Inhibitors of the cleavage of aclacinomycins

Summary The reductive cleavage of the aclacinomycins A (I), Y (II), and B (III) by intact mycelia or subcellular fractions of the producer strain S. spec. AM 33352/F43 is suppressed in the presence of uncouplers, complex-forming agents, detergents, and some metal anions such as chromate. Increased concentration of the latter in complete cultures caused rearrangement of I to III.     

Identification and purification of vitamin K-dependent proteins and peptides with monoclonal antibodies specific for gamma -carboxyglutamyl (Gla) residues

Novel monoclonal antibodies that specifically recognize gamma-carboxyglutamyl (Gla) residues in proteins and peptides have been produced. As demonstrated by Western blot and time-resolved immunofluorescence assays the antibodies are pan-specific for most or all of the Gla-containing proteins tested (factors VII, IX, and X, prothrombin, protein C, protein S, growth arrest-specific protein 6, bone Gla protein, conantokin G from a cone snail, and factor Xa-like proteins from snake venom). Only the Gla-containing light chain of the two-chain proteins was bound. Decarboxylation destroyed the epitope(s) on prothrombin fragment 1, and Ca(2+) strongly inhibited binding to prothrombin. In Western blot, immunofluorescence, and surface plasmon resonance assays the antibodies bound peptides conjugated to bovine serum albumin that contained either a single Gla or a tandem pair of Gla residues. Binding was maintained when the sequence surrounding the Gla residue(s) was altered. Replacement of Gla with glutamic acid resulted in a complete loss of the epitope. The utility of the antibodies was demonstrated in immunochemical methods for detecting Gla-containing proteins and in the immunopurification of a factor Xa-like protein from tiger snake venom. The amino acid sequences of the Gla domain and portions of the heavy chain of the snake protein were determined.     

Cytokines secreted by lymphokine-activated killer cells induce endogenous nitric oxide synthesis and apoptosis in DLD-1 colon cancer cells

IL-2-activated killer lymphocytes (LAK cells) secrete inflammatory cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNFalpha) that can induce nitric oxide (NO) synthesis. We evaluated whether LAK cells could activate NO synthesis in human cancer cells. LAK cells and their culture supernatants induced NO synthesis in DLD-1 colon cancer cells in a dose-dependent manner. NO synthesis was inhibited completely by blocking antibodies to IFN-gamma, demonstrating a key role for this LAK cell cytokine in regulating NO synthesis. The addition of TNFalpha antibodies resulted in partial inhibition. Induction of iNOS mRNA and protein expression in DLD-1 cells was detected. Endogenous NO production inhibited DLD-1 cell proliferation and induced apoptosis, processes that were inhibitable by the NO synthase inhibitor N(G)-monomethyl-l-arginine. Our study has identified a novel, non-contact-dependent LAK cell cytotoxic mechanism: induction of growth inhibition and programmed cell death due to endogenous NO synthesis in susceptible human cancer cells.     

Differences in the denaturation behavior of ribonuclease A induced by temperature and guanidine hydrochloride

Moderate temperatures or low concentrations of denaturants diminish the catalytic activity of some enzymes before spectroscopic methods indicate protein unfolding. To discriminate between possible reasons for the inactivation of ribonuclease A, we investigated the influence of temperature and guanidine hydrochloride on its proteolytic susceptibility to proteinase K by determining the proteolytic rate constants and fragment patterns. The results were related to changes of activity and spectroscopic properties of ribonuclease A. With thermal denaturation, the changes in activity and in the rate constants of proteolytic degradation coincide and occur slightly before the spectroscopically observable transition. In the case of guanidine hydrochloride-induced denaturation, however, proteolytic resistance of ribonuclease A initially increases accompanied by a drastic activity decrease far before unfolding of the protein is detected by spectroscopy or proteolysis. In addition to ionic effects, a tightening of the protein structure at low guanidine hydrochloride concentrations is suggested to be responsible for ribonuclease A inactivation.     

Application of higher order statistics for atrial arrhythmia classification

Atrial fibrillation (AF) and atrial flutter (AFL) are the two common atrial arrhythmia encountered in the clinical practice. In order to diagnose these abnormalities the electrocardiogram (ECG) is widely used. The conventional linear time and frequency domain methods cannot decipher the hidden complexity present in these signals. The ECG is inherently a non-linear, non-stationary and non-Gaussian signal. The non-linear models can provide improved results and capture minute variations present in the time series. Higher order spectra (HOS) is a non-linear dynamical method which is highly rugged to noise. In the present study, the performances of two methods are compared: (i) 3rd order HOS cumulants and (ii) HOS bispectrum. The 3rd order cumulant and bispectrum coefficients are subjected to dimensionality reduction using independent component analysis (ICA) and classified using classification and regression tree (CART), random forest (RF), artificial neural network (ANN) and k-nearest neighbor (KNN) classifiers to select the best classifier. The ICA components of cumulant coefficients have provided the average accuracy, sensitivity, specificity and positive predictive value of 99.50%, 100%, 99.22% and 99.72% respectively using KNN classifier. Similarly, the ICA components of HOS bispectrum coefficients have yielded the average accuracy, sensitivity, specificity and PPV of 97.65%, 98.16%, 98.75% and 99.53% respectively using KNN. So, the ICA performed on the 3rd order HOS cumulants coupled with KNN classifier performed better than the HOS bispectrum method. The proposed methodology is robust and can be used in mass screening of cardiac patients.     

Immunological relationship between different types of bovine intermediate filaments

In order to examine the relationship between the intermediate filaments from Purkinje fibres of the cow heart conduction system and five proposed subclasses of mammalian intermediate filaments, the gel electrophoresis-derived enzyme-linked immunosorbent assay (GEDELISA) has been used to examine the specificity and crossreactivity of our antibodies against the Purkinje fibre intermediate filament protein, skeletin. Bovine tissues known to contain intermediate filaments of the five main subclasses were examined with antiskeletin and with preimmune serum and the specific antiserum absorbed with pure skeletin as controls. The antibodies raised against Purkinje fibre skeletin reacted with all three polypeptides of the "neurofilament triplet", with glial fibrillary acidic protein (GFAP), with smooth muscle desmin and also slightly with some prekeratin subunits and with endothelial vimentin. From studies with monoclonal antibodies and amino acid sequencing, certain regions of all intermediate filaments are suggested to be structurally related. Here we show that Purkinje fibre skeletin seems to share antigenic determinants with the proposed five main classes of intermediate filaments. Our antibody is the first carefully controlled experimentally induced antibody having such properties. This might be due to the special attributes of the intermediate filament system in Purkinje fibres, which themselves have unique properties.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

Limnological studies of the lakes and streams of the upper qu'appelle river system,Saskatchewan, Canada

Summary Physical aspects including geography, geology, climate, soils, vegetation and lake morphometry of the upper Qu'Appelle River basin have been summarized. The importance of multiple use of this area has been stressed.The lakes were studied during the 1959–1969 period. Only Katepwa Lake showed temperature stratification for any length of time. Ice cover persisted for about five months each year. Buffalo Pound Lake had the lowest mean Secchi disc transparency (0.9 m) while Katepwa Lake had the highest (2.1 m). Light transmissibility tended to be lowest during May and September when wind velocities were highest. The 1 % level of light extinction was usually between 1 and 3 meters depth. The yellow portion of the spectrum penetrated the furthest.Buffalo Pound Lake was the least saline (600–700 mg/l TDS) while Last Mountain Lake was most saline (2000–2400 mg/l TDS). Input from Lake Diefenbaker and heavy spring runoffs effected major decreases in all lakes by May 1969. All lakes except Last Mountain were considerably more saline than during the 1937–1941 period. Variations in anions and cations within and between lakes vary with the total dissolved solids. As salinity increases magnesium, sulphate and chloride increase faster proportionally than other ions. The order of concentration of major cations is Na > Mg > Ca > K while the major anions' order of concentration is SO₄ > CO₃.HCO₃ > Cl.Soluble phosphate levels tend to be lowest in Buffalo Pound Lake. Levels increased sharply in Pasqua Lake below sewage outfalls from Moose jaw and Regina. Subsequently levels decreased in each successive downstream lake. Concentrations increased during the period of study. It was estimated that 594 metric tons of orthophosphate were contributed in domestic sewage in 1966. In addition land drainage contributes considerable amounts of phosphate during spring runoff.Nitrogen-bearing compounds vary in similar patterns as soluble phosphate but with smaller fluctuations. Domestic sewage and land drainage contribute significant amounts but of greater importance may be in situ biological fixation.All lakes were basic with usual pH of 8.4–9.0.From a chemical point of view these lakes are distinctly eutrophic.

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Zusammenfassung An fünf Seen des Ober Qu'Appelle Fluszgebietes, Saskatchewan, sind seit 1959 die jahreszeitlichen Veränderungen des Planktons und auch wichtige chemische und physikalische Milieufaktoren studiert worden. In diesem Teil werden die physiographischen Faktoren diskutiert.Dauernde Temperaturschichtung kommt nur im Katepwa See vor. Die 1% Lichtstärke war meistens zwischen 1 und 3 m Tiefe gelegen.Der Salzgehalt der Ober Qu'Appelle Seen liegt zwischen 600 mg/l (Buffalo Pound See) and 2400 mg/1 (Last Mountain See). Die Kationen ordnen sich in der Reihenfolge Na > Mg > Ca > K, die Anionen: SO₄ > CO₃.HCO₃ > Cl. Der pH bleibt meistens zwischen 8.4 and 9.0.Die Phosphatkonzentration ist sehr hoch. Abwasserverunreinigung, verursacht durch die Städte Regina und Moose jaw, ist bedeutsam für Phosphatgehalt in allen Seen auszer Buffalo Pound See. Die Stickstoffkonzentration ist ebenfalls hoch aber schwankt nicht soviel wie das Phosphat.Vom chemischen Standpunkt ausgesehen sind diese Seen sehr eutrophiert.