Biochemical and molecular medicine最新文献

A direct (as opposed to competitive) enzyme immunoassay (EIA) was developed to detect neuron-specific enolase (NSE) in cerebrospinal fluid (CSF). Most common methods of evaluating NSE levels have utilized radioimmunoassay. These are highly sensitive, but cannot be employed in laboratories not equipped or licensed for the use of radioisotopes. The EIA developed here shows sensitivity within the physiological range of values for CSF-NSE (>1 ng/ml) and can be used in laboratories with appropriate densitometric scanning capabilities. The assay was applied to CSF samples obtained from patients with a variety of diagnoses at the time of surgical intervention for their respective disorders. While there were no diagnostically significant differences between the levels of NSE in CSF from patients with different neurological disorders utilized in the development of this procedure, we were able to differentiate between marginally different levels of NSE. We conclude that we have developed a safe, fast, reliable, and sensitive assay for NSE in the CSF that can be used to study NSE levels in a variety of neurological cases.

This investigation was undertaken to clarify the mechanisms of superoxide anion (O₂⁻) generation in rat peritoneal mast cells. Compound 48/80, a typical histamine liberator mediated by calcium influx, elicited O₂⁻generation from the mast cells in a dose-dependent fashion. It was demonstrated by immunohistochemical study and Western blot analysis that the mast cells contained the 47-kDa phagocyte oxidase (p47phox) protein, which was one cytosolic component of the NADPH oxidase system. Arachidonic acid stimulated O₂⁻generation in the mast cells, but other unsaturated fatty acids had no effect. On the other hand, 48/80-induced O₂⁻generation was inhibited by phospholipase A2 inhibitors, such as arachidonyl trifluoromethyl ketone and manoalide. Forskolin, isoprenaline, and dibutyryl cyclic AMP inhibited the O₂⁻generation, and KT-5720, a cyclic AMP-dependent protein kinase (A-kinase) inhibitor, markedly enhanced the O₂⁻generation. These findings suggest that O₂⁻is generated by a NADPH oxidase-like enzyme system in mast cells and that this enzyme system is activated by arachidonic acid released by cytosolic phospholipase A2. Thus, it is regulated by the cyclic AMP-A kinase system.

Control rats and diabetic animals injected with streptozotocin during the neonatal period were either maintained on a standard diet or given access to food supplemented with dehydroepiandrosterone (DHEA, 0.2%) for 11 days before sacrifice. In both control and diabetic rats, DHEA feeding augmented the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase and cytosolic NADP-linked malate dehydrogenase in liver, but not so in either the parotid gland or pancreatic islets. DHEA lowered, in both control and diabetic rats, the ratio betweend-glucose oxidation and utilization and the rate of insulin release in pancreatic islets exposed to a high concentration ofd-glucose, as well as the insulin concentration and insulin/glucose ratio in plasma. These findings support the view that, in diabetes, DHEA, by increasing sensitivity to insulin, may allow islet B-cells to avoid the otherwise unfavorable consequences of chronic hyperactivity.

Human seminal plasma contains two enzyme activities both capable of dephosphorylating all nucleoside 5-monophosphates with different efficiency and specificity. Broad-spectrum soluble 5′-nucleotidase is the object of this paper which deals with the definition of the response of this enzyme to effectors, some physiological and others not naturally occurring. The enzyme did not show any product regulation as all the nucleosides tested caused a moderate effect on the hydrolysis of the substrates. Theophylline and other xanthine derivatives had no effect on enzyme activity, whereas glycerate 2,3bisphosphate, like other soluble 5′-nucleotidases, caused a stimulation of the enzyme, especially toward CMP and UMP. 5-Deoxy-5-isobutylthiadenosine resulted in no inhibition of the hydrolysis of AMP and IMP. The enzyme was affected neither by monovanadate nor by decavanadate, whereas it was strongly inhibited by Ap₅A. Variations in adenylate energy charge did not cause any alteration of the enzyme activity toward AMP and only a slight decrease of the hydrolysis of IMP. These regulatory properties, distinct from those of other soluble 5′-nucleotidases, show that this form, newly isolated from human seminal plasma, is subject to an almost unique, tissue-specific regulation.

The effects of histidine-modifying reagents on human serum butyrylcholinesterase (BChE) were investigated. The commercially available enzyme was further purified by chromatography on a Sepharose CI-6B column prior to use. In the modification studies, we found that the histidine-specific reagents tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCK) did not modify the enzyme; however, they inhibited the enzyme reversibly. The kinetic parameters of enzyme inhibition calculated were α = 10.8, β = 0.26, andK_i= 0.016 mmfor TPCK. TLCK inhibition gave similar kinetic behavior, with α = 41.6, β = 0.065, andK_i= 0.039 mm. Tosyllysine, an analog of TLCK, did not inhibit the enzyme. Removal of TPCK and TLCK by dialysis resulted in significant reactivation of the enzyme. From kinetic studies, it was found that the inhibitions were hyperbolic mixed-type inhibitions. We concluded that the reagents competed with substrate for hydrophobic binding sites and inhibited the enzyme reversibly. On the other hand, in the modification studies with diethyl pyrocarbonate (DPC), it was observed that inactivation of the enzyme was irreversible and time-dependent. In the protection studies, the activity of the enzyme was partially protected from inactivation by DPC even at a 50 mmconcentration of butyrylthiocholine. The results indicate that DPC modifies some essential histidine side chains in BChE, including the functional histidyl residue found at the active site.

The degradation of 3 human natriuretic peptides by human kidney neutral endopeptidase 24.11 has been investigated. The studies revealed that hANP-28 and hCNP-22 are the preferred substrates, whereas hBNP-32 is not. The enzyme has been known to inactivate hANP-28 from cleavage at the Cys-Phe bond at the beginning of its ring structure. Analysis of the cleavage sites of each peptide indicated that the initial cleavage site of hCNP-22 is analogous to that of hANP-28. The Cys-Phe bond of hBNP-32 was insensitive to this enzymatic cleavage. We speculate that the stability of hBNP-32 may result from the insusceptibility of its Cys-Phe bond at the beginning of the ring structure.

Leukotrienes (LT) are potent vasoconstrictors in the pulmonary circulation. We have investigated the synthesis of LTC₄by intrapulmonary arteries and veins of perinatal lambs. Paired vessels of near-term fetal 146 ± 2- and 2- to 7-day-old newborn lambs (eachn= 7), were incubated for 10 min at 37°C in baseline, with 1 μmol/L A32187 or 0.1 mmol/L arachidonic acid. Produced leukotriene C₄was assayed from media by ELISA. Baseline production of leukotriene (ng/mg tissue, means ± SEM) by vessels for arteries was 0.006 ± 0.001 and 0.059 ± 0.009 for fetus and newborn, respectively. In veins, the values were 0.013 ± 0.003 and 0.073 ± 0.007 for fetus and newborn, respectively. On stimulation with the calcium ionophore A23187, production by arteries increased 25-fold in the fetus, but 4-fold in the newborn. The corresponding values for stimulated veins were 37-fold and 9-fold in fetus and newborn, respectively. Generally, production by veins was greater than production by the matching arteries. In all instances, the fetal vessels produced less leukotrienes than the newborn vessels. Western analysis of stimulated and unstimulated vessel membrane protein showed greater expression of 5-lipoxygenase in veins than in arteries (P< 0.05). Our data show that veins produce more LTC₄due to greater expression of 5-lipoxygenase in the vessels and thus suggest that veins of perinatal lamb lungs may be more susceptible to LT-induced vasoreactivity in the perinatal pulmonary circulation. We speculate that a higher production of LTC₄by fetal veins may be necessary to maintain a high venous tone in fetal lungs.

Two nuclear hormone receptor superfamily members, DAX1 and SF1, are required for normal adrenal cortical development. Mutations in DAX1 are responsible for X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism. Steroidogenic Factor 1 (SF1) regulates the expression of a number of steroidogenic genes and a putative SF1 response element (SF1-RE) in the DAX1 promoter which binds SF1 specifically. Therefore, we examined deletions in the DAX1 promoter driving expression of β-galactosidase, with and without coexpression of SF1, in the human adrenocortical carcinoma cell line NCI-H295. We defined the DAX initiation start site and localized the putative SF1-RE at −135 to −143 bp. Loss of the putative SF1-RE region or specific removal of the 9-bp SF1 site resulted in decreased transcriptional activity by 2.3- to 2.5-fold. When cotransfected with 1550 bp of the DAX1 promoter, an SF1-containing expression vector increased the transcriptional activity of the DAX1 promoter by 4-fold. No significant change above baseline occurred when the cells were cotransfected with the 1541-bp fragment containing the entire 1550-bp promoter region minus the 9-bp SF1-RE. We conclude that the SF1-RE is an enhancer element within the DAX1 promoter and speculate that SF1 may be a transcription factor that acts, at least in part, through DAX1 for normal adrenal cortical development.

In order to investigate the spinal muscular atrophy (SMA) disease processes, the expression of the survival motor neuron gene (SMN) has been analyzed in human fetal tissues using RT–PCR andin situhybridization. These studies allowed the detection of SMN RNA in all the examined tissues, with no significant variation between different developmental stages. In particular, SMN mRNA was detected in spinal cord (dorsal and ventral portions), skeletal muscle, lung, heart, kidney, liver, and spleen. Moreover, RT–PCR studies demonstrated that the expression pattern of SMN isoforms was similar to that observed in adult tissues. The present data confirm a housekeeping role for the SMN protein and may have implications on the search for early therapeutic strategies.

We used single-strand conformational polymorphism and direct nucleotide sequencing to identify a novel mutation in the cystathionine β-synthase (CBS) gene of two siblings with homocystinuria. Both patients are heterozygous carriers of the G₉₁₉A transition and the novel mutation which involves a G-to-A transition in the intron 12 splice donor site. Reverse transcription of RNA harvested from transformed lymphocytes followed by PCR showed a normal size product along with two shorter products involving the deletion of either exon 12 alone or both exons 11 and 12. To our knowledge, the skipping of more than one exon through a single base substitution at a splice-donor site has not been previously reported. The normal size splice product was found to have either a G or an A at nucleotide position 919, indicating that normal size mRNA was produced by both alleles.