Detection of IgG and IgM antibodies with ELISA technique in human trichomoniasis

The direct wet mount examination of vaginal secretion, widely applied for the diagnosis of Trichomonas vaginalis infection in woman patients, is rapid and economical, however, the sensitivity of this technique is not so high. In this study enzyme-linked immunosorbent assay (ELISA) was employed for the detection of serum anti-T. vaginalis IgG and IgM antibodies from 30 vaginal trichomoniasis patients and 30 non-infected healthy persons. The results were as follows: 1. Serum ELISA-IgG value was 0.37 +/- 0.134 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.21 +/- 0.054 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgG antibody were 70.0% and 96.7%, respectively. 2. Serum ELISA-IgM value was 0.33 +/- 0.177 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.11 +/- 0.051 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgM antibody were 70.0% and 96.7%, respectively. 3. The ELISA-IgG values showed a significant correlation with ELISA-IgM values (r = 0.77, p less than 0.005). With above results, it is assumed that ELISA is a reliable method for the diagnosis of T. vaginalis infection and simultaneous measurement of serum IgG and IgM with this technique is recommended.     

Maturation-associated loss and incomplete de novo synthesis of the transferrin receptor in peripheral sheep reticulocytes: response to heme and iron

Hemin, but not iron, in the culture medium stimulates the maturation-associated loss of the transferrin receptor from sheep reticulocytes (t1/2 for loss approximately 6 hr) and its appearance in a population of externalized vesicles. A similar pattern is seen with nucleoside binding (a measure of the nucleoside transporter), where hemin increases the loss of binding activity from the cells during culture, concomitant with an increase in nucleoside binding in the externalized vesicles. Sheep reticulocytes retain the ability to synthesize the transferrin receptor, but the 35S-labeled receptors are not detected in released vesicles. Whereas hemin stimulates the loss of 35S-labeled transferrin receptors from the cell (t1/2 for loss approximately 20 hr), nonheme iron is more effective than heme. This difference in response of native and 35S-labeled receptor to hemin and iron supplements appears to be related to the differences in the two classes of receptors. Although the 35S-labeled receptor binds transferrin and both native and 35S-labeled peptides comigrate after chemical deglycosylation, the 35S-receptor is approximately 2 kD smaller than the native receptor and fails to acquire its complete size even when chased for up to 24 hr. Moreover, the 35S-labeled receptor is not expressed at the cell surface, but is retained in a nonrecycling compartment, where it is insensitive to digestion by trypsin at both 0 degrees C and 37 degrees C.     

Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids

Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.     

Light Activation of Pyruvate, Orthophosphate Dikinase in Maize Mesophyll Chloroplasts: A Role for Adenylate Energy Charge

Pyruvate, orthophosphate dikinase (EC 2.7.9.1 [EC] ) was activatedin the light and inactivated following a dark treatment in intactmaize mesophyll chloroplasts. Addition of catalase (100–250units/ml) to the assay medium was necessary to obtain good activationand to keep the enzyme in an active state during illumination.Arsenate and carbonyl cyanide m-chlorophenyl-hydrazone, uncouplersof photophosphorylation, inhibited the activation. Pyruvate,which has been proposed to have a critical role in supportingthe light activation of pyruvate, orthophosphate dikinase, actuallyinhibited the activation. The pyruvate level in the chloroplastsuspension decreased when the enzyme was light-activated. Measurementsof adenylates and pyruvate in the chloroplasts indicated thatthe energy state of the chloroplasts was more important forthe light activation than was the level of pyruvate. ¹Present address: Department of Biochemistry, Faculty of Science,Saitama University, 255, Shimo-Okubo, Urawa, 338 Japan ²Present address: National Institute of Agrobiological Resources,Yatabe, Tsukuba, Ibaraki, 305 Japan (Received May 2, 1989; Accepted October 2, 1989)     

Phosphatidylethanolamine synthesis by castor bean endosperm : membrane bilayer distribution of phosphatidylethanolamine synthesized by the ethanolaminephosphotransferase and ethanolamine exchange reactions

A base exchange reaction for synthesis of phosphatidylethanolamine by the endoplasmic reticulum of castor bean (Ricinus comminus L. var Hale) endosperm has been examined. The calculated Michaelis-Menten constant of the enzyme for ethanolamine was 5 micromolar and the optimal pH was 7.8 in the presence of 2 millimolar CaCl₂. l-Serine, N-methylethanolamine and N,N-dimethylethanolamine all reduced ethanolamine incorporation, while d-serine and myo-inositol had little effect. These inhibitions of ethanolamine incorporation were found to be noncompetitive and ethanolamine also noncompetitively inhibited l-serine incorporation by exchange. The activity of the ethanolamine base exchange enzyme was affected by several detergents, with the best activity being obtained with the zwitterionic defjtergent 3-3-cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate.     

Redox titrations of carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Redox titrations of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum were performed using the reductant CO and the oxidant thionin. Titrations were followed at 420 nm, a wavelength sensitive to redox changes of the iron-sulfur clusters in the enzyme. When CODH was oxidized by just enough thionin to maximize A420, two molecules of CO per mole of CODH dimer (4 equiv/mol) reduced the enzyme fully. Likewise, 4 equiv/mol of thionin oxidized the fully-reduced enzyme to the point where A420 maximized. The four n = 1 redox sites which titrated in this region were designated group I sites. They include at least two iron-sulfur clusters, [Fe/S]A and [Fe/S]B, and two other sites, A' and B'. The [Fe4S4]2+/1+ cluster in CODH is included in this group. [Fe/S]B and B' have reduction potentials (at pH 8) below -480 mV vs NHE; [Fe/S]A and A' have reduction potentials above that value. The reduction potential of either [Fe/S]B or B' is near to the CO/CO2 couple at pH 8 (-622 mV). When CODH was oxidized by more than enough thionin to maximize A420, some of the excess thionin oxidized the so-called group II redox sites. These sites have reduction potentials more positive than group I and do not exhibit changes at 420 nm when titrated. Titration of group II sites required 1-2 equiv/mol. EPR of reduced group II sites exhibited the gav = 1.82 signal. When these sites were oxidized, the only signal present had g values at 2.075, 2.036, and 1.983.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sites of lymph follicle formation in the draining popliteal lymph nodes of mice locally injected with antigenic and mitogenic substances

Our previous studies showed that some antigenic and mitogenic substances, when locally injected into mice, efficiently produced new lymph follicles outside pre-existing follicles in draining lymph nodes, whereas others had virtually no effect. In the present experiments, young adult male mice were injected with several antigens and mitogens in the rear footpad, and the number and development sites of newly produced lymph follicles in the draining popliteal nodes were studied using serial sections of the nodes obtained between 5 and 21 days after injection. In the unstimulated state, each popliteal node contained a limited number of lymph follicles which mostly lay in a portion of the peripheral cortex overlaying the deep cortex (this portion is referred to as the PCOU), whereas a portion of the peripheral cortex extending beyond the deep cortex (referred to as the PCBU) was underdeveloped with only occasional follicles. Mice treated with soluble PHA or fluid tetanus toxoid developed germinal centers in association with existing follicles but failed to produce new follicles. The PCBU of the draining nodes remained underdeveloped, and the number and distribution pattern of lymph follicles within a draining node were comparable to those in the control node. Animals treated with LPS (50 micrograms), Con A, alum-precipitated PHA or alum-precipitated tetanus toxoid produced significantly large numbers of new follicles outside pre-existing follicles in the draining nodes, the new follicles produced in the PCBU being generally more numerous than those in the PCOU. In these draining nodes, the peripheral cortex, comprising a number of follicles, was found to overlie the deep cortex and extend beyond the deep cortex towards the hilar region. In animals given a less effective stimulant, such as ferritin or a smaller dose of LPS (10 micrograms), the draining nodes produced a relatively small number of new follicles, most of which were formed in the PCBU. The present results indicate that in the mouse popliteal node, the PCBU is morphologically underdeveloped under normal conditions, but develops lymph follicles in response to exogenous stimuli more readily than the PCOU, and that substances efficient in inducing follicle formation can be regarded as capable of stimulating the development of the peripheral cortex.     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)