Modeling the performance of immobilized α-chymotrypsin catalyzed peptide synthesis in acetonitrile medium

A model was developed which describes simultaneous reaction and internal diffusion for kinetically controlled, immobilized α-chymotrypsin-catalyzed, oligopeptide synthesis in acetonitrile medium. The model combines the equations that describe the intrinsic kinetics of four different reactions and the physical characteristics of three different support materials, as determined experimentally, to predict the apparent initial activity and nucleophile selectivity of the immobilized biocatalyst. The model is able to predict reasonably well the experimentally observed initial rate and nucleophile selectivity vs. enzyme loading profiles. The reduction in observed initial rate with enzyme loading when fast reactions are carried out with α-chymotrypsin immobilized on celite, and the larger influence of mass transfer limitations on the initial reaction rates than on nucleophile selectivities are correctly predicted by the numerical calculations. The model is general in terms of its application to other systems — enzymes, reactions, support materials and/or kinetic schemes — as long as the intrinsic kinetics and the characteristics of the enzyme and support material are known.     

Age Dynamics of Body Mass and Human Lifespan

Age dynamics of human body mass (0–90 years) is described as a function of periodic damped oscillations. Common regularities are found in the age changes of mass, of entropic equivalent—a parameter equivalent to thermodynamic entropy, and of intensity of natural mortality. It is shown that the mass reflects the biological system thermodynamic state and is measured oppositely directed to the entropy value. The second, third, and fourth extremes of the mass age dynamics correspond to the mean (70–75 years), the commonly accepted maximal (100–110 years), and the maximal known (140–150 years) human lifespan, while the mass oscillations cease at the age associated with the maximal known lifespan—about 145 years.     

Hypergastrinaemia induced by acid blockade evokes enterochromaffin-like (ECL) cell hyperplasia in chicken,hamster and guinea-pig stomach

Summary Treatment of chickens, hamsters and guinea-pigs with large doses of the long-acting antisecretory agent omeprazole for 10 weeks resulted in elevated serum gastrin levels and in increased stomach weight and mass of oxyntic mucosa. Also the antral gastrin cell density was increased. Another striking effect was the hyperplasia of the histamine-producing enterochromaffin-like (ECL) cells — a prominent endocrine cell population with unknown function — in the oxyntic mucosa. Accordingly, the gastric mucosal histamine concentration and rate of histamine formation were increased in all three species. The results suggest that marked and long-lasting suppression of acid secretion leads to elevated serum gastrin levels and diffuse ECL cell hyperplasia not only in the rat, as previously seen, but also in the chicken, hamster and guinea-pig; this hyperplasia is associated with accelerated histamine formation in all three species. The following sequence of events is suggested to occur in mammalian as well as submammalian vertebrates: suppression of acid secretion — hypergastrinaemia — ECL cell hyperplasia.     

Fine structure of the giant aflagellate spermatozoon in pseudostomum quadrioculatum (leuckart) (platyhelminthes,prolecithophora)

The giant aflagellate spermatozoa of P. quadrioculatum are composed of two different parts: a thicker head piece and a more slender tail piece. In the head there exist a large elongated nucleus and an elongated mitochondrial derivative situated in a groove-like cavity of the nucleus. In mature spermatozoa the nuclear material is arranged in many small membrane bounded areas. Both structures, nucleus and mitochondrial derivative, are spirally coiled. The outer part of the membrane in the mitochondrial derivative forms many loop-like foldings. Both organelles continue to the tail in form of two small, helically coiled ribbons; the nucleus is anchored within the mitochondrial derivative by an electron-opaque process. A sheath of spirally-orientated cortical microtubules starting from the tip of the head runs to the tip of the tail under the cell membrane. In addition, a second sheath of tubules occurs in the tail region, these tubules also run parallel to each other, but in the opposite direction to the microtubules of the outer sheath.The possible relations between the structures observed and the motility of the spermatozoa are discussed; in addition, some phylogenetic comments are attempted.Abbreviations c — cerebrum - com — cortical microtubules - cop — copulatory organ - fm — foldings of the mitochondrial membrane - l — lattice - mid — mitochondrial derivative - mt — microtubules - n — nucleus - ne — nuclear envelope - ph — pharynx - pn — protonephidium - rp — ribbon-like nuclear process - te — testis - tt — testis - tt — tip of the tail - vi — vitellarium - vs — vesicula seminalis     

Determination of therapeutic and toxic concentrations of doxepin and loxapine using gas—liquid chromatography with a nitrogen-sensitive detector, and gas chromatography—mass spectrometry of loxapine

A gas—liquid chromatographic procedure is presented for the determination of therapeutic and toxic serum levels of doxepin and loxapine, using a nitrogen—phosphorus-sensitive detector. Amitriptyline is used as the internal standard. The method is accurate, sensitive and specific with no derivatization required prior to analysis. An advantage of the procedure is the small serum sample size needed for analysis and the selectivity and sensitivity of the detector, with the limit of detection being 3 and 2 μg/l for doxepin and loxapine, respectively. Nine cases of doxepin and loxapine misuse are presented. Serum doxepin concentrations ranged from 113 to 439 μg/l, with a loxapine concentration of 192 μg/l observed in one patient. The presence of the tricyclics was identified and confirmed by gas chromatography—mass spectrometry and the mass spectrum of loxapine is reported.     

Impact of air ions of both polarity on evaporation of certain organic and inorganic liquids

Air ions of both polarity, produced by corona electrodes, were used to evaporate to dryness liquid samples of ethyl alcohol (EA), water (W), and carbon tetrachloride (CTC). Drying times were determined with a beta-ray gauge. Ion exposed samples of EA, W, and CTC dried, respectively, 2.3, 3.2, and 5.4 times faster than the corresponding control samples when exposed simultaneously to 0.94×10¹² positive and 1.83×10¹² negative air ions cm^–2s^–1 under the same laboratory conditions. Drying by corona discharge could be explained by three different mechanisms. Electric wind caused by the ionic drag is proposed as the principal driving force for the observed enhancement of evaporation. The decrease in free energy of a dielectric in the presence of an electric field compared to its absence may have increased the escaping tendency of the molecules of the treated liquids. The turbulence in the liquids created by the rotational effect on the dielectric molecules by the electric field may also be a factor in further enhancing the mass transfer rates from the samples.     

Mass Change of Glaciers in Muztag Ata–Kongur Tagh,Eastern Pamir,China from 1971/76 to 2013/14 as Derived from Remote Sensing Data

The assessment of glacier mass budget is crucial for assessing water reserves stored in glaciers. Derived glacier mass changes in the Muztag Ata and Kongur Tagh (MAKT) region in the eastern Pamir, northwestern China, is helpful in improving our knowledge of the dynamics of glaciers under a changing climate in High Mountain Asia. Here, glacier area and mass changes derived from remote sensing data are investigated for the period 1971/76–2013/14 for glaciers in MAKT. We have used ASTER images (2013/14), Cartosat-1 (2014) and Landsat, SRTM (Shuttle Radar Terrain Mission) digital elevation model (DEM) (2000), topographic maps (1971/76) and the first and second Chinese glacier inventories (CGIs). Our results indicated that the glacier area of MAKT decreased from 1018.3 ± 12.99 km² in 1971/76 to 999.2 ± 31.22 km² in 2014 (–1.9 ± 0.2%). Weak area shrinkage of glaciers by 2.5 ± 0.5 km² (0.2 ± 0.1%) happened after 2000 and the period 2009–2014 even saw a slight expansion by 0.5 ± 0.1 km² (0.1 ± 0.0%). The glaciers in this region have experienced an overall loss of –6.99 ± 0.80 km³ in ice volume or –0.15 ± 0.12 m water equivalent (w.e.) a^–1 from 1971/76 to 2013/14. The mass budget of MAKT was –0.19 ± 0.19 m w.e. a⁻¹ for the period ~1971/76–1999 and –0.14 ± 0.24 m w.e. a⁻¹ during 1999–2013/2014. Similar to previous studies, there has been little mass change in the Pamir over recent decades despite such uncertainties. Glacier mass change showed spatial and temporal heterogeneity, with strong mass loss on debris-covered glaciers with an average of –0.32 ± 0.12 m w.e. a⁻¹ from the 1970s to 2013/14.     

Freeliving marine nematodes from East African coasts.Bathyepsilonema anulosum sp.n. andLeptepsilonema richardi sp.n.

Two new epsilonematid nematode species from Kenyan intertidal areas are described:Bathyepsilonema anulosum sp.n. — characterized by the large number of body-annules (118–124); small unispiral amphid, straight ambulatory setae and the very small copulatory thorns of the males — andLeptepsilonema richardi sp.n., — characterized by the sexual dimorphism in the amphid, the ornamentation of the body cuticle and the umbrella shaped capitulum in the males.     

Critical analysis of factors influencing sphaeroplast generation from Saccharomyces cerevisiae

Efficient synthesis of large numbers of viable sphaeroplast from Saccharomyces cerevisiae has been found to be influenced by a number of factors. In this case, Trichoderma harzianum, NCIM 1185, culture filtrate has been used to prepare sphaeroplast from Saccharomyces cerevisiae, NCIM 3288. A method has been devised to isolate large number of viable sphaeroplast from the cell. Detailed analysis of various factors affecting the formation of sphaeroplasts from Saccharomyces cerevisiae has not yet been reported. This study showed critical analysis of various factors which influenced sphaeroplast formation. Most suitable conditions were: Age of the organism in slant — 1 d, cell age in liquid medium — 24 h, time of incubation of cell with 0.3% -mercaptoethanol — 30 min, level of lytic ezyme concentration — 79.2 ml, concentration of cell (dry wt. equivalent) — 0.1262 g, time of contact with lytic enzyme — 25 min, temperature of sphaeroplast formation — 30 °C, phosphate buffer — 25 mM of pH 6.5 and KCl as osmotic stabilizer — 0.7 M.     

Relative efficiency of selection methods to improve a ratio of two traits in Tribolium

Summary Three generations of upward selection for the egg mass/adult weight ratio were carried out in Tribolium castaneum. The experiment involved five lines: E — selected for increased egg mass; W — selected for decreased adult weight; R — selected for egg mass/adult weight directly; L — linear index selected with economic weights of m ₂: -m ₁ egg mass to adult weight (m ₁ and m ₂ are the means for adult weight and egg mass, respectively); NL — nonlinear index selected. Adult weight (7 days after adult emergence) and egg mass (between 7 and 11 days) were measured. The NL, E, and L lines had the greatest observed responses for the ratio; the R and W lines were not effective in improving the egg mass/adult weight ratio. It was expected that the NL line would be superior to the E, L, and R lines, and that the W line would respond the least. Observed response was significant for egg mass in the NL line, and for adult weight in the E, W, and R lines. Strong selection to increase egg mass seems to represent the optimal criterion for the ratio to be improved. The usefulness of nonlinear indices as selection criteria to improve a nonlinear trait, previously found to be optimal for a trait defined as the product of two component traits, appears to hold also for the selective improvement of the ratio of two traits. Serious limitations expected for direct selection of the ratio have been confirmed in this experiment.     

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ubiquity of microbes creates a copious supply of potential contaminants in a laboratory. To ensure experimental success, the number of contaminants on equipment and work surfaces must be minimized. Common among many experiments in microbiology are techniques involving the measurement and transfer of cultures containing bacterial cells or viral particles. To do so without contacting non-sterile surfaces or contaminating sterile media requires (1) preparing a sterile workspace, (2) precisely setting and accurately reading instruments for aseptic transfer of liquids, and (3) properly manipulating instruments, cultures flasks, bottles and tubes within a sterile field. Learning these procedures calls for training and practice. At first, actions should be slow, deliberate, and controlled with the goal being for aseptic technique to become second nature when working at the bench. Here we present the steps for measuring volumes using serological pipettes and micropipettors within a sterile field created by a Bunsen burner. Volumes range from microliters (μl) to milliliters (ml) depending on the instrument used. Liquids commonly transferred include sterile broth or chemical solutions as well as bacterial cultures and phage stocks. By following these procedures, students should be able to: •Work within the sterile field created by the Bunsen burner flame. •Use serological pipettes without compromising instrument sterility.• Aspirate liquids with serological pipettes, precisely reading calibrated volumes by aligning the meniscus formed by the liquid to the graduation marks on the pipette. •Keep culture bottles, flasks, tubes and their respective caps sterile during liquid transfers. •Identify different applications for plastic versus glass serological pipettes. •State accuracy limitations for micropipettors. •Precisely and accurately set volumes on micropipettors. •Know how to properly use the first and second stop on a micropipettor to aspirate and transfer correct volumes.     

Applications of mass spectrometry in screening for new biocatalysts

Each biocatalyst screen is unique, defined by the combination of factors involved in the screen, including the number and type of biocatalysts in the screening collection, substrate chemistry and the type of assay. Advances in the technology surrounding mass spectrometry — in software, in ionization sources and interfaces and in engineering, which allows smaller mass spectrometry systems and narrow bore HPLC — have made the application of this versatile technology in screening assays possible. A mass spectrometric assay provides sensitive, specific, quantitative, high-throughput detection of new biocatalyst activities. Examples of these applications are presented and potential pitfalls are discussed.     

Les ultrastructures nucléaires de la Noctiluque (Dinoflagellé libre) au cours de la sporogenèse

The trophozoït of Noctiluca miliaris has a large nucleus (30 ) with several nucleoli of considerable size that contain DNA fibrillae lying in the interspaces. — Before and during the first sporogenetic divisions, the nucleoli disintegrate, releasing towards the cytoplasma numerous groups of ribonucleic granules passing through the nuclear ampullae. At the end of the sporulation, there are no nucleoli visible in the nuclei and no ampullae. — The nucleoplasm diminishes, as the DNA filaments are built up, to form the meshes of a network which limit the masses of chromatic material that take the shape of chromosomes characterized by regular fibrillar arches, at the 8–16 nuclei stage. In their centre, there is an axial structure which remains intact during the chromosomal segregation; its function during mitosis seems to be important: supplementary layers of arches appear at this level. — The progressive condensation of the chromosomes is correlated to the sporogenetic evolution of the nuclei, not to the different phases of the mitotic cycle. — The karyokinesis is brought about, during early stages, by mere splitting of the chromatic mass and of its envelope, and later one by separation into two lots of chromosomes. The segregation of these chromosomes is effected by partial intervention and growth of the envelope of the nucleus; there is no centromeric structure visible. At the end of divisions, the nucleus is almost entirely formed by its chromosomes. — The nucleolar structure, the karyokinesis, the structure of the nuclear envelope and the chromosomal cycle show the particularly high evolution of Noctiluca, within the Dinoflagellata.     

Individual selection,culture and manipulation of higher plant cells

Summary Due to the heterogeneity in morphology, physiological and morphogenetical capabilities of higher plant cells in mass culture, the development of methods for individually culturing defined cells seemed to be useful and necessary. Individual cell culture represents a powerful tool for studies on the physiology of different cell types, the analysis of differentiation programs, the genetic manipulation of plant cells and cell-cell interactions. An improved microculture system based on a computer-controlled set-up for the efficient selection, transfer and individual culture of defined higher plant cells until regeneration of whole plants is described. Related experimental approaches for individually manipulating higher plant cells under controlled conditions, such as electrofusion of defined pairs of protoplasts and subprotoplasts, cell reconstruction and intranuclear microinjection of protoplasts and karyoplasts — mainly performed with cells of the crop plant Brassica napus L. — are presented.This article is dedicated to the memory of H.-G. Schweiger, initiator and mentor of the experimental single cell approach reviewed herein     

The effect of changes in the respiratory metabolism upon genome activity in Drosophila

Inhibition of hydrogen transfer between NADH and Co Q by rotenone or amytal in salivary gland cells of Drosophila hydei maintained in vitro, results in the activation of a particular group of four loci in the polytene chromosomes (puff formation). The response of these loci to the same treatment is enhanced if Na-malonate is present in the incubation medium. — Three of the loci become active if the glands are kept in a medium supplied with antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide (H QNO), specific inhibitors of the electron transfer between cytochromes b and c. — It was established that a temperature treatment and DNP raise oxygen consumption of the cells to a certain level. Following the same treatments of glands supplied with Na-malate and Na-succinate the raise in oxygen consumption attains a significantly higher level. Under these conditions no response is observed at the genome level. — Whereas DNP, which uncouples oxidative phosphorylation and enhances the respiratory chain reactions, does induce the initiation of puff formation, oligomycin, which inhibits oxidative phosphorylation and suppresses the respiratory chain reactions, is ineffective in initiating puff formation at the specific loci. However, if oligomycin is supplied to the medium in combination with KCN which inhibits the cytochrome oxidase activity, three of the four loci become active. — The presence in the medium of substances which may act as hydrogen acceptors, e.g. menadione or methylene blue, can also result in activation of the chromosome loci. — These results are interpreted as indications for the existence of a regulatory mechanism between mitochondrial respiratory metabolism and the activity of a particular group of genome loci.     

Bioanalysis of erythromycin 2'-ethylsuccinate in plasma using phase-system switching continuous-flow fast atom bombardment liquid chromatography—mass spectrometry

A specific method for the determination of erythromycin 2'-ethylsuccinate (EM-ES) in plasma is described. The method involves a liquid—liquid extraction procedure followed by the analysis of extracts using phase-system switching (PSS) continuous-flow fast atom bombardment (CF-FAB) liquid chromatography—mass spectrometry (LC—MS). In PSS EM-ES is enriched after analytical separation on a short trapping column, from which it is desorbed to the LC—MS interface. In this way, favourable mobile phases can be used for the LC separation and for the MS detection. Using the PSS approach a flow-rate reduction from 1.0 ml/min in the LC system to 15 μl/min going into the mass spectrometer was achieved without splitting. The determination limit for EM-ES was 0.1 μg/ml.     

Quantification of D-amino acids in human urine using GC-MS and HPLC

Summary The relative amounts of free D-amino acids (D-AA) in the urine of seven healthy volunteers (age 27 to 49 years) were determined using chiral phase (Chirasil-L-Val) capillary gas chromatography in conjunction with selected ion monitoring mass spectrometry. The absolute amounts of free D-AA were determined by pre-column derivatization of the amino acids witho-phthaldialdehyde andN-isobutyryl-L-cysteine followed by high-performance liquid chromatographic separation and fluorescence detection of the isoindol derivatives formed. The following most abundant D-AA were found (highest and lowest absolute and relative amounts): D-Ser (379.8 — 30.1µMol/L; 56.5 — 19.0%), D-Ala (53.8 — 7.6µMol/L; 19.6 — 5.7%), D-Thr (5.8 — 0.25µMol/L; 3.4 — 1.0%), D-Val (3.7 — 0µMol/L; 4.2 — 0%), and D-Phe (3.5 — 0.35µMol/L; 4.8 — 1.4%).     

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

Homodimeric and expanded behaviour of trimethylamine dehydrogenase in solution at different temperatures

Earlier studies using x-ray crystallography have shown that trimethylamine dehydrogenase (TMADH) from methylotropic bacteria exists as homodimers in the crystalline state. In this present hydrodynamic study we show that this is true also in dilute solution conditions and investigate the degree of swelling or relaxation of the protein in solution. Analytical ultracentrifugation was used to determine the molar mass and to investigate whether the homodimeric nature of this molecule in crystal form — as visualized by x-ray crystallography — is reproduced in dilute solution at temperatures between 4 and 40°C. The globular solution structure determined at 4 and 40°C is in good agreement with crystallographic data although trimethylamine dehydrogenase was found to be either more asymmetric in solution — or highly hydrated —, a phenomenon found to increase with temperature. In agreement with the crystallographic structure, the enzyme sediments as a homodimer with a molar mass of (163,000±5,000) g/mol. The concentration dependence of the sedimentation coefficient in the range of 0–1 mg/ml, indicates that no association or dissociation occurs. These findings are additionally supported by sedimentation equilibrium data in the concentration range of 0 to 1.8 mg/ml. Finally, from the sedimentation coefficient distribution at various temperatures, it was concluded that the enzyme is conformationally flexible and assumes an even more expanded structure at higher temperatures which is in good agreement with the hydrodynamic calculations performed. Correspondence to: S. E. Harding     

Qualitative and quantitative analysis of some synthetic, chemically acting laxatives in urine by gas chromatography—mass spectrometry

A method for the qualitative and quantitative simultaneous analysis of dioxyanthraquinone, desacetyl-Bisacodyl, phenolphthalein and Oxyphenisatin in human urine using gas chromatography—mass spectrometry (GC—MS) has been developed. The compounds were extracted from urine at pH 7.5 with diethyl ether using Extrelut extraction columns, followed by evaporation and trimethylsilylation.The method used electron beam ionization GC—MS employing a computer-controlled multiple-ion detector (mass fragmentography). The recovery from urine for the various compounds was between 80% and 100%. The detection limit for these compounds was in the range 0.01–0.05 μg/ml of urine.The method proved to be suitable for measuring urine concentrations for at least four days after administration of a single oral low therapeutic dose of the laxatives to sixteen healthy volunteers.