Antigen-specific Th1 but not Th2 cells provide protection from lethal Trypanosoma cruzi infection in mice

Infection with Trypanosoma cruzi results in the development of both type 1 and type 2 patterns of cytokine responses during acute and chronic stages of infection. To investigate the role of Th1 and Th2 subsets of CD4(+) T cells in determining the outcome of T. cruzi infection in mice, we have developed T. cruzi clones that express OVA and have used OVA-specific TCR-transgenic T cells to generate OVA-specific Th1 and Th2 cells. BALB/c mice receiving 10(7) OVA-specific Th1 cells and then challenged with OVA-expressing T. cruzi G-OVA.GPI showed significantly lower parasitemia and increased survival in comparison to mice that received no cells. In contrast, recipients of OVA-specific Th2 cells developed higher parasitemias, exhibited higher tissue parasitism and inflammation, and had higher mortality than recipients of Th1 cells after infection with T. cruzi G-OVA.GPI. Mice receiving a mixture of both Th1 and Th2 OVA-specific cells also were not protected from lethal challenge. The protective effect of the OVA-specific Th1 cells was OVA dependent as shown by the fact that transfer of OVA-specific Th1 or Th2 cells failed to alter the course of infection or disease in mice challenged with wild-type T. cruzi. Immunohistochemical analysis of OVA-specific Th1 and Th2 cells at 4, 15, and 30 days postinfection revealed the persistence and expansion of these cells in mice challenged with T. cruzi G-OVA.GPI but not in mice infected with wild-type T. cruzi. We conclude that transfer of Ag-specific Th1 cells but not Th2 cells protect mice from a lethal infection with T. cruzi.     

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

Antimicrobial alkaloids from Zanthoxylum tetraspermum and caudatum

Two benzophenanthrene alkaloids, 8-acetonyldihydronitidine and 8-acetonyldihydroavicine were isolated from Zanthoxylum tetraspermum stem bark along with liriodenine, sesamin, lichexanthone and (+)-piperitol-gamma,gamma-dimethylallylether. The species endemic to Sri Lanka, Z. caudatum, contained sesamin, savinin, liriodenine, decarine and 8-O-desmethyl-N-nornitidine. 8-Acetonyldihydronitidine and 8-acetonyldihydroavicine showed significant antibacterial activity while the former along with liriodenine was strongly antifungal. Savinin exhibited potent spermicidal activity. Both savinin and sesamin exhibited significant insecticidal activity.     

Rapid isolation of plant Ty1-copia group retrotransposon LTR sequences for molecular marker studies

The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identified a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers specific for three pea LTRs have been used to reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.     

Purification of muscle enzymes by pseudoaffinity chromatography

A method based on pseudoaffinity chromatography has been developed for the separation of lactate dehydrogenase (LDH), pyruvate kinase (PK) and aldolase from rabbit muscle extract using cross-linked guar (CLG) and cross-linked pectin (CLP) as the matrices, and dyes as the ligands. Screening of several dyes revealed that dyes No. 1014 and No. 1015, immobilized on CLG and CLP displayed a higher affinity for LDH and PK. Aldolase was not retained on any of the dye columns. It was observed that 1014-CLP and 1014-CLG columns retained 90% and 55% LDH activities, respectively, whereas 1015-CLP and 1015-CLG retained 83% LDH and 72% PK. A coupled-column system comprising 1014-CLP and 1015-CLP or 1014-CLG and 1015-CLG could separate LDH, PK, and aldolase from a mixture of these enzymes, as well as from rabbit muscle extract. Enzymes were found to be homogeneous on polyacrylamide gel electrophoresis. The method has been found to be simple and economical.     

Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

Non-iron metalloporphyrins: potent antibacterial compounds that exploit haem/Hb uptake systems of pathogenic bacteria

A new group of potent antibacterial compounds, non-iron metalloporphyrins (MPs), is described. MPs possess a strong antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and mycobacteria. Anaerobically grown bacteria and microorganisms that do not respire and/or express haem uptake systems were resistant to MPs. Antibacterial activity of MPs was not affected by known antibiotic resistance mechanisms operating in bacteria. The most potent MP against Y. enterocolitica, methicillin-resistant S. aureus and M. smegmatis was gallium protoporphyrin IX (Ga-PPIX). When tested alone, Ga ions and metal-free porphyrins had approximately 100-fold higher minimum inhibitory concentration (MIC) values for these organisms. Ga-PPIX was not degraded by MP-sensitive bacteria, indicating that the whole molecule is responsible for antibacterial activity. MPs are antibacterial 'Trojan horses', as they exploit haem transport systems of Gram-negative bacteria as portals of entry into the cell. Bacterial mutants in superoxide dismutases, catalases and stationary-phase sigma factors were hypersensitive to Ga-PPIX. The extreme sensitivity of sod mutants to MPs and the requirement for active respiration for MP activity suggests that these compounds stimulate the production of reactive oxygen radicals in bacteria. Ga-PPIX was not toxic to primary human fibroblasts, several established cell lines and experimental animals at concentrations > 100-fold higher than the MIC for sensitive bacteria.     

The anhydrous milk fat, ghee, lowers serum prostaglandins and secretion of leukotrienes by rat peritoneal macrophages

Ghee, the anhydrous milk fat, is one of the most important sources of dietary fat in India. Male Wistar rats were fed diets containing 2.5, 5.0 and 10 wt% ghee for a period of 8 weeks. The diets were made isocaloric with groundnut oil. The results showed that serum thromboxane levels decreased by 27-35%, and 6-keto-prostaglandin F1alpha by 23-37% when ghee was incorporated at level of 10% in the diet. Prostaglandin E2 levels in serum and secretion of leukotrienes B4, C4 and D4 by peritoneal macrophages activated with calcium ionophore decreased when increased amounts of ghee from 2.5 to 10% were included in the diet. Arachidonic acid levels in macrophage phospholipids decreased when incremental amounts of ghee were fed to rats. These studies indicate that ghee in the diet not only lowers the prostaglandin levels in serum but also decreases the secretion of leukotrienes by macrophages.     

Purification and characterization of an extracellular polygalacturonase from the thermophilic fungus,Thermomyces lanuginosus

A polygalacturonase was purified from the thermophilic fungus, Thermomyces lanuginosus to apparent homogeneity by ultrafiltration, acetone precipitation and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60 °C. The apparent K_M with potassium pectate was 0.67 mg/ml and the V_max was 7.2 × 10⁵ mol/min/mg protein. The apparent molecular weight of the enzyme was 59 kDa and it contained approximately 10% carbohydrate. The enzyme was completely stable at room temperature (32 ± 3 °C) and retained about 50% activity at 50 °C for 6 h. The zymogram of the purified enzyme revealed two activity bands, one of which was a major one. Polyclonal antibodies raised against the enzyme did not show any immunological relatedness with other mesophilic polygalacturonases.