Polyamines in Trichomonas vaginalis

Trichomonas vaginalis was grown in a modified Bushby's medium and putrescine, spermidine and spermine levels were determined in extracts from 24- and 48-h cultures and also in the culture media. All three polyamines were present in T. vaginalis extracts; the putrescine level and putrescine/spermidine ratio were much higher than those reported for other protozoa or for mammalian tissues. There were no significant differences between 24-h and 48-h amine levels per mg protein in these extracts, but amine levels per cell were higher at 24 than at 48 h. The spent culture media had a much higher putrescine content than corresponding uninoculated media and it was concluded that T. vaginalis secreted putrescine into the culture medium.     

Allelic association with SNPs: metrics, populations, and the linkage disequilibrium map

Comparison of different metrics, using three large samples of haplotypes from different populations, demonstrates that rho is the most efficient measure of association between pairs of single nucleotide polymorphisms (SNPs). Pairwise data can be modeled, using composite likelihood, to describe the decline in linkage disequilibrium with distance (the Malecot model). The evidence from more isolated populations (Finland, Sardinia) suggests that linkage disequilibrium extends to 427-893 kb but, even in samples representative of large heterogeneous populations, such as CEPH, the extent is 385 kb or greater. This suggests that isolated populations are not essential for linkage disequilibrium mapping of common diseases with SNPs. The in parameter of the Malecot model (recombination and time), evaluated at each SNP, indicates regions of the genome with extensive and less extensive disequilibrium (low and high values of in respectively). When plotted against the physical map, the regions with extensive and less extensive linkage disequilibrium may correspond to recombination cold and hot spots. This is discussed in relation to the Xq25 cytogenetic band and the HFE gene region.     

Assessment of the role of "enkephalinase" in cholecystokinin inactivation

Cholecystokinin octapeptide and the C-terminal tetrapeptide are hydrolysed by a highly purified preparation of "enkephalinase" (EC 3.4.24.11). In both cases the Asp-PheNH2 bond is hydrolysed and the Gly4-Trp5 bond of the octapeptide is also cleaved, though more slowly. Evaluated from the appearance of Phe-NH2, the Km for the hydrolysis of the octapeptide by the purified peptidase is 57 microM and that for the tetrapeptide 65 microM. The apparent affinities of these peptides for the enzyme in striatal membranes are similar. The importance of this hydrolysis in the inactivation of endogenous cholecystokinin was assessed by studying the fate of cholecystokinin immunoreactivity released from slices of rat cerebral cortex and striatum by depolarization with potassium. In the absence of any peptidase inhibitor only 16% of the peptide released from the tissue was recovered in immunoreactive form in the medium, indicating that endogenous cholecystokinin octapeptide is, like other neuropeptides, rapidly and extensively hydrolysed following release. Selective inhibition of "enkephalinase" by Thiorphan (DL-3-mercapto-2-benzylpropanoyl glycine) did not significantly alter the recovery from slices of cerebral cortex and had only a very slight effect in the case of striatal slices. This suggests that, while cholecystokinin octapeptide is a substrate for "enkephalinase", this enzyme plays a less important (if any) role in the inactivation of endogenous cholecystokinin than for the opioid peptides.     

Substance P and somatostatin metabolism in sympathetic and special sensory ganglia in vitro

Mechanisms regulating the content of the putative peptide transmitters, substance P and somatostatin, were examined in several neuronal populations in culture. Substance P levels increased more than 25-fold within 48 h in sympathetic neurons in the explanted rat superior cervical ganglion, and remained elevated for 4 weeks. Identity of the peptide was authenticated by combined high pressure liquid chromatography-radioimmunoassay. Veratridine prevented the increase of substance P in vitro, and tetrodotoxin blocked the veratridine effect, suggesting that sodium ion influx and membrane depolarization prevent peptide elevation. Veratridine (or potassium)-induced membrane depolarization released substance P into the culture medium through a calcium-dependent process. Consequently, at least some veratridine effects are attributable to release and subsequent depletion of ganglion peptide. However, the inhibitory effects of veratridine were far greater than could be accounted for by the quantity of peptide released, suggesting a separate influence on net synthesis (synthesis less catabolism) of substance P. Viewed in conjunction with previous in vivo studies, our observations suggest that trans-synaptic impulses, through the mediation of postsynaptic sodium flux, release substance P from sympathetic neurons and also regulate intracellular peptide metabolism. To determine whether the processes regulating substance P in sympathetic neurons reflect generalized mechanisms, a different peptide, somatostatin, was examined in sympathetic neurons; moreover, substance P was examined in a different neuronal population, special sensory neurons in the nodose ganglion. Substance P levels increased significantly in both sympathetic and sensory neurons after explantation, and somatostatin levels increased in sympathetic neurons. In each instance, the increase was dependent upon the presence of the calcium ions. Moreover, these increases were all prevented by veratridine, in a tetrodotoxin-sensitive manner. Our observations suggest that common regulatory mechanisms govern peptide transmitter metabolism in diverse neuronal populations.     

A reversed-phase high-performance liquid chromatographic assay for the determination of N-acetylcysteine in aqueous formulations

A stability-indicating assay is described for the determination of N-acetylcysteine in aqueous pharmaceutical formulations. The sample is diluted to an appropriate concentration with dilute aqueous orthophosphoric acid. An aliquot of the solution, containing added l-tyrosine as an internal standard, is chromatographed using a 10-mum C(18) stationary phase with dilute orthophosphoric acid (pH 2.0) containing 0.5% w/v of sodium perchlorate as the mobile phase. The assay, which has a relative standard deviation of about 0.8%, can also be used as a test for related impurities in N-acetylcysteine. It is also suitable for determining the N-acetylcysteine content of the drug substance.     

Analysis of neomycins A, B and C by high-performance liquid chromatography with post-column reaction detection

The analysis of the antibiotics neomycins A, B and C was investigated. The separation of the components was studied using reversed-phase and reversed-phase ion-pair chromatography. The optimum separation was obtained utilizing a Lichrosorb RP-2 column with a mobile phase consisting of 75 mg/l sodium dodecyl sulphate, 0.5M Na2SO4 and 0.015 M sodium acetate buffer at pH 7.0. Using this mobile phase, baseline separation was obtained for all three compounds in approximately 20 min. Detection was via post-column derivatization of the analytes with ortho-phthalaldehyde in the presence of mercaptoethanol to form fluorescent iso-indole products. This system is applied to the analysis of a number of formulated products containing neomycin.     

Handling of biological samples in the determination of the anti-neoplastic drug mitomycin C

A study to ascertain suitable conditions for handling biological samples from patients, treated with the antibiotic mitomycin C (MMC), with the objective of improving the accuracy and reliability of the determination is described. Situations frequently occurring in medical practice are simulated to optimize procedures for reliable and reproducible sampling, sample treatment and determination of MMC. Continuation of drug partitioning in whole blood after sampling can be prevented by immediate cooling in ice before the separation of plasma from cells. The adjustment of the pH of urine samples is shown to be particularly important since a low urinary pH causes decomposition of MMC; moreover, it may decrease extraction recovery. Furthermore, long-term exposure of samples to daylight induces drug decomposition. Frozen storage of plasma and urine samples for periods greater than 3 weeks is to be avoided as this results in a considerable drop in MMC concentration. Repeated cycles of freezing and thawing are shown to have no effect upon the analytical results (6 cycles tested). The analysis of extracts of biological samples may take place up to at least 24 h after their preparation without measurable loss of analyte.     

Measurement of 5-HIAA levels in ventricular CSF (by LCEC) and in striatum (byin vivo voltammetry) during pharmacological modifications of serotonin metabolism in the rat

Summary The relationship between the concentrations of 5-hydroxyindoleacetic acid (5-HIAA) in the CSF and in the striatum has been evaluated in the rat by measuring the levels of this metabolite in ventricular CSF (by liquid chromatography coupled with electrochemical detection) and in the striatal extracellular fluid (byin vivo voltammetry) after administration of inhibitors of serotonin synthesis or degradation. Pargyline, NSD 1015 and-propyldopacetamide all caused an exponential decline of 5-HIAA in both CSF and striatum. For a given drug, the rate constants for 5-HIAA disappearance were identical in the CSF and in the striatal extracellular fluid. These results confirm the view that CSF 5-HIAA may serve as a good index of brain serotonin turnover.     

A single-strip mini-paper chromatographic method for rapid purity-control of 99mTc-labeled radiopharmaceuticals

A single-strip miniaturized paper chromatographic method (Mini-PC) was developed using 80%–90% acetone solvent for rapid purity-control of ^99mTc-radiopharmaceuticals. Routine ^99mTc-radiopharmaceuticals (eight kinds of kit made agents) and diluted agents (in which radiochemical impurities might be formed) were analyzed by Mini-PC and other methods. This showed that, compared with the other methods, the Mini-PC technique is useful for the simple and rapid routine analysis of radiochemical impurities of kit made ^99mTc-radiopharmaceuticals.