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Biomedical subjects

A J Lloyd

Publications and source records attributed to A J Lloyd.

32 records · Page 2Linked to original sources
PubMed1995

A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity.

We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.

Acylation↗
PubMed1995

Tyrosinaemia type 1 and glutathione synthetase deficiency: two disorders with reduced hepatic thiol group concentrations and a liver 4-fumarylacetoacetate hydrolase deficiency.

Thiol groups are important components of proteins and their oxidation can lead to a substantial loss of protein function. Patients with two apparently unrelated inborn errors of metabolism, tyrosinaemia type 1 and glutathione synthetase deficiency, have been reported to show reduced cell glutathione concentrations. We have found that not only glutathione but also protein thiol concentrations are reduced in the liver in tyrosinaemia type 1 patients. We also report a case of glutathione synthetase deficiency with a substantial deficiency of liver 4-fumarylacetoacetate hydrolase and provide evidence that glutathione, or some small-molecular-weight thiol, is essential for maintaining stability of this enzyme in vitro. Our results suggest that the availability of thiol groups may modify the phenotype of tyrosinaemia type 1 and that liver 4-fumarylacetoacetate hydrolase deficiency may be a secondary complicating factor in some forms of glutathione synthetase deficiency.

Amino Acid Metabolism, Inborn Errors↗
PubMed1994

A point mutation in Euglena gracilis chloroplast tRNA(Glu) uncouples protein and chlorophyll biosynthesis.

The universal precursor of tetrapyrrole pigments (e.g., chlorophylls and hemes) is 5-aminolevulinic acid (ALA), which in Euglena gracilis chloroplasts is derived via the two-step C5 pathway from glutamate charged to tRNA(Glu). The first enzyme in this pathway, Glu-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNA(Glu) (Glu-tRNA) to glutamate 1-semialdehyde (GSA) with the release of the uncharged tRNA(Glu). The second enzyme, GSA-2,1-aminomutase, converts GSA to ALA. tRNA(Glu) is a specific cofactor for the NADPH-dependent reduction by GluTR, an enzyme that recognizes the tRNA in a sequence-specific manner. This RNA is the normal tRNA(Glu), a dual-function molecule participating both in protein and in ALA and, hence, chlorophyll biosynthesis. A chlorophyll-deficient mutant of E. gracilis (Y9ZNalL) does not synthesize ALA from glutamate, although it contains GluTR and GSA-2,1-aminomutase activity. The tRNA(Glu) isolated from the mutant can still be acylated with glutamate in vitro and in vivo. Furthermore, it supports chloroplast protein synthesis; however, it is a poor substrate for GluTR. Sequence analysis of the tRNA and of its gene revealed a C56-->U mutation in the resulting gene product. C56 is therefore an important identity element for GluTR. Thus, a point mutation in the T loop of tRNA uncouples protein from chlorophyll biosynthesis.

Aldehyde Oxidoreductases↗
PubMed1992

Conditions associated with normal levels of prostate-specific antigen and elevation of prostatic acid phosphatase.

Thirteen cases are reported with normal levels of prostate-specific antigen and elevated prostatic acid phosphatase (PAP). Conditions that were associated with an elevated PAP were myeloproliferative syndromes (four cases), metastatic nonprostatic carcinoma (six cases), prostatic carcinoma (one case), tuberculosis with a concurrent lupus-like syndrome (one case), and osteomyelitis (one case). The inclusion of PAP in the initial investigation of cases with suspected prostatic disease results in a decreased specificity.

Acid Phosphatase↗
PubMedDate not supplied

Varicella susceptibility and vaccination strategies in young adults.

BACKGROUND: Varicella infection causes substantial morbidity in young adults. Most military basic trainees are 18 to 21 years old, yet the Army has no varicella vaccination policy. We therefore determined varicella susceptibility in a population of Army basic trainees, examined variables that might predict antibody status, and developed a vaccination strategies model. METHODS: Fifteen-hundred ninety-five trainees completed a demographic and historical questionnaire. Varicella antibody status was determined on 1201 volunteers. These data plus information from the literature were used to construct a decision tree of vaccination strategies that was applied to the total population of Army basic trainees in 1995 (n = 65,298). RESULTS: Fifty (4.2 percent) of 1201 soldiers were antibody negative. Trainees who lived with no or 1 sibling while growing up were most likely to be seronegative (P < 0.01). The positive predictive value of a history of varicella was 98.5 percent, whereas the negative predictive value of a negative history of varicella was 23 percent. In the vaccination strategies model, serologically testing soldiers with a negative history of varicella and vaccinating those without protective antibodies was the most cost-effective approach. CONCLUSIONS: In young adults a positive varicella history accurately predicts immunity, but verification of a negative history with antibody testing is recommended before vaccination.

Adolescent↗