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The GenTechnique project at Washington State University uses a networked learning environment for molecular genetics learning. The project is developing courseware featuring animation, hyper-link controls, and interactive self-assessment exercises focusing on fundamental concepts. The first pilot course featured a Web-based module on DNA replication with mentoring and evaluation conducted via e-mail and discussion software. (Author/AEF)

De novo synthesis and the secretion of cellobiase from Neocallimastix frontalis EB188 were studied. Cellobiase was secreted rapidly in cellulose switch cultures. Chemical inhibition of protein synthesis and radiotracer studies suggested secretion was dependent on nascent protein synthesis. Inhibitors of protein glycosylation also inhibited secretion. An 85,000 dalton protein (and several others) represented the principal differences in de novo synthesis between cellulose- and glucose-switched cultures. Concentrations of the 85,000 daltons protein increased with culture incubation time and ultimately accounted for 7% of the total protein secreted. This protein was purified by gel electrophoresis separations and was determined to be a cellobiase. Secretion of this cellobiase was correlated with its radiolabeling. The possibility of cellobiase induction representing a specialized gene control system in Neocallimastix frontalis EB188 is discussed. [ABSTRACT FROM AUTHOR]

Protein and cellulase secretion in Neocallimastix frontalis EB188 was studied. Induction of secretion in this ruminal fungus was dependent on the amount of medium cellulose and on de novo protein synthesis. Medium glucose repressed secretions and diminished the utilization of medium cellulose. Exogenously added cyclic nucleotides failed to derepress glucose repression. Medium glycerol had no effect on protein and cellulase secretion, would not support cell growth, and was nontoxic. [ABSTRACT FROM AUTHOR]

Extracellular cellulase induction in the ruminal fungus Neocallimastix frontalis isolate EB188 was followed. Glucose media-established cultures produced cellulase when switched to a variety of cellulose-containing media. High levels of cellulase and xylanase activities were present in cultures switched to sigma cell 100, solka floc, avicel, sisal fiber, and wheat straw, but not those switched to glucose, carboxymethylcellulose, or wood chips. Several assay substrates were used to show differential cellulase induction as well as β-glucosidase activity. Cellulases hydrolyzed short oligosaccharides and released glucose from insoluble cellulose. Cellobiase activity was also indicated. Cellulase activity tolerated brief exposure to high temperature, was insensitive to certain metal ions, and possessed pH optima between 5.0 and 6.5. [ABSTRACT FROM AUTHOR]

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.

The aim of the present study was to analyze the protein composition of ductal breast carcinoma and the surrounding normal tissue in individual patients using comparative 2D proteomics and mass spectrometry to detect candidate disease biomarkers for diagnosis and prognosis. Samples of normal and cancerous tissue obtained form 28 patients were analyzed. Chaperonins and cytoskeletal proteins predominated among the 11 proteins for which major changes in abundance were detected. Of these 11 proteins with an altered expression, 2 had a decreased expression and 9 had an increased expression. In addition, the abundance of a few cytokeratins was also altered; however, they were not capable of serving as specific circulatory biomarkers. The proteins which we observed to exhibit an altered expression in infiltrating ductal breast carcinoma may be exploited as novel targets for therapeutic interventions or represent novel diagnostic/prognostic markers for the early detection of aggressive tumors, particularly those with multridrug-resistant phenotypes during the earlier stages of the disease.

The diatom test is widely used by forensic pathologists as proof of drowning, notwithstanding some criticisms mainly concerning the occurrence of false-positive results (presence of diatoms in the tissues of subjects who died from causes other than drowning). The aim of the present study was to verify the claimed inaccuracy of the method caused by an excessive rate of false-positives related to inadvertent exposure to diatoms of the general population. The study was carried out to investigate the presence of diatoms in the tissues (lungs and sternum) of subjects who died from causes other than drowning. Two groups of cadavers that underwent an autopsy at the Institute of Forensic Medicine of the University of Verona were included in the study. Group A comprised 45 individuals who died from causes other than drowning, whereas Group B comprised 20 bodies which had been recovered from water. The extraction of the diatoms was performed by incubation of samples in nitric acid for 48 hours at 60°C. The analysis of the samples from Group A showed the absence of diatoms in both lung and sternum samples. In Group B all lung samples showed the presence of diatoms, whereas only six sternum samples were shown to contain diatoms. The difference between Groups A and B was statistically highly significant. The absence of diatoms in the samples collected from Group A falsified the hypothesis that false-positive results from the diatom test may occur due to diatoms entering living bodies through the respiratory and/or digestive tracts via air, water or food, supporting the validity of the diatom test as proof of drowning.

Background: Bladder cancers have different angiogenic pathways distinguishing not only papillary from solid tumours, but even papillary superficial from papillary invasive ones, thus representing selective targets for antiangiogenic drugs., Methods: The bacterial wall component tecogalan, inhibiting basic fibroblast growth factor (bFGF), the fumagillin derivative TNP-470, inhibiting vascular endothelial growth factor (VEGF), the distamycin A derivative PNU153429, and the tetracycline minocycline were administered to nude mice injected with the human bladder cancer cell lines 639V, causing bFGF-expressing papillary superficial tumours, or T24, causing VEGF-expressing papillary invasive tumours., Results: Tecogalan had no effect even on 639V tumour growth, where bFGF was unaffected. TNP-470 only had an effect on T24 tumours, delaying tumour appearance and growth and lowering VEGF; these effects were augmented by adding minocycline. PNU153429 had no effect on 639V tumours, and a slight effect on T24 tumours., Conclusion: TNP-470 may represent a selective drug for the treatment of VEGF-expressing invasive papillary bladder tumours., (Copyright 2005 S. Karger AG, Basel.)

The region 6q27 from human chromosome 6 has been reported to contain one or more tumor suppressor genes on the basis of cytogenetic, molecular and functional studies. We have recently carried out a detailed analysis of a candidate gene from 6q27 to evaluate its putative role as a tumor suppressor gene involved in ovarian cancer pathogenesis. The RNASET2 gene was shown to behave as a class II tumor suppressor and abolish the tumorigenic potential of an ovarian cancer-derived cell line. In this study, we have started the cellular and biochemical characterization of RNASET2 and showed that it is a secreted glycoprotein. Moreover, we have extended our previous studies by evaluating the effect of RNASET2 on the metastatic behavior of the highly-invasive ovarian cancer cell line HEY3MET2. From such analysis, RNASET2 was found to significantly decrease the metastatic potential of this cell line in vivo. Moreover, RNASET2-mediated suppression of tumorigenesis and metastasis was not affected by a double point mutation targeted to the putative ribonuclease catalytic sites, suggesting that tumor suppression by RNASET2 is not mediated by its ribonuclease activity. The potential biological implications of this unexpected finding are discussed in relation to the current evolutionary models.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) immunohistochemical expression was analyzed in 75 human bladder tumor specimens, where the expression of some angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PDECGF), and tumor progression markers, such as epidermal growth factor receptor (EGFr), p16, mutated p53, and normal pRB, were also analyzed. The results were then compared to the clinical and pathological characteristics of the disease. PPAR gamma was expressed more significantly in papillary tumors than in solid cancers, and its presence was associated with statistical significance to low incidence of tumor recurrence or progression. This significant association was observed also when PPAR gamma was expressed in the presence of PDECGF, which resulted, when considered alone, to an angiogenic factor typical of solid cancers and appeared related to poor prognosis. In the presence of bFGF, on the contrary, PPAR gamma expression no longer resulted to a significant association with low incidence of tumor recurrence or progression, suggesting a possible worsening role of this angiogenic factor, typical of papillary cancers, in its interaction with PPAR gamma.

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.

The phylogenetic position of eleven 14-3-3 proteins from five protozoal species was tested relative to other eukaryotic 14-3-3 versions representing many of the previously described isoforms. The protozoal proteins, four from Entodinium caudatum, three from Entameoba histolytica and four from apicomplexan parasites formed clusters closer to the plant and animal epsilon isoforms than to the animal beta, gamma/eta, sigma/theta, and zeta isoforms. This extends the preliminary findings of Wang and Shakes (1996) but data from a wider range of genera are still required to strengthen our hypothesis that the protozoan isoforms may constitute novel isoforms of the 14-3-3 family.

A lambda phage cDNA expression library was constructed from washed suspensions of the rumen ciliate protozoan, Entodinium caudatum, which had been maintained in an isolated, monofaunated sheep. The library was screened using an anti-E. caudatum antiserum raised in rabbits against sonically disrupted protozoa, DNA sequences for two centrins or caltractins, a subfamily of the EF-hand Ca(2+)-modulated proteins which are closely related, highly conserved cytoskeletal proteins, were identified and characterised. Their phylogenetic position was established relative to other centrin gene sequences. The two proteins showed homology to Paramecium tetraurelia centrins: 50 and 52% identities and 71 and 75% similarities in the protein sequence, over 99 and 110 amino acids fragments. Codon usage and indices revealed the E. caudatum follows universal codon usage, but with a restricted number of codons, and has a low G&C content.

Platelet aggregation is accompanied with reorganization of contractile systems. This event involves tyrosine phosphorylation of various proteins. When tyrosine phosphorylation is induced by an intracellular trigger as vanadate, a phosphorylated cytosolic 60 Kd protein acts as a nucleating center on which new actin filaments grow. When aggregation is induced by an extracellular signal as ristocetin, actin filaments of the membrane-associated skeleton and the 60 Kd protein bound to this structure migrate toward the cytosol and only in this soluble state the 60 Kd protein undergoes tyrosine phosphorylation and consequently triggers reorganization of contractile systems.

A method that combines glucose oxidase with [U-14C]glucose to measure subnanomolar to decamillimolar concentrations of oxygen is presented. The method is inexpensive and easy to use. It is independent of sample pH and enzyme inhibitors, including detergents and metal ions. This method can be directly applied to gaseous or aqueous samples and multiple assays can be run simultaneously.

In aerated water solutions Fe(II)Cytochrome c is slowly oxidized to Fe(III)Cytochrome c by molecular oxygen, from which superoxide anion radicals are produced (kobs = 2.7 x 10(-4) min-1 at 37 degrees C and pH = 7.3). The biological importance of this reaction has been evidenced by kinetic investigations in the presence of scavengers. In the presence of Superoxide dismutase the oxidation rate is strongly enhanced (kobs = 3 x 10(-3) min-1 at 37 degrees C). Catalase and mannitol reduce the rate constant values by 50% and 25% respectively either in the presence or in the absence of Superoxide dismutase: differences between rate constants correspond to the differences in stoichiometric redox ratios indicating that hydrogen peroxide and hydroxyl radicals are formed subsequently to the production of superoxide anion radicals.

Collagen addition to platelets suspended in Calcium-free medium induces slow shape change followed by fast aggregates formation. Time courses of membrane phospholipids metabolism and arachidonic acid oxidative metabolism indicate that phospholipase C is the immediate target of the stimulus, and subsequently phospholipase A-2 is activated by synergistic action of released calcium and protein kinase C.

The von Willebrand Factor-Ristocetin adduct activates Platelets aggregation and secretion. Acetylsalicylic acid inhibits Platelets activation by two distinct mechanisms indicating that the adduct activates Platelets by triggering at least two distinct intraplatelet metabolic pathways. The first starts from the activation of Phospholipase A-2 that produces Arachidonic acid, which, in turn, undergoes the metabolic pathway leading to Thromboxane A-2; this pathway can be blocked by the intraplatelet Acetylsalicylic acid by irreversible inactivation of Cyclooxygenase but it is insensitive to the extra-platelet Acetylsalicylic acid. The second pathway is triggered by intact von Willebrand Factor, but not by the Acetylsalicylic acid-treated one; it is insensitive to intraplatelet Acetylsalicylic acid and therefore unrelated to the Arachidonic acid metabolism.

Ristocetin induces a conformational change on von Willebrand Factor (vWF) similar to that due to the interaction with the subendothelium, by which the former can interact with the Glycoprotein-1 B (GPIB) of the platelet membrane and trigger aggregation and granule content secretion. Platelet Rich Plasma (PRP) treated with Acetyl Salicylic Acid (ASA) loses completely the aggregability induced by addition of Ristocetin whereas ASA-treated and successively Washed Platelets (AWP) supplemented with normal plasma (PPP) give an aggregation and a secretory response to Ristocetin similar to that given by PRP; similarly normal Washed Platelets (WP) supplemented with ASA-treated plasma (APPP) give identical aggregation, and secretion by Ristocetin addition. Ours results indicate that the Ristocetin-vWF complex can trigger two distinct intraplatelet metabolic pathways. A first well known way starts from the activation of Phospholipase A-2 (PL-A2), by which arachidonic acid is produced, that, in turn, undergoes the metabolic pathway leading to Thromboxane A-2; this pathway can be blocked by the intraplatelet ASA by irreversible inactivation of Cyclooxygenase, but it is insensitive to the extra-platelet ASA. A second, independent metabolic pathway, can be triggered by intact vWF, but not by the ASA treated one. It is insensitive to intraplatelet ASA and therefore unrelated to the arachidonic acid metabolism. This pathway could start from the activation of Phospholipase C (PL-C).

A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.

Methods are described that allow extraction of high molecular weight DNA from germinated conidia of Neurospora crassa. By labelling DNA with ribonucleosides, early conidia were shown to be active in DNA synthesis. These cells when treated with the enzyme Zymolyase became fragile and could be readily lysed with ionic detergents to release high molecular weight DNA. The DNA extracted from Zymolyase treated cells on to alkaline sucrose gradients sedimented as a heterogeneous species of up to 150 x 10(6) molecular weight. A minor DNA species (presumably mitochondrial) of 20 x 10(6) molecular weight comprised 2-7% of the total. The identity of the DNA was confirmed by sensitivity to DNAase, the diphenylamine assay and TLC. Sedimentation patterns were unaffected by protease digestions and no anomalous high speed rotor effects were evident. Isopycnic gradients suggested that the DNA released was uncomplexed with either protein or carbohydrates. Sepharose chromatography of extracted, RNAase-treated Zymolyase lysate resulted in clearly separate high molecular weight DNA and RNA-protein elution profiles. UV light preferentially inhibited nuclear DNA synthesis and drastically reduced the size and amount of nascent DNA being synthesized in the excision defective uvs-2 mutant. Sites in parental DNA sensitive to Micrococcus luteus UV endonuclease were measured in cells made permeable with Triton X-100.

Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.

A method is described to isolate transcriptionally active Nicotiana tabacum L. cv. Xanthi chromatin from suspension-cultured cells. Enzymatic preparation of protoplasts with solubilization of the plasma membrane, Triton X-100 and homogenization resulted in chromatin free from cellular debris. Incroporation of [(3)H]uridine triphosphate into RNA increased for more than 30 min at 30° C. Transcriptional activity was maximally stimulated at 10 mM MgCl2, 200 mM (NH4)2SO4 and 150 mM KCl. The in-vitro synthesized RNA was found to contain 3.8% polyadenylated RNA. The results of digestion studies with ribonuclease, heat and detergent inactivation studies, α-amanitin and actinomycin D inhibitor effects, as well as dependency on ribonucleotide triphosphates, showed that the activity recorded in the tobacco chromatin was bona-fide enzymatic RNA transcription. There also was marked stimulation of transcriptional activity when exogenous DNA was added to the assay mixture.