Application of FT-IR spectroscopy for control of the medium composition during the biodegradation of nitro aromatic compounds

Previous studies showed that cabbage leaf extract (CLE) added to the growth medium can noticeably promote the degradation of nitro aromatic compounds by specific consortium of bacteria upon their growth. For further development of the approach for contaminated soil remediation it was necessary to evaluate the qualitative and/or quantitative composition of different origin CLE and their relevance on the growth of explosives-degrading bacteria. Six CLE (different by species, cultivars and harvesting time) were tested and used as additives to the growth medium. It was shown that nitro aromatic compounds can be identified in the FT-IR absorption spectra by the characteristic band at 1,527 cm⁻¹, and in CLE by the characteristic band at 1,602 cm⁻¹. The intensity of the CLE band at 1,602 cm⁻¹ correlated with the concentration of total nitrogen (R ² = 0.87) and decreased upon the growth of bacteria. The content of nitrogen in CLE differed (0.22–1.00 vol.%) and significantly influenced the content of total carbohydrates (9.50–16.00% DW) and lipids [3.90–9.90% dry weight (DW)] accumulated in bacterial cells while the content of proteins was similar in all samples. Though this study showed quantitative differences in the composition of the studied CLE and the response of bacterial cells to the composition of the growth media, and proved the potential of this additive for remediation of contaminated soil. It was shown that analysis of CLE and monitoring of the conversion of nitro aromatic compounds can be investigated by FT-IR spectroscopy as well as by conventional chemical methods.     

Immune interactions in malaria co-infections with other endemic infectious diseases: implications for the development of improved disease interventions

Today targeted research efforts are in progress with the goal to develop vaccines, microbicides, new drugs and alternative treatments for some of the neglected infectious diseases (NIDs). Until now the world is far from having effective cures and/or prophylactic vaccines in place. People living in endemic areas generally are more skewed towards a Th2 profile (i.e. anti-inflammatory) that could greatly affect the induction of an inflammatory Th2 type response needed to combat many infectious microorganisms. Despite this, very little is today known about how co-infections with NID can affect the outcome of the different diseases and the possibilities for prophylactic vaccination and treatment. Thus, if we are to intervene successfully to eradicate infections or prevent immune pathology either by vaccination or other immune intervention therapies it will be crucial to understand how co-infections with different pathogens affect the adaptive immunity and the establishment of immunological memory The aim of this paper is to review what is known about co-infection with malaria and certain other pathogens.     

Sex-specific effects of handling time on an index of immune activity in zebra finches

Recently, there has been considerable interest in the role of the immune system in shaping life-history evolution, sexual selection strategies, and indexes of individual quality. The most frequently used assay of immune function, particularly in avian field studies, is the phytohemagglunitin (PHA) skin test. PHA is injected subcutaneously into the wing web, and the magnitude of the resultant swelling has traditionally been interpreted as an index of an individual's cell-mediated immunocompetence. The test follows one of two protocols: the traditional two-wing injection protocol, with one wing web injected with PHA and the other with phosphate-buffered saline (PBS), or the simplified one-wing protocol that omits the PBS injection. In this technical comment, we alert researchers to the importance of considering handling time when performing the PHA test. We show that zebra finches (Taeniopygia guttata) subjected to the two-wing protocol had a lower wing-web swelling than individuals injected in one wing. In males, handling time explained over 50% of the variation in an individual's skin swelling response; females were relatively unaffected by handling time. We suggest that caution should be exercised when comparing the magnitude of wing-web swelling across studies in which the alternate protocol was followed. In addition, the recording of handling time, and its inclusion in subsequent statistical analyses, may aid in the detection of subtle differences across treatments.     

Cerebral malaria is frequently associated with latent parasitemia among the semi-immune population of eastern Sudan

The accurate diagnosis of malaria starts with clinical suspicion, confirmed by reliable laboratory results. A hospital-based study, described here, was carried out in a malaria mesoendemic area in eastern Sudan, where the inhabitants are semi-immune to malaria, and the fever threshold of parasitemia is not above the detection level of microscopy. Thus, we hypothesized that patients with symptoms highly suggestive of cerebral malaria (CM), but aparasitemic by microscopy, may have submicroscopic parasitemia. Patients in our malaria clinic were screened by microscopy, and 120 individuals were selected for the study, including febrile patients with and without microscopically detectable parasitemia, and apparently healthy individuals. In the two former groups there were patients with severe anemia and deep coma. Polymerase chain reaction (PCR) for parasite detection and ELISA tests for measuring serum antibody levels were carried out on all blood samples. A majority of the febrile patients who were parasite negative by microscopy showed the presence of a Plasmodium falciparum infection by PCR. The occurrence of P. falciparum infection with parasitemia below the detection level of microscopy was recognized more often in patients with CM symptoms than in those with severe malarial anemia (SMA), and in older rather than younger patients. Patients clinically suspected (CS) of having CM ((CS)CM) mostly were infected with a single clone, and a large proportion of them acquired antibodies (Abs) against merozoite surface protein (MSP) antigens (Ags). The therapeutic response to quinine treatment was comparable between patients with (CS)CM and CM. In conclusion, uniquely in this setting, CM can be associated with sub-patent parasitemia; thus, a diagnostic tool more sensitive than microscopy is needed.     

Plasma factors required for human apolipoprotein A-II dimerization

Although plasma high-density lipoproteins (HDL) have been implicated in several cardioprotective pathways, the physiologic role of apolipoprotein (apo) A-II, the second most abundant of the HDL proteins, remains ambiguous. Human apo A-II is distinguished from most other species by a single cysteine (Cys6), which forms a disulfide bond with other cysteine-containing apos. In human plasma, nearly all apo A-II occurs as disulfide-linked homodimers of 17.4 kDa. Although dimerization is an important determinant of human apo A-II metabolism, its mechanism and the plasma and/or cellular sites of its dimerization are not known. Using SDS-PAGE and densitometry we investigated the kinetics of apo A-II dimerization and observed a slow (t(1/2) = approximately 10 days), second-order process in Tris-buffered saline. In 3 M guanidine hydrochloride, which disrupts apo A-II secondary structure and self-association, the rate was 3-fold slower. In contrast, lipid surfaces that promote apo A-II alpha-helix formation and lipophilic interaction profoundly enhanced the rate. Reassembled HDL increased the second-order rate constant k(2) by 7500-fold, unilamellar 1-palmitoyl-2-oleoylphosphatidylcholine vesicles increased k(2) 850-fold, and physiological concentrations of human serum albumin increased k(2) 220-fold. Thus, while dimerization of apo A-II in aqueous buffer is too slow to account for the high fraction of dimer found in plasma, lipids and proteins "catalyze" dimer formation, a process that could occur either intracellularly prior to secretion or in the plasma compartment following secretion. These data suggest that formation of disulfide links within or between polypeptide chains can be controlled, in part, by coexisting lipids and proteins.     

Thymic shared antigen-2: a novel cell surface marker associated with T cell differentiation and activation

Thymic shared Ag-2 (TSA-2) is a 28-kDa, glycophosphatidylinitosol-linked cell surface molecule expressed on various T cell and thymic stromal cell subsets. It is expressed on most CD3-CD4-CD8-, CD4+CD8+, and CD3highCD4-CD8+ thymocytes but is down-regulated on approximately 40% of CD3highCD4+CD8- thymocytes. Expression on peripheral TCR-alphabeta+ T cells is similar to that of CD3+ thymocytes, although a transient down-regulation occurs with cell activation. Consistent with the recent hypothesis that emigration from the thymus is an active process, recent thymic emigrants are primarily TSA-2-/low. TSA-2 expression reveals heterogeneity among subpopulations of CD3highCD4+CD8- thymocytes and TCR-gamma delta+ T cell previously regarded as homogenous. The functional importance of TSA-2 was illustrated by the severe block in T cell differentiation caused by adding purified anti-TSA-2 mAb to reconstituted fetal thymic organ culture. While each CD25/CD44-defined triple-negative subset was present, differentiation beyond the TN stage was essentially absent, and cell numbers of all subsets were significantly below those of control cultures. Cross-linking TSA-2 on thymocytes caused a significant Ca2+ influx but no increase in apoptosis, unless anti-TSA-2 was used in conjunction with suboptimal anti-CD3 mAb. Similar treatment of mature TSA-2+ T cells had no effect on cell survival or proliferation. This study reveals TSA-2 to be a functionally important molecule in T cell development and a novel indicator of heterogeneity among a variety of developing and mature T cell populations.     

Tumour inhibitory effects of TCGF/IL-2/-containing preparations

Summary Supernatants from ConA-stimulated rat spleen cell cultures and from cultures of PMA-stimulated murine lymphoma subline EL-4TF were found to contain TCGF and to inhibit growth of a transplantable, MC-induced sarcoma MC11 in syngeneic mice. Tumour-inhibitory effects of the supernatants were dependent on local and repeated administration. Prior to use of the supernatants obtained from PMA-stimulated EL-4TF cell cultures, the dialysable PMA had to be removed; contamination with PMA was found to abolish the tumour-inhibitory effect of the supernatants and to produce enhancement of tumour growth. A significant tumour-inhibitory effect has also been obtained with partially purified TCGF prepared from culture supernatants of cloned EL-4TF cells by ammonium sulphate precipitation, ion-exchange (FPLC) chromatography, and AcA 44 Ultrogel filtration.Abbreviations TCGF T cell growth factor - IL-2 interleukin 2 - Con A concanavalin A - ³H-TDR tritiated thymidine - PMA 4--phorbol-12--myristate-13--acetate - NK natural killer cells - B10 C57BL10/ScSnPH mice - FPLC fast protein liquid chromatography - BSA bovine serum albumin - OVA ovalbumin - SBI trypsin inhibitor from soybean - Cyt C cytochrome C - OD optical density - PBS phosphate-buffered saline - MC 3-methylcholanthrene     

Decreased Synthesis of Glycosphingolipids in Cells Lacking Vimentin Intermediate Filaments

We are studying defects in glycosphingolipid synthesis in cells lacking vimentin intermediate filaments (vimentin−). Sugars can be incorporated into glycolipids whose ceramide is synthesized eitherde novo(pathway 1) or from sphingoid bases salvaged from hydrolysis of sphingolipids (pathway 2) and into glycolipids recycling from the endosomal pathway through the Golgi (pathway 3). Vimentin− embryonic fibroblasts, obtained from vimentin-knockout mice, incorporate less sugar into glycolipids than vimentin+ fibroblasts. Using two inhibitors of ceramide synthesis, β-chloroalanine and fumonisin B1, we found the major defect in synthesis to be in pathway 2 and not inde novosynthesis. We used two additional approaches to analyze the functions of pathways 2 and 3. First, we used exogenous glucosylthioceramide ([¹⁴C]C₈-Glc-S-Cer), a synthetic, nonhydrolyzable glycosphingolipid, as a precursor for synthesis of larger glycolipids. Vimentin− SW13 cells and embryonic fibroblasts glycosylated [¹⁴C]C₈-Glc-S-Cer less extensively than their vimentin+ counterparts. Second, we used chloroquine to inhibit the hydrolysis of sphingolipids in endosomes and lysosomes. Chloroquine markedly decreased the incorporation of sugars into glycolipids larger than glucosylceramide. The defect in glycolipid synthesis in vimentin− cells probably results from impaired intracellular transport of glycolipids and sphingoid bases between the endosomal/lysosomal pathway and the Golgi apparatus and endoplasmic reticulum. Intermediate filaments may accomplish this function by contributing to the organization of subcellular organelles and/or by binding proteins that participate in transport processes.     

Plasmodium falciparum: differential parasite reactivity of rabbit antibodies to repeated sequences in the antigen Pf155/RESA

For selection of immunogens capable of inducing high levels of antibodies reactive with the Plasmodium falciparum antigen Pf155/RESA, rabbits were immunized with synthetic peptides corresponding to sequences based on the repeat subunits EENVEHDA and (EENV)2 from the C-terminus of this antigen. The antibodies obtained were analyzed with regard to binding to synthetic peptides in ELISA and to reactivity with parasite antigens by immunofluorescence or immunoblotting. All antisera reacted with both the peptides EENVEHDA and (EENV)2 as well as with Pf155/RESA. Antibody fractions specific for each of the two peptides were prepared by affinity chromatography on insolubilized peptides. Strong reactivity with antigens in the membrane of erythrocytes infected with early stages of the parasite as well as reactivity with Pf155/RESA in immunoblotting correlated with reactivity of antibody with (EENV)2. Antibody preparations reactive with EENVEHDA and depleted of (EENV)2 reactivity showed only a weak reactivity with Pf155/RESA but reacted also with P. falciparum polypeptides of 250, 210, and 88 kDa. In immunofluorescence, these antibodies stained mainly the intraerythrocytic parasite. Both EENVEHDA- and (EENV)2-specific antibodies inhibited merozoite reinvasion in P. falciparum in vitro cultures, the latter antibodies being the most efficient. This study defines the specificity and cross-reactivity with other P. falciparum antigens of antibodies to the C-terminal repeats of Pf155/RESA.     

Arylamidases of rat liver and chemically induced hepatomas. 1. Subcellular distribution of L-leucine. 2. Naphthylamidase-active antigens.

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using L-leucine 2-naphthylamide as substrate. Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase). The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal and cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.