Hepatic microsomal alterations during chronic trypanosomiasis in the field vole, Microtus montanus

The field vole, Microtus montanus, was used as a model system to evaluate the chronic effects of infection by Trypanosoma brucei gambiense on hepatic mixed-function oxidase activity. At day 28 post inoculation there was a 97% increase in liver wet weight per g body weight. A portion of the increase (21%) was accounted for by tissue edema which occurred after day 14 of infection. Total hepatic cytochrome P-450 content and related total tissue mixed-function oxidase activities were decreased to about 60% of control levels at day 28 post inoculation. The decrease in total tissue mixed-function oxidase activity was partly due to a small decrease in microsomal protein per cell, and partly to a large decrease in cytochrome P-450 concentration in the endoplasmic reticulum. Although the decrease in total liver monooxygenase activity in several substrates roughly paralleled the loss in cytochrome P-450 content, several other microsomal enzyme markers not related to cytochrome P-450 monooxygenation were elevated in proportion to total liver microsomal protein content. The results suggest that in M. montanus during trypanosomiasis, there is proliferation of hepatic cells with normal content of endoplasmic reticulum. Furthermore, there appears to be selective toxicity for hepatic cytochrome P-450 and related monooxygenase activities. This may compromise the animals' ability to metabolize and dispose of other drugs to which the animal may be exposed in the course of infection.     

Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the ¹²⁵I-proteins and ¹²⁵I-antigens compared with those of uninfected RBCs. New ¹²⁵I-proteins of M_r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M_r 230 000 and 72 000 bands being quantitatively the major new components. New ¹²⁵I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M_r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact ¹²⁵I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact ¹²⁵I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.     

Trypanosoma brucei brucei: Patterns of glycolysis at 37°C in vitro

In the absence of mammalian cells, freshly isolated monomorphic bloodstream forms of Trypanosoma brucei brucei maintain a constant and high level of aerobic glycolysis in vitro for at least 4 h at 37°C when suspended in RPMI medium 1640 containing 20% heat-inactivated and dialyzed fetal calf serum and 25 mM Hepes at an initial pH of 8. In the absence of nutrients other than glucose, salts and protein, some cell death and a decrease in the rate of glycolysis are observed. In the absence of protein, extensive cell death and a decrease in the rate of glycolysis are seen. These observations may be useful in the design of short-term in vitro metabolic studies with T. b. brucei.     

Purine and pyrimidine metabolizing activities in Trichomonas vaginalis extracts

Sixty-one purine and pyrimidine metabolizing activities were assayed in extracts of Trichomonas vaginalis. Of these, 43 were detected and quantitated. The only phosphoribosyltransfer activity observed was with uracil. No such activity was observed with adenine, guanine, hypoxanthine, xanthine or orotic acid. The rate of nucleoside cleavage was increased dramatically by the addition of inorganic phosphate. In addition, the extracts could catalyze the synthesis of ribonucleosides from the bases adenine, hypoxanthine, guanine and uracil but not cytosine, thymine or orotic acid, in the presence of ribose 1-phosphate. These data suggest that T. vaginalis contains primarily nucleoside phosphorylases instead of nucleoside hydrolases. Adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine, uridine, thymidine and GMP were phosphorylated in the presence of ATP. No nucleoside phosphotransferase activity was detected. Deamination of guanine, adenosine, deoxyadenosine, cytidine and deoxycytidine but not adenine was observed. These data suggest that salvage of adenine and guanine for ribonucleotide synthesis in T. vaginalis occurs via a phosphorylase/kinase pathway instead of through a phosphoribosyltransferase pathway which predominates in mammalian cells. In contrast, the pyrimidine base uracil can be converted to UMP via both a phosphoribosyltransferase or a phosphorylase/kinase pathway, analogous to that in mammals.     

Cell-free and cellular synthesis of chromogranin A and B of bovine adrenal medulla

We have studied the cell-free and cellular synthesis of chromogranins A and B, two immunologically distinct protein families of adrenal chromaffin granules. Two cell-free systems (wheat germ and reticulocyte lysate) were used for translating messenger RNA isolated from bovine adrenal medulla. Two primary translation products could be immunoprecipitated in case of chromogranin A. In the presence of microsomes the two chromogranin A precursors (pre-chromogranins A) were converted into a single protein, apparently by the removal of different signal peptides. For chromogranin B only one precursor (pre-chromogranin B) was translated. In isolated chromaffin cells only one chromogranin A protein was synthesized which corresponded to the processed cell-free translation product. During prolonged incubation this protein became slightly larger and more acidic, probably due to glycosylation in the Golgi region. Chromogranin B is post-translationally converted to a significantly more acidic protein. It is concluded that proteolytic breakdown of newly synthesized chromogranin A and B in chromaffin granules is a slow process comparable to that of the enkephalin precursors. It is not yet known what function these chromogranins have and whether breakdown to smaller subunits is necessary for any function to evolve.     

Proteinases of Leishmania mexicana amastigotes and promastigotes: Analysis by gel electrophoresis

The proteinases of Leishmania mexicana mexicana amastigotes and promastigotes have been analysed by electrophoresis on polyacrylamide gels containing denatured haemoglobin. Eleven bands of activity were detected indicating multiple proteinases. There were significant quantitative and qualitative differences between the proteinases of the two developmental forms. Four, B–E, were present in both forms but were of much higher activity in the amastigote. There were two major activities in promastigotes, A and D. The other proteinases, F–K, were of lower activity; I and K were not detected in promastigotes. All proteinases were active optimally at pH 4.0. Most of them, including the major proteinases A–E, were thiol proteinases since they were stimulated by 1 mM dithiothreitol and were sensitive to inhibitors such as HgCl₂, leupeptin, antipain and iodoacetic acid.     

Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

On the DNA content and ploidy of trypanosomes

We have determined the nuclear and kinetoplast DNA content of two trypanosomatids by quantitative absorption and fluorescence cytophotometry of individual Feulgen-pararosaniline stained cells. For the insect trypanosomatid Crithidia fasciculata we find nuclear and kinetoplast DNA contents of 0.095 and 0.032 pg per non-replicating cell. For the African trypanosome Trypanosoma brucei these values are 0.097 and 0.004 pg. A sub-population of T. brucei cells with two kinetoplasts and one nucleus was found to contain 0.181 pg/nucleus. The DNA values of bloodstream form T. brucei and the procyclic culture form were not significantly different.In DNA-DNA renaturation experiments the haploid amount of DNA in T. brucei was previously found to be 0.041 pg/nucleus (Borst, P., Fase-Fowler, F., Frasch, A.C.C., Hoeijmakers, J.H.J. and Weijers, P.J. (1980) Mol. Biochem. Parasitol. 1, 221–246). Our data, therefore, indicate that T. brucei is diploid. No sub-population of haploid cells was observed in T. brucei grown in rats or in culture.     

Inhibition of the in vitro growth of Plasmodium falciparum by D vitamins and vitamin D-3 derivatives

D vitamins are effective inhibitors of the in vitro intraerythrocytic growth of Plasmodium falciparum. Disappearance of the parasitemia was observed after 48 h contact between infected cells and 5 x 10(-6) M 1 alpha-hydroxycholecalciferol, 5 x 10(-5) M 25-hydroxycholecalciferol (25-OH-D-3), 1 alpha, 25-dihydroxycholecalciferol or 2.5 x 10(-4) vitamin D-2 and D-3. A 48 h pretreatment of healthy erythrocytes with 5 x 10(-5) M 25-OH-D-3 did not change their susceptibility to invasion by the parasite and their ability to support the growth of P. falciparum. Ionomycin, a calcium ionophore, and EGTA prevented parasite development at concentrations greater than 2 x 10(-7) M and 4 x 10(-4) M, respectively, but did not antagonize the inhibitory activity of 25-OH-D-3. Addition of 25-OH-D-3 for 12 or 24 h duration to synchronized cultures, showed that the drug had a schizonticidal action, but was without effect when parasites were in the ring form.     

Control of pyrimidine biosynthesis in the Ascaris ovary: regulatory properties of glutamine-dependent carbamoyl-phosphate synthetase and copurification of the enzyme with aspartate carbamoyltransferase and dihydroorotase

Glutamine-dependent carbamoyl-phosphate synthetase, the first enzyme of the de novo biosynthetic pathway for pyrimidine nucleotides, was purified about twenty-fold from 105 000 x g supernatant of the Ascaris ovary homogenate. The enzyme activity was feedback-inhibited by UDP and UTP while it was stimulated by 5-phosphoribosyl 1-pyrophosphate. Most of the catalytic and regulatory properties of the Ascaris synthetase were similar to those of the mammalian synthetase. A significant difference is that the Ascaris enzyme was more strongly inhibited by UDP than by UTP whereas the mammalian enzyme is more sensitive to UTP than to UDP. The Ascaris enzyme was also inhibited by other various nucleoside diphosphates, such as dUDP, dADP and CDP, generally more strongly than by the corresponding nucleoside triphosphates. Aspartate carbamoyltransferase and dihydroorotase, the second and third enzymes of the pathway, were also demonstrated in the supernatant fraction. These two enzymes were copurified with the synthetase and the relative activities of the three enzymes remained nearly constant (1:850-890:50-60) throughout the purification. In a sucrose gradient centrifugation, the enzymes cosedimented as a single peak with a sedimentation coefficient (s20,w) of about 32 S under the condition used. These results strongly suggest that the enzymes exist as a multienzyme complex similar to those found in higher animals. The activity of the carbamoyltransferase was insensitive to nucleotides and related compounds. These results indicate that the synthetase plays a key role in the control of pyrimidine biosynthesis in the Ascaris ovary.     

Purification of two structurally and morphologically distinct populations of rat neurohypophysial secretory granules.

A method for the subcellular fractionation of the neurosecretory granules of the rat Neurol lobe has been developed which employs density gradients of sucrose and metrizamide which are isoosmotic at 360m osmolality (osmol/kg solvent) (0.30 M sucrose). This procedure produces two populations of granules: one with an isopycnic density of ρ ≈ 1.13 containing granules of dia. 172 ± 2 (S.E.M.) nm and a second with density of ρ ≈ 1.11 containing granules of dia. 197 ± 9nm. The two populations contain oxytocin, vasopressin and the neurophysins. The denser granules are insensitive to osmotic change while the less dense particles behave like perfect osmometers. When compared to the tissue homogenate these fractions were purified 4–5-fold in relation to the mitochondria and 7-fold in relation to lysosomes. Chromatography of the gradient fractions on CPG 10–3000 porous glass beads results in further purification.In tissue from the neurohypophysis fixed as a whole, granules located in the nerve terminals and in dilated axonal segments (swellings) measure 173 ± 2 nm and 188 ± 2 nm respectively. It is proposed that one of the populations of granules isolated on the iso-osmotic gradient is derived from the nerve terminals whereas the other is derived from the axonal swellings. We suggest that the second population is formed from the first by a yet unspecified process.     

Development of cholinergic neurones in cultures of rat superior cervical ganglia. Role of calcium and macromolecules

Superior cervical ganglia from 3-day-old rats were grown in vitro for 14 days. Addition of an extract of rat heart to the culture medium led to no change in the activity of tyrosine hydroxylase but a doubling of the activity of choline acetyltransferase in the ganglia.To see whether this effect was due to a change in the concentration of unbound Ca²⁺, the calcium-binding capacity of extracts of adult rat heart was determined in a dialysis-binding study. Both cardiac extracts and serum lowered the levels of free calcium in the medium, but by relatively small amounts. When ganglia were grown in media containing 0, 2.5 and 6 mm Ca²⁺, there was no change in the ratio of choline acetyltransferase activity to tyrosine hydroxylase activity, although the activities of both enzymes increased at lower Ca²⁺ concentrations.When superior cervical ganglia were grown in Rose chambers under cellophane strips, the choline acetyltransferase/tyrosine hydroxylase ratio was higher than when superior cervical ganglia were loose in the medium. When a larger number of ganglia was grown under cellophane this further increased the ratio, while positioning of the ganglia so that substances could diffuse out from under the cellophane decreased the choline acetyltransferase/tyrosine hydroxylase ratio. When ganglia from 3-day-old rats were grown side by side with ganglia from 3-week-old rats, there was an even greater effect on the ratio of the two enzyme activities.We conclude that the ability of cardiac extracts to increase the ratio of choline acetyltransferase activity to tyrposine hydroxylase activity in ganglia is not due to changes in the Ca²⁺ concentration in the medium, but to the presence of a non-dialysable macromolecule. A similar factor also seems to be secreted by the ganglion itself.     

Molecular and immunological characterization of subtilisin like serine protease, a major allergen of Curvularia lunata

Serine protease from numerous sources have been identified and characterized as major allergens. The present study aimed to clone, express and characterize a serine protease from Curvularia lunata. cDNA library screening identified partial protease clones. A clone showed significant homology to subtilisin like serine proteases from Aspergillus and Penicillium species. Full length sequence was generated by RACE PCR, subcloned in pET vector, protein expressed in Escherichia coli and purified from inclusion bodies yielding 0.5 mg/L of culture. Bioinformatic analysis identified serine protease motifs of subtilase family, catalytic triad and N-glycosylation sites on the primary sequence. The protein resolved at 54-kDa on SDS-PAGE and was recognized as a major allergen on immunoblot with 13/16 C. lunata sensitive patients’ sera in ELISA and immunoblot. Recombinant protein reacted with rabbit polyclonal antibodies against alkaline serine proteases from C. lunata. Recombinant protein required 50-56 ng of same protein for 50% inhibition of IgE binding in competitive ELISA. In addition, 13 of 16 patients’ samples showed significant basophil histamine release upon stimulation with purified recombinant protein. In conclusion, a 54 kDa major allergen of C. lunata was cloned, expressed, characterized and showed biological activity. It has potential to be used in molecule based approach for allergy diagnosis and therapy.     

Olive oil polyphenols extracts inhibit inflammatory markers in J774A.1 murine macrophages and scavenge free radicals

Here we evaluate the olive oil antiradical and anti-inflammatory potential through its polyphenols extracts and examine the influence of olive maturity on olive oil quality properties, polyphenols composition and biological potentials. Samples have been obtained from minor Tunisian olive cultivars (Chemchali, Fouji and Zarrazi) at different maturity indices. Principal quality properties were evaluated and polyphenols analysis was carried out by Folin Ciocalteu reagent and HPLC-UV-MS. Antiradical activity was examined by DPPH and FRAP scavenging assays while J774A.1 murine macrophages were used to evaluate anti-inflammatory potential by analyzing NO production with Griess reagent method and iNOS and COX-2 expression by cytofluorimetric analysis. Our results revealed that quality characteristics, total phenol content, as well as phenolic compound concentrations were significantly affected by the olive maturity levels. On the other hand, the polyphenols extracts showed an interesting radical scavenging capacity and a potential ability to inhibit inflammatory markers at 90% for NO release and 75% for iNOS expression. Thus, our study establishes that olive oil through its polyphenols extracts has a substantial antiradical and anti-inflammatory potential. Likewise a lot of attention should be attributed to olive ripening level in order to decide the optimum harvesting time.     

A rapid quantitative staphylococcal co-agglutination assay. Utilization for the assay of bovine factor VIII-related antigen

A simple and rapid technique to measure bovine factor VIII-related antigen has been developed which utilizes protein A-bearing staphylococci and monospecific rabbit antiserum to bovine factor VIII. Staphylococci coated with a specific antibody agglutinate when they are mixed with the specific antigen. We have used an aggregometer to detect an quantitate the agglutination of the antibody-coated staphylococci. The assay has been optimized with respect to amount of antiserum needed for coating staphylococci, concentration of antibody-coated staphylococci, pH and ionic strength of the assay system, and stirring speed of the aggregometer. The staphylococcal co-agglutination assay as monitored by an aggregometer is at least 10 times more sensitive than the conventional slide agglutination method, and can detect as little as 0.1 microgram/ml of factor VIII antigen. It however, cannot be used to quantitate factor VIII-related antigen in plasma, since plasma contains some components which can non-specifically agglutinate staphylococci.     

Quantitative analysis of the expression of the glutamate–aspartate transporter and identification of functional glutamate uptake reveal a role for cochlear fibrocytes in glutamate homeostasis

There are several subtypes of fibrocyte in the spiral ligament and spiral limbus of the cochlea that may contribute to fluid homeostasis. Immunocytochemical data suggest that these fibrocytes possess the glutamate–aspartate transporter, GLAST, as do supporting cells around the hair cells. However, functional glutamate uptake has not been demonstrated in fibrocytes. We used confocal and post-embedding immunogold electron microscopy to confirm that GLAST is expressed in adult fibrocytes of CD-1 mice with a relative expression: spiral limbus fibrocytes>type II>V>IV>I spiral ligament fibrocytes. Because they were sparsely present in most samples, type III fibrocytes were assessed only in one sample where their GLAST levels were similar to type I. Type II, type V and spiral limbus fibrocytes have many fine cellular processes that increase their surface area, those of the latter two coming into direct contact with perilymph, and type V fibrocytes contain the most glutamate. These data imply that glutamate uptake occurs in the fibrocytes. We assessed uptake of d-aspartate (a glutamate analogue) together with GLAST expression immunocytochemically and electrophysiologically. d-Aspartate accumulated into GLAST expressing fibrocytes in vitro and evoked currents blockable by the GLAST inhibitor d,l-threo-β-benzyloxyaspartate (TBOA), similar to those of supporting cells around inner hair cells. Currents were strongest in spiral limbus fibrocytes, progressively lower in type V and type II fibrocytes, and were negligible in type I fibrocytes in accordance with the relative expression levels of GLAST. We conclude that in addition to their known homeostatic functions, fibrocytes, in particular spiral limbus, type II and type V fibrocytes play a role in glutamate homeostasis in the cochlea.     

Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach

Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the changes in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/microg of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions.     

A novel model of drug hapten-induced hepatitis with increased mast cells in the BALB/c mouse

Clinical evidence suggests that idiosyncratic hepatitis following administration of halogenated volatile anesthetics is mediated by autoimmune responses. No murine model to study mechanisms of anesthetic-induced or any other form of drug-induced idiosyncratic hepatitis exists. Anesthetics are believed to trigger hepatitis by covalently linking a trifluoroacetyl (TFA) chloride hapten to hepatic proteins, forming haptenated self-proteins. To test this hypothesis, we developed a hapten-induced model of hepatitis by immunization with syngeneic S100 liver proteins covalently coupled to TFA (TFA-S100). We found that TFA-S100 induced hepatitis was more severe than disease induced by S100 plus adjuvants or by the adjuvant alone and was characterized by neutrophil, mast cell, and eosinophil infiltration. TFA-specific IgG1 antibodies directly correlated with hepatitis, whereas S100 autoantibodies did not. TNF-alpha, IL-1beta, and IL-6 released from splenocytes collected 2 weeks after TFA-S100 inoculation were increased resembling the elevated serum cytokines reported in patients with autoimmune hepatitis (AIH). Three weeks after inoculation, the peak of hepatitis, we noted decreased numbers of Kupffer cells and lower levels of IL-6 and IL-10 in the liver, cytokines produced by Kupffer cells. This is the first report, to our knowledge, of a hapten-induced model of hepatitis with immune and autoimmune features.