Breakage by hydrodynamic shear of the bonds between cohered ends of lambda-DNA molecules

The rate of breakage by hydrodynamic shear of the cohered ends of λ-DNA molecules has been observed for the circular monomers, joined half molecules, and joined quarter molecules, in a capillary apparatus with known flow parameters. The rate constant for breakage has been measured as a function of shear stress, temperature, ionic strength, and molecular length. There is a large temperature coefficient, with an activation energy of 120 ± 20 kcal./mole. The values of d ln k/dG, where k is the rate constant for breaking and G is shear gradient, in aqueous solution at 25°C. are about 3.8 ± 0.3 × 10^?4 see. The shear stresses needed for breakage of joined quarter molecules and of circular monomers, respectively, are about equal, and about half that needed for breakage of joined half molecules. The rate of breakage at a given shear stress increases with decreasing ionic strength, approximately as [Na⁺]^?1.6. Self-protection effects are not observed for opening of circular monomers at a DNA concentration of 5 μg./ml. but are observed for breakage of joined half molecules at concentrations down to 0.5 μg./ml. The large temperature coefficient which is approximately equal to that of the thermal dissociation of the cohered ends is interpreted to mean that shear breakage is a mechanically assisted thermal reaction in which the thermal fluctuations provide most of the free energy of activation for breakage. A detailed model for this interpretation is presented. The self-protection effect implies that those molecules which break are not average molecules but exceptional ones which, due to some fluctuation, are more fully extended in the flow field.     

Flow properties of high-molecular-weight DNA solutions: viscosity, recoil, and longest retardation time

Viscosity η, recoil Γ, and longest retardation time τ_1R were measured for buffered aqueous solution of T2 DNA (M = 1.3 × 10⁸) and cell lysattes of Bacillus subtiles W23 (DNA M = 2 × 10⁹) as a function of concetration, shear stress σ, and time of shear t, using a cartesian-diver-suspended couette flow system. For the T2 solution, η decreased with increasing σ, Γ(t) going through a maximum with t. For the B. subtiles lysates, the preponderant rheological component of which is chromatin DNA, above a critical σ, the η, Γ(t), and τ_1R increased t over 15 to 45 minutes duration to values up to several times larger, indicating the formation of intermolecular aggregates. The onset of such effects and their significance in th e measurement of molecular properties by hydrodynamic means is disscussed.     

Sedimentation Coefficient and Fragility under Hydrodynamic Shear as Measures of Molecular Weight of the DNA of Phage T5

T5 DNA molecules resemble fragments of T2 DNA of molecular weight 84 × 10⁶ with respect to sedimentation coefficient and susceptibility to breakage under hydrodynamic shear. The sedimentation coefficient falls by the same factor when either T2 or T5 DNA is broken at its characteristic critical shear rate. At a given high rate of shear, both DNA's are broken into fragments exhibiting the same sedimentation coefficient. It follows that 84 × 10⁶ is a proper estimate of the molecular weight of T5 DNA, and that particles of phage T5, like those of T2, contain a single DNA molecule.     

Effects of tree characteristics and substrate condition on critical breaking moment of trees due to heavy flooding

To elucidate tree breakage conditions with different breaking mechanisms, i.e., moment by drag force, local scour, and degradation of the substrate around trees, field surveys were conducted after a flood event (September 2007 flood due to Typhoon 9) in the Tamagawa River, Japan. Trees in a river have two main breaking mechanisms during a flood event, moment by fluid force and erosion of the substrate. Moment by fluid force causes two breaking phenomena, trunk damage (bending, breakage) and overturning. Trunk bending or breakage can be expressed as a function of d ^c , where d is the trunk diameter at breast height and the power c equals 3 for trunk bending or breakage, and approximately 2 for overturning. Smaller diameter trees experienced trunk breakage, but larger trees were overturned. The range for these two breaking patterns changes with the substrate condition. If severe scouring has occurred, the threshold for overturning moment can be quite small. Tree overturning occurred mostly on the bank side of the gravel bar; however, some trees, especially Robinia pseudo acacia and Morus bombycis, were overturned if the substrate was a thin deposited soil or silt layer on gravel. The roots were anchored in the small-particle deposited layer in that case. As for the erosion of the substrate, the tree-breaking patterns can be classified into three types depending on the relationship between the nondimensionalized bed shear stress of d ₅₀ and d ₈₄, the representative grain diameters at which 50 and 84% of the volume of the material, respectively, is finer. The nondimensionalized shear stress of d ₈₄ is an important parameter for discussing the rehabilitation of the gravel bed bar. The boundary region for tree overturning can be changed by the effects of plant cover and debris attachment.     

Effects of Sucrose and Citric Acid on the Viscometric Behavior of Aqueous Methylcellulose Solution

The flow of the mixture of methylcellulose (MC)—sucrose—citric acid—water was essentially thixotropic. The viscometric behaviors could be expressed by the power law, S = KDⁿ, where S was shearing stress and D rate of shear. The differential viscosity (ηd) of the mixture was strongly dropped by the addition of citric acid. Sucrose had the tendency to increase ηd of the mixtures except in the case of high sucrose concentration. From the results obtained, it has been concluded that citric acid tends to break the intermolecular hydrogen bonds of MC molecules, and that sucrose tends to keep the hydrogen bonds.     

Shear inactivation of cellulase of Trichoderma reesei

Inactivation of the cellulase of Trichoderma reesei (EC 3.2.1.4) by shear, is of sufficient magnitude to merit consideration in the design of equipment for the enzymatic hydrolysis of cellulose. The inac inactivation constant, k_d, is a function of the flow rate of the enzyme solution through a fine capillary tube. k_d increased slowly at low shear stress, and much more rapidly when the shear stress was greater than 15 dynes cm^?2.     

Effects of shear flow on photosynthesis in a dilute suspension of microalgae

This study investigates the effects of shear stress on photosynthesis in dilute suspensions of Spirulina platensis and Chlorella by measuring the oxygen production rate using a coaxial, double-rotating-cylinder apparatus that generates Couette shear flow. Our device enables up to 0.6 Pa shear stress to be applied, which has the hydrodynamic effect of generating the algal motion and acutely augmenting the oxygen production rate of Spirulina, primarily because the surface area of algae exposed to illumination is increased. However, there is shear-flow limitation on any increase in oxygen production, and the shear stress at maximum oxygen production rate tends to decrease with increasing temperature. The comparative study with Chlorella showed the reverse relationship between oxygen production and shear stress, and the cause of this difference is discussed in terms of several factors such as size, shape, hydrodynamic stress capacity and others.     

Flow dichroism of DNA: a new apparatus and further studies

A new apparatus for the study of flow dichroism of macromolecules is described. The flow is down a long, narrow channel and an unpolarized light beam propagates along the flow direction. For a molecule such as DNA, in which the transition moments of the chromophores are perpendicular to the axis of orientation, an increase of absorbance is observed during flow. The apparatus is best suited for macromolecules which are readily orientable or at high shear gradients so that the extinction angle is close to 0°. The apparatus has the following advantages: dilute macromolecule solutions can be used; high shear gradients are easily obtained; only small volumes of solution are needed. The flow can be stopped rapidly so that relaxation times for disorientation can be studied. The flow dichorism of native, two-stranded DNA has been measured for the molecular weight range of 0.6 × 10⁶ to 125 × 10⁶, and for the shear gradient range (in aqueous solution at 25°C) from 200 sec^?1 to 21000 sec^?1. At a fixed gradient the dichroism increases with molecular weight, but the curve is concave downwards. At a given molecular weight the dichroism increases with increasing shear gradient, but the curve is concave downwards. When the solvent viscosity and temperature are varied, the dichroism is a function of η〈G〉/T showing that the orientation is due to hydro-dynamic shear stress and that the flexibility of DNA in a flow field is not due to local denaturation. The Zimm-Rouse theory with no parameters taken from flow optical data predicts the correct order of magnitude of the dichroism but the experimentally observed shear gradient and molecular weight dependence do not fit the theory. This is an expected result, since the theory is believed to be applicable only at small distortions and extensions of the macromolecule.     

Immunoglobulins in the Eastern Carolines

Serum samples from Micronesian populations on the Pingelap, Mokil, Ponape and Kusaie islands were tested for the immunoglobulin G (IgG) allotypes, Gm (1, 2, 3, 5, 6, 13, 14, 21), and for Inv (1). All four populations have the Gm phenogroups, Gm^1,21, Gm^1,2,21, Gm^1,3,5,13,14, and Gm^1,5,6. The Ponapeans have Gm^1,5,13,14 also. Pedigree analysis shows that the Gm^1,5,6 phenogroup in the Pingelap and Mokil populations is derived from the single offspring of a member of a crew of a whaling ship and that the Gm^1,2,21 phenogroup was introduced by three non-native individuals. The Gm allotypes indicate that the Ponapean and Kusiean populations also have phenogroups from other races, and historical data show that there has been adequate opportunity for this to have occurred. Only the phenogroups Gm^1,21 and Gm^1,5,13,14 appear to be endemic to eastern Micronesia.     

Gammaglobulin groups (Gm and Inv) of various Southern African populations

Data are presented on the distribution of the Gm and Inv groups in approximately 3500 individuals belonging to a number of diverse Southern African populations. The indigenous peoples show the presence of the Gm alleles known to occur in Negroes (Gm^{1, 5, 13, 14}, Gm^{1, 5, 6, 14} and Gm^{1, 5, 6}) but the Bushmen possess some of them in very low frequencies and have, in addition and in appreciable frequencies the Gm¹ and Gm^{1, 13} alleles which have not been reported as occurring in West African populations. The distribution of the Gm^{1, 13} allele in various Bantu-speaking tribes of the sub-continent reveals a marked cline, increasing from north to south along the eastern seaboard. The correlation between the frequency of Gm^{1, 13} and the Khoisan morphological, features present in a number of the tribes, and with the linguistic evidence which has been used to group them is high. The Bushmen possess a Gm^{1, 5} allele and may also have a Gm^{1, 5, 13, 14, 17, 21} allele. A Gm^{1, 2, 5, 13, 14, 17} allele seems to be present in the Bantu. Its presence in Eastern New Guinea would also appear to be indicated by the population data presented here.     

The Multistep Process of Homotypic Neutrophil Aggregation: A Review of the Molecules and Effects of Hydrodynamics

Homotypic adhesion of neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes are supported by L-selectin and β₂-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand the hydrodynamic stresses. Using cone and plate viscometry to apply a uniform linear shear field to suspensions of neutrophils and flow cytometry to quantitate the size distribution of aggregates formed over the time course of formyl peptide stimulation, we conducted a detailed examination of the affect of shear rate and shear stress on the kinetics of cell aggregation. The efficiency of aggregate formation was fit from a mathematical model based on Smoluchowski's two-body collision theory. Over a range of venular shear rates (400–800 s^-1), β90% of the single cells are recruited into aggregates ranging from doublets to groupings larger than sextuplets. Adhesion efficiency fit to the kinetics of aggregation increased with shear rate from β20% at 100s^-1 to a maximum level of β80% at 400 s^-1. This increase to peak adhesion efficiency was dependent on L-selectin and β₂-integrin, and was resistant to shear stress up to β7 dyn/cm². When L-selectin was blocked with antibody, β₂-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s^-1). Aggregates formed over the rapid phase of aggregation remain intact and resistant to shear up to 120 s. At the end of this plateau phase of stability, aggregates spontaneously dissociate back to singlets. The rate of cell disaggregation is linearly proportional to the applied shear rate. The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to firm but reversible adhesion.     

Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti‐Leishmania activities. In the present study, the total syntheses of backbone‐cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)^2,15]‐Gm (one disulfide bond) and [Thr^2,6,11,15,d ‐Pro⁹]‐Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu¹ and Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60 °C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N‐terminal amino acid pGlu¹ and the amidated C‐terminal tripeptide Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr^2,6,11,15,d ‐Pro⁹]‐Gm analogue remains the best active Gm‐derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanical stress tolerance of two microalgae

Aim of the present work is quantifying the mechanical stress generated by some major process equipment used in massive microalgae culturing plants (centrifugal and air-lift pumps, and nozzles) and highlighting its effects on the microalgal population. Two microalgal species were used as test cases: Chlorella vulgaris (unicellular) and Scenedesmus dimorphus 1237 (colonial). The evaluation of the shear effect on algal growth was carried out through measurement of absorbance, photosynthetic activity (oxygen evolution) and variable chlorophyll fluorescence. Cell aggregate development/breakage was effectuated by visual inspection and light scattering. The use of centrifugal pumps for culture recycling strongly affected the growth of C. vulgaris, while nozzles effects were confined to aggregate breakage of S. dimorphus. The analysis of experimental data is supported by the consideration of hydrodynamic stress calculated by: shear rate, shear stress, stress volumes/times, energy dissipation rates, and turbulence microscale size.     

DNA single-strand breakage in mammalian cells induced by redox cycling quinones in the absence of oxidative stress

Quinone-induced cell death is often attributed to oxidative stress during which the formation of DNA strand breaks is thought to play an important role. In this study, extensive DNA damage was observed in human chronic myelogenous leukemic cells (K562) exposed for 15 minutes to low concentrations (15–100 μM) of the redox cycling quinones 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) and menadione. However, DNA strand breakage and cell death could not be attributed to oxidative stress as the intracellular level and redox status of the reducing equivalents NADP(H) and GSH were unaffected. The intracellular level of NAD⁺ was found to correlate well with the extent of DNA repair (r = 0.93, P < 0.02) and cell proliferation (r = 0.96, P < 0.01) in cells exposed to the quinones. In contrast, a significant decrease in the level of intracellular ATP was only observed in cells exposed to menadione (50–100 μM). These results suggest that redox cycling quinones are capable of inducing DNA damage in mammalian cells by a mechanism that does not involve oxidative stress. Following DNA damage, cell death is dependent on the availability of NAD⁺, which may be key to the rapid repair of strand breaks. © 1995 John Wiley & Sons, Inc.     

Determination of shear stress thresholds in toxic dinoflagellates cultured in shaken flasks: Implications in bioprocess engineering

Marine dinoflagellates are potentially important innovative sources of high-value toxins in biomedical, toxicological and chemical research programs. However, little is known about the difficulties related to dinoflagellate cultures. In this article, we demonstrate that the shear sensitivity of cells may be one of the main causes. The red-tide Protoceratium reticulatum, a producer of yessotoxins, was used to examine the effect of hydromechanical shear stress associated with intermittent fluid agitation on cell growth. Shaken flasks, widely used in biotechnological process research, were used as model bioreactors, as hydrodynamic shear stress is relatively easy to quantify in them. Intermittent turbulence regime was characterized by three key operating variables: shear stress, cycle time or shaking frequency, and fraction of time shaken per agitation cycle. The light/dark cycle was also used as another variable. Cell damage depended on the combination of the above-mentioned variables. A damage threshold was observed at an average shear stress of approximately 0.16 mN m⁻² (equivalent shear rate of 0.12 s⁻¹). Cell damage from exposure to average detrimental shear stress was also shown to be greater in the dark than in the light period. Preliminary experiments demonstrated that dinoflagellates are also much more sensitive to bubbling than the majority of common fragile microalgae. Although slight toxicity of Pluronic F-68 was observed at a concentration of 0.05% (w/v), this protective medium additive considerably reduced cell breakage. On the other hand, no cell adaptation to stronger shear stress was observed. Finally, the implications of the proposed approach for the hypothetical mass culture of dinoflagellates in bioreactors were also thoroughly assessed.     

Effect of Concentration and Temperature on Flow Properties of Alyssum homolocarpum Seed Gum Solutions: Assessment of Time Dependency and Thixotropy

Effects of different shear rates (14, 25, and 50 s⁻¹), gum concentrations (3%, 3.5%, and 4%), and temperatures (5–65 °C) on flow properties of Alyssum homolocarpum seed gum solutions were investigated using a rotational viscometer. The experimental data were fitted with three time-dependent rheological models, namely second-order structural kinetic model, Weltman model, and first-order stress decay model with a non-zero stress value. The rate constant and extent of viscosity strongly depended on the shear rate, gum concentration, and temperature. It was found that A. homolocarpum seed gum samples exhibited shear thinning and thixotropic behavior for all concentrations and temperatures. The amount of structural breakdown decreased with shear rate, but it did not have a general trend with concentration and temperature. The extent of thixotropy increased with increasing gum concentration and decreased with increasing temperature and shear rate. In this work, the decay rate constant generally increased with increasing shear rate; however, it did not have any trend with concentration and temperature.     

Gm and Km(Inv) frequencies in two Roumanian populations

Summary Serum samples from 170 unrelated individuals from the Suceava District of Roumania and from 199 unrelated individuals from Bucharest, Roumania were tested for Gm(1, 2, 3, 5, 6, 13, 14, 17, 21) and Km(1)[Inv(1)]. Selected samples were also tested for Gm(15) and Gm(16).The frequencies of the three common Caucasoid haplotypes, Gm ^{3, 5, 13, 14}, Gm ^{1, 17, 21}, and Gm ^{1, 2, 17, 21} in these two populations were found to be similar to those in neighboring Slavic states and Hungary. Racial admixture was evidenced by the presence of the Gm ^{1, 13, 15, 16, 17} and Gm ^{1, 3, 5, 13, 14} haplotypes, which are primarily Mongoloid, and the Gm ^{1, 5, 13, 14, 17} haplotype which is primarily Negroid.Comparisons of these data with those from earlier studies of populations from Central Europe indicate that the frequency of the Gm ^{3, 5, 13, 14} haplotype within this region is high and essentially uniform. Published data for several blood group systems also indicate essentially uniform distributions of frequencies in this region. It is suggested that this region may be the center of a clin that radiates from it.Post-Doctoral Fellow supported by NIH Training Grant Gm07004.     

Photolithotrophic cultivation of <Emphasis Type="Italic">Laminaria japonica</Emphasis> gametophyte cells in a silicone tubular membrane-aerated photobioreactor

Gametophyte cells of brown algae Laminaria japonica were employed both in a modified silicone tubular membrane-aerated photobioreactor (bubble-less cultivation mode) and a bubble-column photobioreactor (bubbling cultivation mode), to study different gas–liquid mixing modes on cell growth rate and cell physiological status. With an inoculum density of 50 mg DCW l⁻¹, in modified artificial Pacific seawater (APSW) medium at 13°C, light intensity of 60 μE m⁻² s⁻¹, light cycle of 16/8 h L/D, and aeration rate of 60 ml min⁻¹, the specific growth rates were 0.082 d⁻¹ for bubble-less mode and 0.070 d⁻¹ for bubbling mode with biomass, in the form of dry cell density, increasing 10.9 and 6.8 times, respectively, during the 36 days’ photolithotrophic cultivation. The specific oxygen evolution rate under bubble-less mode was 39.6% higher than under bubbling mode on the 18th day. The gametophyte cells grew in cell aggregates with clump sizes, at day 36, of 1.5 mm and 0.5 mm diameter under bubble-less and bubbling mode respectively and cell injury percentages of 5.1% and 21.1%, respectively. The silicone tubular membrane-aerated photobioreactor was better suited for the cultivation of fragile macroalgal gametophyte cells due to the absence of hydrodynamic shear stress caused by fluid turbulence and the presence of a bubble-less gas supply.     

Processing character of the action of wheat endonucleases WEN1 and WEN2. Kinetic parameters

The wheat seedling endonucleases WEN1 and WEN2 dependent on Mg²⁺, Ca²⁺, and S-adenosyl-L-methionine (SAM) and sensitive to the substrate DNA methylation status have an expressed processing action. The enzymes hydrolyze DNA at a few subsequent stages: first, they split λ phage DNA specifically at CNG-sites (WEN1) with liberation of large fragments; second, they hydrolyze these fragments to 120–140 bp oligonucleotides that finally are hydrolyzed to very short fragments and mononucleotides. Initial stages of DNA hydrolysis may proceed in the absence of Mg²⁺, but subsequent hydrolysis stages are very strongly stimulated by Mg²⁺. It cannot be ruled out that modulation of enzymatic activity with Mg²⁺ and probably with DNA fragments formed is associated with reorganization of the structure of eukaryotic (wheat) endonucleases with respective changes in their catalytic properties and site specificity of action. Michaelis constant value for WEN1 endonuclease on hydrolysis of methylated λ phage DNA containing Cm⁵CWGG and Gm⁶ATC sites is four-fold lower compared with that observed on hydrolysis of unmethylated λ phage DNA. This may indicate that affinity of WEN1 enzyme to methylated DNA is higher than that to unmethylated DNA. In the presence of SAM, the Michaelis constant for WEN2 on the DNA hydrolysis stage characterized by formation of 120–140 bp fragments is decreased, but for WEN1 it is increased by 1.5–2.0-fold. This means that SAM inhibits WEN1 but stimulates WEN2. Thus, wheat endonucleases WEN1 and WEN2 differ significantly in affinities to substrate DNAs with different methylation status, in velocities of DNA hydrolysis, and time of production of DNA fragments of similar length. It seems that the investigated plant endonucleases can hydrolyze DNA in the nucleus as well to both large and very short fragments including mononucleotides, that is, in particular, essential for utilization of cell nucleic acid material during apoptosis.     

Investigation on the Anticancer and Antibacterial Mechanism of Thiazoline/Imidazoline Derived Palladium(II) Complexes

Biological activities of a series of palladium(II) complexes (M1–M9) bearing N^∩N, N^∩S, and N^∩O chelating ligands are reported. The palladium complexes were tested for their cytotoxic properties against human cervical cancer (HeLa) cells and antibacterial activity against Gm^+ve and Gm^–ve bacteria. Among the palladium complexes studied (M1-M9), the complex M5, M8, and M9 were found to be more effective in inhibiting the proliferation of HeLa cells. Hence, these complexes were further investigated for their potential role in cellular damage and apoptosis. DCFDA staining, Rhodamine 123 staining and DNA cleavage assay revealed that complex M5, M8 and M9 induced apoptotic cell death in HeLa cells through ROS generation, DNA damage and mitochondrial depolarization. Computational and titration studies also indicated strong electrostatic interaction with DNA groove. Most of the complexes exhibited good antibacterial activity against both Gm^+ve and Gm^−ve bacteria. The antibacterial activity of the compounds could not be correlated with their anticancer activity indicating a differential mechanism at their effective concentrations. The detailed study on the antibacterial mechanism of the most potent complex M7 revealed that it exerted its antibacterial activity by inhibiting the function of FtsZ and perturbing the localization of the Z-ring at the mid cell.