerevisiae N-myristoylproteins.

Acyl Coenzyme A↗
PubMed1992

4-oxatetradecanoic acid is fungicidal for Cryptococcus neoformans and inhibits replication of human immunodeficiency virus I.

Candida albicans and Cryptococcus neoformans are major causes of systemic fungal infections, particularly in patients with acquired immunodeficiency syndrome. Metabolic labeling studies revealed that these organisms synthesize a small number of N-myristoylproteins, the most prominent being 20-kDa ADP-ribosylation factors (Arfs). C. albicans Arf has approximately 80% identity with the essential Arf1 and Arf2 proteins of Saccharomyces cerevisiae. [3H]Myristic acid analogs with oxygen for -CH2- substitutions at C4, C6, C11, and C13 are incorporated into cellular N-myristoylproteins, phospholipids, and neutral lipids produced by these three yeasts during exponential growth at 30 degrees C in complex media. Analog- and organism-specific differences in the efficiency of labeling of proteins and lipid classes were observed. The effects of oxatetradecanoic acids with oxygen for -CH2- substitutions at C3-C13 on C. neoformans, C. albicans, and S. cerevisiae were assessed during mid-log phase growth at 30 degrees C. A single dose of 3-oxa-, 4-oxa-, 5-oxa- or 6-oxatetradecanoic acid (O3-O6, final concentration = 300 microM) was able to inhibit growth of C. neoformans in the order O4 greater than O5 greater than O3 approximately O6. The other compounds were inactive. 4-Oxatetradecanoic acid was fungicidal, producing a 10,000-fold reduction in viable cell number 1 h after administration and continued suppression of cell growth for 7 h. A clear dose response was observed over a concentration range of 100-300 microM. 4-Oxatridecanoic acid was 100-fold less potent in reducing cell viability than 4-oxatetradecanoic acid but more potent than 5-oxatridecanoic acid. O4 produced approximately 10-100-fold reductions in the viability of C. albicans and S. cerevisiae at 300-500 microM, respectively, whereas O5 and O6 were less active. Since N-myristoylation of the Pr55gag polyprotein precursor produced by human immunodeficiency virus I (HIV-I) is essential for its assembly, we also assessed the antiviral effects of 4-oxatetradecanoic acid. O4 is able to produce a 50% reduction in the replication of HIV-I in acutely infected human T-lymphocyte cell lines at a concentration of 18 microM. Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I.

ADP-Ribosylation Factor 1↗
PubMed1992

Substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Analysis of fatty acid analogs containing carbonyl groups, nitrogen heteroatoms, and nitrogen heterocycles in an in vitro enzyme assay and subsequent identification of inhibitors of human immunodeficiency virus I replication.

Covalent attachment of myristic acid (C14:0) to the amino-terminal glycine residue of a variety of eukaryotic cellular and viral proteins can have a profound influence on their biological properties. The enzyme that catalyzes this modification, myristoyl-CoA-protein N-myristoyltransferase (NMT), has been identified as a potential target for antiviral and antifungal therapy. Its reaction mechanism is ordered Bi Bi with myristoyl-CoA binding occurring before binding of peptide and CoA release preceding release of myristoylpeptide. Perturbations in the binding of its acyl-CoA substrate would therefore be expected to have an important influence on catalysis. We have synthesized 56 analogs of myristic acid (C14:0) to further characterize the acyl-CoA binding site of Saccharomyces cerevisiae NMT. The activity of fatty acid analogs was assessed using a coupled in vitro assay system that employed the reportedly nonspecific Pseudomonas acyl-CoA synthetase, purified S. cerevisiae NMT, and octapeptide substrates derived from residues 2-9 of the catalytic subunit of cyclic AMP-dependent protein kinase and the Pr55gag polyprotein precursor of human immunodeficiency virus I (HIV-I). Analysis of ketocarbonyl-, ester-, and amide-containing myristic acid analogs (the latter in two isomeric arrangements, the acylamino acid (-CO-NH-) and the amide (-NH-CO)) indicated that the enzyme's binding site is able to accommodate a dipolar protrusion from C4 through C13. This includes the region of the acyl chain occurring near C5-C6 (numbered from carboxyl) that appears to be bound in a bent conformation of 140-150 degrees. The activities of NMT's acyl-CoA substrates decrease with increasing polarity. This relationship was particularly apparent from an analysis of a series of analogs in which the hydrocarbon chain was terminated by (i) an azido group or (ii) one of three nitrogen heterocycles (imidazole, triazole, and tetrazole) alkylated at either nitrogen or carbon. This inverse relationship between polarity and activity was confirmed after comparison of the activities of the closely related ester- or amide-containing tetradecanoyl-CoA derivatives. Members from all of the analog series were surveyed to determine whether they could inhibit replication of human immunodeficiency virus I (HIV-I), a retrovirus that depends upon N-myristoylation of its Pr55gag for propagation. 12-Azidododecanoic acid was the most active analog tested, producing a 60-90% inhibition of viral production in both acutely and chronically infected T-lymphocyte cell lines at a concentration of 10-50 microM without associated cellular toxicity.

Acyltransferases↗
PubMed1992

Delayed-onset sensitization emerges after dishabituation in developing Aplysia.

A recent study of the development of nonassociative learning in the siphon withdrawal reflex of Aplysia showed that dishabituation (facilitation of an habituated response) and sensitization (facilitation of a nonhabituated response) emerge according to different developmental timetables: dishabituation precedes sensitization by approximately 60 days (Rankin & Carew, 1988). Both forms of facilitation of the reflex were observed within 90 s of an electrical shock to the tail. However, more recent work by Marcus and colleagues (1988) in adult animals revealed that sensitization can have a delayed onset of 20-30 min after a strong tail shock. Since the developmental study of Rankin and Carew (1988) only tested the reflex for 10 min after tail shock, it is possible that sensitization was in fact present at earlier developmental stages, but was undetected. To examine this question, in the present study we utilized a longer (40-50 min) post-shock observation period to determine whether delayed-onset sensitization is exhibited in juvenile Aplysia, and if so, when it is expressed during development. In our first experiment, we found that Early Stage 12 juveniles (80-95 days after metamorphosis) showed significant delayed-onset sensitization 30-50 min after a strong tail shock. In a second experiment, we found that delayed-onset sensitization was absent in Stage 11 animals (20-70 days after metamorphosis). Thus delayed-onset sensitization emerges in Early Stage 12. The fact that the birthdate of delayed-onset sensitization is at least 30 days after that of dishabituation (Rankin & Carew, 1987, 1988) supports the hypothesis that these two forms of nonassociative learning may have at least partly different underlying mechanisms.

Animals↗