Assay for L-pipecolate oxidase activity in human liver: detection of enzyme deficiency in hyperpipecolic acidaemia.

A direct assay method is described for L-pipecolate oxidase. The assay uses NaHSO3 to trap the L-alpha-amino [3H]adipate delta-semialdehyde (AAS) formed as a direct reaction product of L-pipecolate oxidase from L-[3H]pipecolic acid. The adduct so formed was separated from the substrate on Dowex 50 (H+) column. The product was identified as [3H]AAS by amino acid analysis after breaking down the adduct by boiling under acidic conditions. The assay is simpler and more specific than fluorometric methods; it is also more sensitive, requiring at most 16 micrograms of liver peroxisome-enriched protein per assay. We have used this assay procedure to detect L-pipecolate oxidase in skin fibroblasts obtained from a control subject and from patients of hyperpipecolic acidaemia and Zellweger syndrome and found that this enzyme activity is present in the control, but absent or decreased in the patients with the peroxisomal disorders.     

l-2-Hydroxyglutarate dehydrogenase: identification of a novel enzyme activity in rat and human liver. Implications for l-2-hydroxyglutaric acidemia

In this paper we studied the degradation of l-2-hydroxyglutarate in tissues from rat and man in order to try and find the underlying basis for the accumulation of this metabolite in l-2-hydroxyglutaric patients. The results show that l-2-hydroxyglutarate is not degraded by an oxidase but via a dehydrogenase which was found to be present in liver only. This newly identified enzyme activity was characterized kinetically, although the nature of the reaction product remains to be identified.     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

A Plasma Long Noncoding RNA Signature for Early Detection of Lung Cancer

The early detection of lung cancer is a major clinical challenge. Long noncoding RNAs (lncRNAs) have important functions in tumorigenesis. Plasma lncRNAs directly released from primary tumors or the circulating cancer cells might provide cell-free cancer biomarkers. The objective of this study was to investigate whether the lncRNAs could be used as plasma biomarkers for early-stage lung cancer. By using droplet digital polymerase chain reaction, we determined the diagnostic performance of 26 lung cancer–associated lncRNAs in plasma of a development cohort of 63 lung cancer patients and 33 cancer-free individuals, and a validation cohort of 39 lung cancer patients and 28 controls. In the development cohort, 7 of the 26 lncRNAs were reliably measured in plasma. Two (SNHG1 and RMRP) displayed a considerably high plasma level in lung cancer patients vs. cancer-free controls (all P?<?.001). Combined use of the plasma lncRNAs as a biomarker signature produced 84.13% sensitivity and 87.88% specificity for diagnosis of lung cancer, independent of stage and histological type of lung tumor, and patients' age and sex (all P?>?.05). The diagnostic value of the plasma lncRNA signature for lung cancer early detection was confirmed in the validation cohort. The plasma lncRNA signature may provide a potential blood-based assay for diagnosing lung cancer at the early stage. Nevertheless, a prospective study is warranted to validate its clinical value.     

Alkyl alkanethiolsulfonate sulfhydryl reagents: β-Sulfhydryl-modified derivatives of l-cysteine as substrates for trypsin and α-chymotrypsin

Derivatives of l-cysteine and the A chain of bovine insulin have been chemically modified at the cysteinyl β-sulfhydryl by certain sulfhydryl-specific alkyl alkanethiolsulfonate reagents. The alkanethiolation products possess mixed-disulfide side chains structurally similar to the side chains of lysine and phenylalanine and hence were studied here as substrates for trypsin and α-chymotrypsin, respectively. Kinetic parameters were obtained for the enzyme-catalyzed hydrolyses of the modified l-cysteine analogs and of specific reference amino acids which were derivatized analogously at both the α-amino and α-carboxyl groups and assayed identically. For both enzymes it was found that the specificity constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for analog esters compare favorably with those for specific reference esters, whereas specificity constants for analog amides compare much less favorably with those for specific reference amides. This discrepancy is largely a consequence of the k_cat values for the analog amides being relatively much lower than the corresponding values for the reference amides. Consistent with this trend, no detectable enzyme-catalyzed hydrolysis of the amide bonds at the sites of modified cysteine residues in the A chain of bovine insulin was observed. It is proposed that the predominant kinetic consequence of the mixed-disulfide side chains of the alkanethiolated cysteine moieties is a decrease in the acylation rate constants, k₂, arising from an increase in the transition-state free energies of acylation.     

Different timing of increases in activities of four X-chromosome linked enzymes during the cell cycle of synchronized human lymphoblasts

A permanent human lymphoblast culture was synchronized with repetitive thymidine blocks, and the changes in the levels of activity of four X-chromosome-linked enzymes were followed during the cell cycle. The four enzymes studied were phosphoglycerate kinase (PGK), α-galactosidase (α-Gal), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and glucose-6-phosphate dehydrogenase (G6PD). The levels of PGK and α-Gal activities increased simultaneously in G1 and in S, while HGPRT and G6PD increased close together in middle and late S. Therefore, different control mechanisms may be involved in the increases of the activities of these two sets of enzymes.     

A Prediction Model Based on Biomarkers and Clinical Characteristics for Detection of Lung Cancer in Pulmonary Nodules

Lung cancer early detection by low-dose computed tomography (LDCT) can reduce the mortality. However, LDCT increases the number of indeterminate pulmonary nodules (PNs), whereas 95% of the PNs are ultimately false positives. Modalities for specifically distinguishing between malignant and benign PNs are urgently needed. We previously identified a panel of peripheral blood mononucleated cell (PBMC)-miRNA (miRs-19b-3p and -29b-3p) biomarkers for lung cancer. This study aimed to evaluate efficacy of integrating biomarkers and clinical and radiological characteristics of smokers for differentiating malignant from benign PNs. We analyzed expression of 2 miRNAs (miRs-19b-3p and -29b-3p) in PBMCs of a training set of 137 individuals with PNs. We used multivariate logistic regression analysis to develop a prediction model based on the biomarkers, radiographic features of PNs, and clinical characteristics of smokers for identifying malignant PNs. The performance of the prediction model was validated in a testing set of 111 subjects with PNs. A prediction model comprising the two biomarkers, spiculation of PNs and smoking pack-year, was developed that had 0.91 area under the curve of the receiver operating characteristic for distinguishing malignant from benign PNs. The prediction model yielded higher sensitivity (80.3% vs 72.6%) and specificity (89.4% vs 81.9%) compared with the biomarkers used alone (all P < .05). The performance of the prediction model for malignant PNs was confirmed in the validation set. We have for the first time demonstrated that the integration of biomarkers and clinical and radiological characteristics could efficiently identify lung cancer among indeterminate PNs.     

Simultaneous measurements of testosterone and three 5a -reduced androgens in the venous effluent of immature rat testes in situ

Simultaneous measurements were made by radioimmunoassay of testosterone, 17β-hydroxy-5 a -androstan-3-one (DHT), 5 a -androstane-3 a, 17 β -diol (3a-diol), and 5 a-androstane-3 β, 17β-diol (3β-diol) in the testicular venous plasma (TVP) and peripheral plasma (PP) of 30, 45, and 55 day old rats. At 30 days of age, the preponderant androgen in both plasmas was 3a-diol but testosterone predominated by day 55. Testosterone levels increased with age in both TVP (6.39, 15.08, and 54.93 ng/ml on days 30, 45, and 55 respectively) and PP (0.13, 0.56, and 1.02 ng/ml on days 30, 45 and 55 respectively) whereas 3a-diol concentrations decreased in TVP (48.07 ng/ml, day 30; 24.85 ng/ml, day 55) though not peripherally (range: 0.41–0.52 ng/ml). DHT was low in both TVP and PP and appeared to rise only slightly although the increase was not statistically significant. Levels of 3β-diol remained low and unchanged. These observations suggest that the total androgen content of the venous effluent from the prepubertal rat test is is quite high and that significant changes in peripheral interconversions of androgens are occurring during sexual maturation.