UDP-Gal: <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.     

Induction of NADP-dependent malic dehydrogenase activity in brain of singi fish, heteropneustes fossilis (bloch), by 3,5,3′-triiodothyronine

For elucidation of thyroid hormone-induced responsiveness of fish brain, various doses (0.012, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2 and 4 μg/g) of triiodothyronine (T₃) were injected in Singi fish, Heteropneustes fossilis (Bloch), for 3 consecutive days and the changes in cytosolic NADP-dependent malic enzyme (ME, EC 1.1.1.40) activity in whole brain tissue were determined. Compared to the control, the ME activity increased with lower doses (0.012, 0.025 and 0.05 μg/g) and decreased with higher doses (1, 2 and 4 μg/g) of T₃, showing a biphasic nature of thyroid hormone action. The enzyme activity remained unaltered with 0.1, 0.25 and 0.5 μg of T₃/g in comparison to the control. Immersion of the fishes in cycloheximide-containing medium (0.5 mg/l) inhibited the T₃ (0.025 μg/g)-induced rise in ME activity. On the other hand, the NAD-dependent cytosolic malate dehydrogenase (EC 1.1.1.37) activity and the total protein content of brain cytosol remained unaltered with all doses of T₃ used. The thyroid hormone specificity of cytosolic NADP-dependent malic enzyme in fish brain is thus documented.     

Plant regeneration from mesophyll protoplasts of Diplotaxis muralis,a wild crucifer

Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2–7.1×10⁵ protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS₂ medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.Abbreviations BAP 6-benzylaminopurine - GA₃ Gibberellic acid - A Kao and Michayluk, 1981 - KM Kao and Michayluk, 1975 - MK₃ Modified K₃ - M8P Modified 8P - MS Murashige and Skoog, 1962 - NAA 1-naphthalene acetic acid - PE Plating efficiency     

NMR characterisation of a triple stranded complex formed by homo-purine and homo-pyrimidine DNA strands at 1:1 molar ratio and acidic pH.

Homo-purine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC) oligomers have been designed such that they are complementary in parallel orientation. When mixed in a 1:1 molar ratio, the system adopts an antiparallel duplex at neutral pH with three mismatched base pairs. On lowering the pH below 5.5, a new complex is formed. The NMR results show the coexistence of a intermolecular pyrimidine.purine:pyrimidine DNA triplex and a single stranded oligopurine at this pH. The triplex is stabilized by five T.A:T, four C+.G:C and two mismatched triads, namely, C+.G-T and T.A-C. This triplex is further stabilized by a Hoogsteen C+.G base-pair on one end. Temperature dependence of the imino proton resonances reveals that the triplex dissociates directly into single strands around 55 degrees C, without duplex intermediates. Parallel duplexes are not formed under any of the conditions employed in this study.     

Fluorescent multiplexing of 3D spheroids: Analysis of biomarkers using automated immunohistochemistry staining platform and multispectral imaging

In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non–small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells.     

A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome)

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.     

Enhancement of catalase activity by repetitive low-grade H2O2 exposures protects fibroblasts from subsequent stress-induced apoptosis

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H2O2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m2) or H2O2 (7.5mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H2O2. Cell viability was also significantly better preserved in VST cells than VC cells after UV or H2O2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H2O2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.     

Crisscross enzymatic reaction between the two molecules in the active dimeric P69 form of the 2'-5' oligodenylate synthetase

2'-5' oligoadenylate (2-5 (A)) synthetases are major components of the antiviral pathways induced by interferons. In the presence of double-stranded RNA, they polymerize ATP to form 2-5 (A) oligomers that, in turn, activate the latent ribonuclease RNase L, causing mRNA degradation. These enzymes, unlike other nucleotidyl transferases, catalyze 2'-5', not 3'-5', phosphodiester bond formation between substrates bound to the acceptor and donor sites. Moreover, unlike other members of this extended family, the P69 isozyme of 2-5 (A) synthetase functions as a homodimer. Here, we report that the need for P69 dimerization is because of a crisscross enzyme reaction joining two substrate molecules bound to two opposite subunits. Consequently, although homodimers of mutants in the previously identified acceptor site, the donor site, or the catalytic site were inactive, selective heterodimers of the mutants were active because of subunit complementation. The catalytic site had to be present in the same subunit that contained the acceptor site, whereas the donor site had to be provided by the other subunit. These results allowed us to design a mutant protein that acted as a dominant-negative inhibitor of wt P69 but not of another isozyme of 2-5 (A) synthetase.