Peutz-Jeghers Syndrome: Confirmation of Linkage to Chromosome 19p13.3 and Identification of a Potential Second Locus, on 19q13.4

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease with variable expression and incomplete penetrance, characterized by mucocutaneous pigmentation and hamartomatous polyposis. Patients with PJS have increased frequency of gastrointestinal and extraintestinal malignancies (ovaries, testes, and breast). In order to map the locus (or loci) associated with PJS, we performed a genomewide linkage analysis, using DNA polymorphisms in six families (two from Spain, two from India, one from the United States, and one from Portugal) comprising a total of 93 individuals, including 39 affected and 48 unaffected individuals and 6 individuals with unknown status. During this study, localization of a PJS gene to 19p13.3 (around marker D19S886) had been reported elsewhere. For our families, marker D19S886 yielded a maximum LOD score of 4.74 at a recombination fraction (theta) of .045; multipoint linkage analysis resulted in a LOD score of 7.51 for the interval between D19S886 and 19 pter. However, markers on 19q13.4 also showed significant evidence for linkage. For example, D19S880 resulted in a maximum LOD score of 3.8 at theta = .13. Most of this positive linkage was contributed by a single family, PJS07. These results confirm the mapping of a common PJS locus on 19p13.3 but also suggest the existence, in a minority of families, of a potential second PJS locus, on 19q13.4. Positional cloning and characterization of the PJS mutations will clarify the genetics of the syndrome and the implication of the gene(s) in the predisposition to neoplasias.     

Fibronectin Fibrils and Growth Factors Stimulate Anchorage-Independent Growth of a Murine Mammary Carcinoma

Stromal cells are important regulators of mammary carcinoma growth and metastasis. We have previously shown that a 3T3-L1 adipocyte cell line secretes hepatocyte growth factor (HGF), which stimulates proliferation of a murine mammary carcinoma (SP1) in monolayer cultures (DNA Cell Biol.13, 1189–1897, 1994). We now examine the role of growth factors and the extracellular matrix protein fibronectin in stimulation of anchorage-independent growth of SP1 cells. Purified transforming growth factor-β (TGF-β) stimulated significant colony growth in soft agar cultures, whereas HGF had a lesser effect. Analysis by confocal microscopy revealed that carcinoma cell colonies contained extracellular microfibrils composed of fibronectin. Partial depletion of fibronectin from 7% FBS/agar cultures reduced the number of colonies; colony growth could be recovered by adding back exogenous fibronectin. Addition of the 70-kDa N-terminal fragment of fibronectin, which inhibits fibronectin fibril formation, reduced growth of SP1 cell colonies, but an 85-kDa fragment containing the cell binding domain did not inhibit colony growth. These findings indicate that deposition of extracellular fibronectin fibrils is necessary, but not sufficient, for anchorage-independent growth of SP1 mammary carcinoma cells; growth factors are also required. SP1 cells had less fibronectin mRNA and secreted less fibronectin protein under anchorage-independent conditions than under anchorage-dependent conditions, as determined by Northern blotting and immunoprecipitation analysis. Thus, both growth factors (HGF and TGF-β) and fibronectin may be important regulators of paracrine stimulation by stromal cells of anchorage-independent growth of mammary carcinoma cells.     

Functional Expression of a Thermostable Endoglucanase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> RCKK in <Emphasis Type="Italic">Pichia pastoris</Emphasis> X-33 and Its Characterization

Thermostable cellulases offer several advantages like higher rates of substrate hydrolysis, lowered risk of contamination, and increased flexibility with respect to process design. In the present study, a thermostable native endoglucanase nEG (EC 3.2.1.4) was purified and characterized from T. aurantiacus RCKK. Further, it was cloned in P. pastoris X-33 and processed for over expression. Expression of recombinant endoglucanase (rEG) of molecular size ~?33 kDa was confirmed by SDS-PAGE and western blotting followed by in gel activity determination by zymogram analysis. Similar to nEG, the purified rEG was characterized to harbor high thermostability while retaining 50% of its initial activity even after 6- and 10-h incubation at 80 and 70 °C, respectively, and exhibited considerable stability in pH range 3.0–7.0. CD spectroscopy revealed more than 20% β-sheets in protein structure consistently when incubated upto 85 °C as a speculated reason for protein high thermostability. Interestingly, both nEG and rEG were found tolerant up to 10% of the presence of 1-ethyl-3-methylimidazolium acetate [C2mim][OAc]. Values of the catalytic constants K_m and V_max for rEG were recorded as 2.5 mg/ml and 303.4 µmol/mg/min, respectively. Thermostability, pH stability, and resistance to the presence of ionic liquid signify the potential applicability of present enzyme in cellulose hydrolysis and enzymatic deinking of recycled paper pulp.     

Temporal distribution,source apportionment,and pollution assessment of metals in the sediments of Beas river,India

Metal pollution in the fluvial sediments of the rivers has become a severe problem throughout the world. Lower valleys with agricultural land are usually receiving diverse contaminants from various sources that are characterized by constant, bioaccumulative and harmful contaminants. One example is of the Beas river (India) which shows the distribution, contamination, and pollution status of metals in the sediments. In the present study, a cluster analysis and principal component analysis were conducted to assess the impact of anthropogenic activities in sediment metals. Results of contamination factor indicate that there is low contamination, whereas enrichment factor showed that 28.5% metals showed significant pollution during pre-monsoon season, 42.8% metals showed moderate to high pollution during post-monsoon season, and 42.8% metals showed moderate to extreme pollution during the winter season. Pollution index and modified pollution index showed that sediments in the winter season are slightly and critically polluted. The ecological risk factors indicate that 23.8% metals posed moderate to high ecological risk in the sediments.     

Seedling and adult‐plant stage resistance of a world collection of barley genotypes to stripe rust

Barley stripe rust caused by Puccinia striiformis f.sp. hordei (PSH) is one of the major diseases in barley production regions worldwide. A total of 336 barley genotypes with diverse genetic backgrounds targeted for low‐input barley production were tested for seedling and adult‐plant stage resistance against six PSH races (0S0, 0S0‐1, 1S0, 4S0, 5S0 and 7S0) originated from India. The seedling resistance was evaluated by inoculating the barley genotypes with six races separately under controlled conditions in Shimla, India. The same barley genotypes were evaluated for adult‐plant stage resistance in the Agricultural Research Station (ARS) of Rajasthan Agriculture University, Durgapura, Rajasthan, India. Out of the 336 barley genotypes tested for seedling resistance, 119 (35.4%), 101 (30.1%), 87 (25.9%), 100 (29.8%), 91 (27.1%) and 70 (20.8%) genotypes were resistant to races 0S0, 0S0‐1, 1S0, 4S0, 5S0 and 7S0, respectively. In the field, 102 (30.3%) genotypes showed the resistance response of which 18 (5.3%) genotypes were highly resistant to PSH. Barley genotypes AM‐14, AM‐177, AM‐37, AM‐120, AM‐300, AM‐36, AM‐103, AM‐189, AM‐291, AM‐275 and AM‐274 showed resistance response to all six races at seedling and adult‐plant stages. Seedling resistance reported in the current study is effective against the newly emerged race 7S0 and previously reported five races in India. Therefore, resistant barley genotypes identified in the current study provided effective protection against all six races at seedling and adult‐plant stages. The stripe rust resistance identified in the current studies may be potential donors of stripe rust resistance to barley breeding programmes in India and elsewhere.     

5-Hydroxy-7-Methoxyflavone Triggers Mitochondrial-Associated Cell Death via Reactive Oxygen Species Signaling in Human Colon Carcinoma Cells

Plant-derived compounds are an important source of clinically useful anti-cancer agents. Chrysin, a biologically active flavone found in many plants, has limited usage for cancer chemotherapeutics due to its poor oral bioavailability. 5-Hydroxy-7-methoxyflavone (HMF), an active natural chrysin derivative found in various plant sources, is known to modulate several biological activities. However, the mechanism underlying HMF-induced apoptotic cell death in human colorectal carcinoma cells in vitro is still unknown. Herein, HMF was shown to be capable of inducing cytotoxicity in HCT-116 cells and induced cell death in a dose-dependent manner. Treatment of HCT-116 cells with HMF caused DNA damage and triggered mitochondrial membrane perturbation accompanied by Cyt c release, down-regulation of Bcl-2, activation of BID and Bax, and caspase-3-mediated apoptosis. These results show that ROS generation by HMF was the crucial mediator behind ER stress induction, resulting in intracellular Ca²⁺ release, JNK phosphorylation, and activation of the mitochondrial apoptosis pathway. Furthermore, time course study also reveals that HMF treatment leads to increase in mitochondrial and cytosolic ROS generation and decrease in antioxidant enzymes expression. Temporal upregulation of IRE1-α expression and JNK phosphorylation was noticed after HMF treatment. These results were further confirmed by pre-treatment with the ROS scavenger N-acetyl-l-cysteine (NAC), which completely reversed the effects of HMF treatment by preventing lipid peroxidation, followed by abolishment of JNK phosphorylation and attenuation of apoptogenic marker proteins. These results emphasize that ROS generation by HMF treatment regulates the mitochondrial-mediated apoptotic signaling pathway in HCT-116 cells, demonstrating HMF as a promising pro-oxidant therapeutic candidate for targeting colorectal cancer.     

Structural Plasticity of the Protein Plug That Traps Newly Packaged Genomes in Podoviridae Virions

Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal “trimer of hairpins” tip. Although the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than 200 genomes of P22-like phages and prophages. In this paper, we used x-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620, and Sf6. In all cases, we found that the N-terminal tip is poorly structured, in stark contrast to the compact trimer of hairpins seen in gp26 crystallized at acidic pH. Hydrogen-deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy, and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer of hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome packaging, and a postejection trimer of hairpins, which forms upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature.     

Effect of refining on pulp surface charge accessible to polydadmac and FTIR characteristic bands of high yield kraft fibres

Studies were carried out to determine the surface charge and total charge of refined softwood high yield kraft pulps at different levels by titration methods. The results showed that the surface charge was highly dependent on the degree of refining. However, the total fibre charge was not affected by refining. The change of the fibre surface charge during refining monitored using ATR-FTIR characteristic bands was within 1700-1300 cm(-1). Reliable PLS1 calibration models could be established to correlate the FTIR spectral data and the surface charge of the refined pulps.