A 7.1 kb linear DNA molecule of Theileria parva has scrambled rDNA sequences and open reading frames for mitochondrially encoded proteins.

Theileria parva, an intralymphocytic protozoan parasite of cattle, contains a linear 7.1 kb DNA element with terminal inverted repeat sequences. The molecule is transcribed into low molecular weight RNA, and both DNA strands encode short stretches of unique sequences, usually < 100 nucleotides, which are similar to large (LSU) or small (SSU) ribosomal subunit RNA. Phylogenetically conserved conformational rRNA domains were assembled from the discontinuous rDNA sequences using comparative secondary structure modelling. For example, a minimum of four predicted sequences, two derived from each DNA strand, is required to assemble domain V of LSU rRNA which participates in peptidyl transferase activity. The discontinuities in the identified rRNA domains fall within regions of no known functional significance. Hence, it is likely that the element encodes fragmented rDNA genes and the mature rRNA is unconventional, consisting of several fragments of RNA, primarily held together by intermolecular and intramolecular base pairing. The element also has ORFs for components of the last two mitochondrial electron transport enzyme complexes. The structure of the parasite DNA element, its protein coding capacity and scrambled rDNA gene sequences, are reminiscent of the mitochondrial genome of Chlamydomonas reinhardtii. We propose that the 7.1 kb element is equivalent to the mitochondrial DNA of T. parva, although a number of its features are unusual for this family of extrachromosomal DNA molecules.     

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.     

A novel cyclomaltodextrin glucanotransferase from Bacillus firmus that degrades raw starch

Summary An alkalophilic Bacillus firmus secreting the enzyme cyclomaltodextrin glucanotransferase was isolated from soil. The enzyme attacked raw starch to produce cyclodextrins. Maximum cyclodextrins were produced from tapioca starch followed by potato and corn starch. About 49 % of tapioca starch (at 10 and 50 g/l) was converted to cyclodextrins. The main reaction products were and -cyclodextrins with 40 % and 8 % yield respectively. On prolonged incubation small amount of -cyclodextrin was also produced. The ratio of cyclodextrins was dependent on the initial substrate concentration as well as reaction time.NCL communication number 6203     

Cloning and expression inEscherichia coli of the sucrase gene fromBacillus subtilis

Summary A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank ofE. coli harboring recombinant cosmids representative of theB. subtilis genome. It was shown that thesacA gene is located in a 2 kbEcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2 kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for inB. subtilis andE. coli. Complementation of asacA mutation was observed in Rec⁺ and Rec^- strains ofB. subtilis. Expression of sucrase was also demonstrated inE.coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic inE. coli since the presence of the recombinant plasmids did not allow the transport of [U¹⁴C] sucrose and the growth of the cells.It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport ofB. subtilis.     

Development of sero-diagnostic and molecular tools for the control of important tick-borne pathogens of cattle in Africa

Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan Africa. ILRI is focussing on developing a range of products, such as vaccines, diagnostics and decision support services to underpin improved control programmes against these diseases. We have developed three highly sensitive and specific enzyme linked immuno-assays (ELISAs), which allow precise diagnosis of Theileria parva, Babesia bigemina and Anaplasma marginale. These tests have been standardised and validated using defined experimental and field infection sera. Parasite specific recombinant antigens and monoclonal antibodies against bovine immunoglobulins as secondary antibodies have played an important role in in enhancing the sensitivity and specificity of the assays. They have been further evaluated in on-farm longitudinal sero-epidemiological studies to define infection dynamics and disease risks in various farming systems in Kenya and Uganda. In addition, DNA-based tests for differentiation of Theileria species and characterisation of Theileria parva stocks have been developed. These tests have been derived through physical mapping and sequencing of key elements of the T. parva genome, which include repetitive and telomeric regions, minisatellite sequences, antigen genes and a number of random DNA sequences. These tools are currently being deployed in conjunction with field immunisation programmes to determine the biological impact of introducing live vaccines of T. parva on population dynamics.     

Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574

The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L(-1)) and achieved a critical micelle concentration (CMC) value of 120 mg L(-1).     

Characterization of the Theileria parva sporozoite proteome

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC–MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact.     

Exploiting Phosphate-Starved cells of <Emphasis Type="Italic">Scenedesmus</Emphasis> sp. for the Treatment of Raw Sewage

Phosphate depletion is one of the favorable ways to enhance the sewage water treatment with the algae, however, detailed information is essential with respect to internal phosphate concentration and physiology of the algae. The growth rate of the phosphate-starved Scenedesmus cells was reduced drastically after 48 h. Indicating cells entered in the stationary phase of the growth cycle. Fourier Transform Infrared analysis of phosphate-starved Scenedesmus cells showed the reduction in internal phosphate concentration and an increase in carbohydrate/phosphate and carbohydrate/lipid ratio. The phosphate-starved Scenedesmus cells, with an initial cell density of, 1 × 10⁶ cells mL^?1 shows 87% phosphate and 100 % nitrogen removal in 24 h. The normal Scenedesmus cells need approximately 48 h to trim down the nutrients from wastewater up to this extent. Other microalgae, Ankistrodesmus, growth pattern was not affected due to phosphate starvation. The cells of Ankistrodesmus was able to reduce 71% phosphate and 73% nitrogen within 24 h, with an initial cell density of, 1 × 10⁶ cells mL^?1.