Alkaline phosphatase of cancerous larynx tissue in comparison with the placental enzyme. Biochemical and histochemical studies.

Tissue specimens of squamous cell carcinoma of the larynx from twenty patients were processed for histological and histopathological characterization. A histochemical study of alkaline phosphatase (ALP) was carried out using the simultaneous azo coupling method, and biochemical studies were performed using disodium phenylphosphate as substrate. Full-term, normal placentae were used for comparison. The specific activity of ALP from cancerous laryngeal tissue was 8.9 mKAU/mg protein compared with 154.7 mKAU/mg protein in the placenta. The ALP was localized histochemically in tumor cells (tumor-specific), blood vessels (vascular) and fibrous tissue (interstitial). The tumor-specific phosphatase was sensitive to inhibition by L-phenylalanine, L-leucine and to a lesser degree by L-tryptophan and levamisole. Placental ALP, on the other hand, was completely inhibited by levamisole, more resistant to leucine and more sensitive to phenylalanine and tryptophan. Biochemical estimation of ALP in cancerous laryngeal tissue combined with inhibition studies revealed that the tumor-specific activity of ALP constitutes 15% of the total ALP activity while the major isoenzyme was the vascular ALP, and around one-third of ALP activity was attributed to the interstitial enzyme. The characterization and localization of these isoenzymes are described and compared with that of the placenta. The significance and implications of the above findings are presented.

Heat-stable alkaline phosphatase. A putative tumor marker of head and neck squamous cell carcinoma.

Serum total alkaline phosphatase (AP) activity and heat-stable AP (HSAP) were investigated in patients with uncontrolled squamous cell carcinoma of the head and neck before and after treatment. No significant differences in AP activity were seen between normal subjects and cancer patients. However, the HSAP fraction of the total AP activity was significantly elevated prior to treatment and the level declined and remained low during successful treatment, while it increased with tumor progression or recurrences. Heat-stable AP was found to be a useful tumor marker of potential usefulness in the management of patients with squamous cell carcinoma of the head and neck.

Concomitant natural infection with L. donovani and L. major: a case report from Iraq.

This is a case report of kala-azar with cutaneous leishmaniasis. Upon admission, the patient had fever, hepatosplenomegaly and an ulcer on her cheek. The patient responded to Pentostam. Isoenzyme studies of parasite isolates from the bone marrow and from the cutaneous lesion revealed that these were L. donovani and L. major, respectively. This is the first report in Iraq of a proven concomitant infection with two species of leishmania parasites.

Axenic cultivation of amastigotes of Leishmania donovani and Leishmania major and their infectivity.

Two clones of promastigotes, one of Leishmania donovani and one of L. major, and an uncloned stock of L. major were axenically transformed to heat-shock amastigotes, at 35 and 37 degrees C, respectively. Of the four different culture media tested, a relatively cheap, liquid medium, RBLM, was found to be the best, both for the transformation of the promastigotes and the serial, axenic cultivation of the amastigotes. In an experiment of 30 days duration, serial cultivation, in an atmosphere with 5% CO2, was possible by subculturing every three days. There were significant differences in virulence in BALB/c mice between axenically-cultured amastigotes and promastigotes, both in terms of the weights, lengths and parasite burdens of the spleens of mice infected intraperitoneally (ip) with L. donovani or L. major and of the appearance, type and size of the cutaneous lesions which developed in mice given L. major by intradermal inoculation.

Solubilization and kinetic characterization of mitochondrial adenosine triphosphatase from Leishmania donovani promastigotes.

Oligomycin-sensitive particulate ATPase (MB ATPase) from L. donovani promastigotes was solubilized by chloroform treatment. Polyacrylamide gel electrophoresis revealed several protein bands, with the major one possessing ATPase activity. The solubilized enzyme had Mg2+-ATPase and Ca2+-ATPase but no K+-dependent alkaline phosphatase activity. The Mg2+-ATPase activity was stimulated by monovalent cations and was not sensitive to oligomycin. Hence it is referred to as F1 ATPase. It had optimum activity at pH 7.6 and 30 degrees C. The Arrhenius plot for MB ATPase was biphasic with activation energies (Ea) of 16.2 and 3.4 kcal mol-1, while F1 ATPase exhibited a linear plot with Ea = 10.1 kcal mol-1. Lineweaver-Burk plots were biphasic with Km values of 0.17 and 1.25 mM for MB ATPase and 0.18 and 1.33 mM for F1 ATPase. The enzyme could be preserved at -15 degrees C in Tris-SO2-(4)-EDTA-ATP-glycerol (t1/2 = 20 days).

Isolation and identification of coenzyme Q from Leishmania donovani.

Crystalline coenzyme Q (CoQ) was obtained from Leishmania donovani promastigotes by extraction of the unsaponifiable material with hexane, chromatography on a Florisil column and crystallization from ethanol. Crystalline CoQ in ethanol revealed an ultraviolet-peak, at 275 nm, which disappeared upon reduction with NaBH4. Polarographic and mass fragmentographic measurements were characteristic of CoQ homologs. Reversed-phase thin-layer chromatography revealed that it is CoQ9.

Regulation of aerobic fermentation in Leishmania donovani promastigotes by NADP+-dependent malic enzyme.

NADP+-dependent malic enzyme (decarboxylating) was extracted from Leishmania donovani promastigotes with Triton X-100. The enzyme was specific for NADP+ and did not decarboxylate oxaloacetate (OA). The substrate activity relationship was hyperbolic for both L-malate and NADP+, and Km values were calculated as 0.18 and 0.12 mM, respectively. The enzyme exhibited a broad pH optimum of 7.5-8.0. Pyruvate, NADPH and OA inhibited the reaction in a competitive manner with apparent Ki values of 0.2, 0.04 and 0.04 mM, respectively, while oxalate inhibition was of the mixed type. The kinetic results obtained indicate that malic enzyme is involved in the regulation of carbon flow towards aerobic fermentation, complete oxidation of dicarboxylic acids or biosynthetic purposes.

Numerical taxonomy of some Old World Leishmania species.

Iraqi Leishmania stocks originating from human cutaneous and visceral infections, others from the sandfly Sergentomyia baghdadis, and one visceral stock from a dog, were compared among themselves and with Old World reference stocks, by electrophoresis of 11 enzymes. 79 unit characters emerged. Paired comparison technique and complete-linkage cluster analysis revealed heterogeneity within the L. donovani complex. The Iraqi L. donovani stocks appear to be more closely related to an Ethiopian reference L. donovani than to an L. donovani infantum. Two Indian visceral stocks were linked to the L. donovani complex and the L. tropica complex by similarity indices of the same range. The results show a marginally closer association between L. tropica and L. donovani complexes than between L. tropica and L. major.

Purification and properties of cytochrome c555 from Leishmania donovani.

A hemoprotein present in minute quantities in Leishmania donovani promastigotes was isolated, purified and spectrally characterized as cytochrome c555. Physicochemical characterization revealed electrophoretic mobility of 1.6 X 10(-5) cm2 V-1 s-1, an isoelectric point (pI) of 9.9, a relative molecular weight (Mr) of 11.9 kDa and half-wave potential (Ep) of -1.058 V. Values obtained were compared with those of horse heart cytochrome c subjected to the same analyses. Heme c555 was also prepared and spectrally characterized. The results indicated that leishmanial cytochrome c555 is similar but not identical to its crithidial counterpart.

Affinity separation and partial characterization of serologically active Leishmania donovani antigens.

Leishmania donovani-soluble antigens capable of antibody production in rabbits were separated from the total antigenic make-up of the parasite by adsorption on to anti-Leishmania immunoglobulins coupled to CNBr-activated Sepharose 4B (ALIG). Immune rabbit sera were produced either by intravenous inoculation of living promastigotes or by intradermal and subcutaneous inoculation of soluble antigens. Approximately four times as much soluble antigen was bound to ALIG produced by i.v. than i.d. and s.c. inoculation although the percentage recovery was less. The antigenic fractions recovered were subjected to immunoelectrophoresis, isoelectric point and molecular weight determination. The results of affinity chromatography indicate high antigen-antibody interaction affinity, the recovery of at least 28 antigens (with 0.15 M NaCl, pH 11.0) and complete loss of serological activity (determined by immunoelectrophoresis) on treatment with 8 M urea.

Comparative diagnostic study of kala azar.

Sera from 58 children with hepatosplenomegaly were tested for kala azar by gel diffusion immunoelectrophoresis, countercurrent immunoelectrophoresis and micro-ELISA. The results are compared with those from bone marrow cultures: 40 cases were diagnosed as kala azar by the four immunological techniques and Leishmania parasites were grown in 37 of 40 bone marrow cultures. The techniques used are discussed in terms of sensitivity, specificity and simplicity. The micro-ELISA technique was the most sensitive and convenient to carry out and is strongly recommended for use in routine diagnosis and epidermiological surveys.

Isoenzyme characterization of Leishmania species from Iraq.

Leishmaniasis in Iraq takes both the visceral and cutaneous forms. The causative organisms are identified according to the electrophoretic variation of the enzymes MDH, GPI, 6PGD, PGM and IDH. The 37 visceral stocks investigated fall into two groups which differ only with respect to GPI. The six cutaneous stocks were divided into three groups. Group 3 represents Leishmania major, while groups 4 and 5 refer to L. tropica showing intraspecific variation with regard to 6PGD.

The primary isolation of Leishmania donovani from Iraq on different culture media.

Leishmania donovani was isolated by culturing bone marrow aspirates from kala-azar patients, on three types of culture media. The isolation was successful in 88% of the cases. The effects of pH, D-glucose, L-proline and antibiotic concentration in the isolation were investigated. The semi-solid medium used (pH 7.4) with L-proline, was better than the modified NNN media for the primary isolation.

Kinetics of lactate dehydrogenase and other enzyme studies in human serum in leukemia.

The activity of some of the clinically important enzymes was investigated in leukemic sera at 37 degrees, using the Beckman Enzyme Activity Analyzer were found to be slightly elevated in some untreated cases of leukemia (1.), while ALP was found to be frequently elevated. Untreated patients with l. had normal or below normal SCPK activity. The most characteristic and significant rise in activity, was found to be associated with SLDH and SHBDH in most cases of acute l. (86%) and in CML, while any elevation observed in CLL, was very slight. The general kinetic parameters of SLDH and SHBDH, were investigated at 37 degrees in acute leukemic patients. These included optimum substrate concentrations (NADH, pyruvate, and 2-oxobutyrate), the rate of pyruvate and 2-oxobutyrate reduction, substrate-velocity relationship, Km (pyruvate), Km (NADH), Km (2-oxobutyrate) as well as the effect of temperature and pH on the kinetics of the reaction. These kinetic characteristics were found to be differently affected by the leukemic process.

Further studies on lactate dehydrogenase from serum of leukemic individuals.

The kinetic properties of SLDH were investigated in untreated patients with acute leukemia at 37 degrees. SLDH was found to be inhibited by high pyruvate concentrations to a degree which depended on the pH of the reaction mixture; the degree of inhibition of normal SLDH at pH 7.5 was much higher than leukemic SLDH. 0.4M citrate was found to activate SLDH reaction rate at low pyruvate concentrations, while it was inhibitory at high pyruvate concentrations; the degree of inhibition being greater for leukemic SLDH than for normal SLDH. Citrate was also found to be a non-competitive inhibitor with respect to NADH and the value of Ki was determined. Methanol (3-4M), acted as a strong inhibitor to SLDH. The inhibition was found to be purely non-competitive with respect to pyruvate. Ki was calculated to be four times higher for leukemic SLDH than for normal SLDH.