The density of protein precipitates and its effect on centrifugal sedimentation

Isoelectric soya-protein precipitate densities were measured for mean particle sizes ranging from 3.4-65 mum by gradient centrifugation, centrifugation in water-immiscible solvents, tracerdilution, gravity sedimentation of isolated particles. Coulter counter volume determination, and a comparison of Coulter counter and centrifugal sedimentation size distributions. The immiscible system and tracer dilution methods were both found to be unreliable due to experimental uncertainties. The Coulter counter volume measurement indicated the existence of a density-size relationship with the aggregate density decreasing as the size increased. Comparison with sedimentation measurements showed that the Coulter counter measures 80% of the total aggregate volume for 6-mum particles. The relation between aggregate density (rho(a), kg m (-3)) and size (d, mum) was measured for isoelectric soya protein and casein precipitated by ammonium sulfate, using a comparison of the Coulter counter size distribution and centrifugal sedimentation. The functions were described for soya by \documentclass{article}\pagestyle{empty}\begin{document}$$ \rho _a - 1004 = 246d;{ - 0.408} $$\end{document} and for casein by \documentclass{article}\pagestyle{empty}\begin{document}$$ \rho _a - 1136 = 31d;{ - 0.441} $$\end{document} The gradient centrifugation method measured the buoyant density of hydrated protein precipitate which was independent of size, and is consistent with an aggregate structure consisting of primary particles. However, the aggregate structure was not described for all sizes by the theoretical cubic packing of hard-sphere primary particles, nor by the successive random addition of primary particles. The density-size functions indicated up to a fivefold difference in Stokes settling velocities compared to those calculated assuming a constant density difference.     

Molecular analysis of glycogen storage disease type Ia in Iranian Azeri Turks: identification of a novel mutation

Glycogen storage diseases (GSDs) are caused by abnormalities in enzymes that are involved in the regulation of gluconeogenesis and glycogenolysis. GSD I, an autosomal recessive metabolic disorder, is the most common GSD and has four subtypes. Here, we examined GSD Ia caused by the defective glucose-6-phosphatase catalytic (G6PC) gene. We investigated the frequency of GSD Ia and clarified its molecular aspect in patients with the main clinical and biochemical characteristics of GSD, including 37 unrelated patients with a mean age of three years at the time of diagnosis. All patients belonged to the Azeri Turkish population. Hypoglycaemia and hypertriglyceridaemia were the most frequent laboratory findings. Mutations were detected by performing direct sequencing. Mutation analysis of the G6PC gene revealed that GSD Ia accounted for 11% in GSD patients with involvement of liver. Three patients were homozygous for R83C mutation. In addition, a novel stop mutation, Y85X, was identified in a patient with the typical features of GSD Ia.     

Enzyme loading of electrically homogeneous human red blood cell ghosts prepared by dielelctric breakdown.

Human red blood cell ghosts were prepared by electrical haemolysis at 0 degrees C in isotonic solutions using a discharge chamber which was part of a high voltage circuit. The size distribution of the ghosts was normally distributed, the modal (=mean) volume was approx. 115 mum3, performing the electrical haemolysis in the following solution: 105 mM KCI, 20 mM NaCL, 4mM MgCl2, 7.6 mM Na2HPO4, 2.94 mM NaH2PO4, 10 mM glucose, pH 7.2. Resealing was carried out at o degrees C for 10 min (after the haemolytic step) and then for further 20 min at 37 degrees C. The mean volume of the ghost preparation could be changed by variation of the phosphate concentration in the above solution replacing a part of NaCl by phosphate (5 mM phosphate: 94 mum3, 15 mM phosphate: 135 mum3). The breakdown voltage of the ghost cell membranes measured with a hydrodynamic focusing Coulter Counter depends on the mean volume (94 mum3 = 1.04 V, 134 mum3 = 1.36 V). On the other hand, the breakdown voltage is constant throughout each size distribution pointing to an "electrically homogeneous" ghost preparation. The sensitiviity of the Coulter Counter to detect electrical inhomogeneities in the membranes of a ghost population is demonstrated by dielectric breakdown measurements of an apparently normally distributed ghost preparation containing two different "electrically homogeneous" ghost population i.e. with two different breakdown voltages. The ghost cells obtained by electrical haemolysis in the above solution containing 10mM phosphate were fairly impermeable to sucrose and behave like an ideal osometer. It is further demonstrated that ghost cells can be loaded with enzymes (e.g. urease) and drugs using this technique and that these loaded ghost cells can be used as bioactive capsules for clinical application.     

Measurement of gastric blood flow with radioactive microspheres.

The validity of using radioactive microspheres (15 +/- 5-mum diameter) to measure gastric blood flow and its partition between gastric wall layers was investigated in anesthetized dogs with a chambered segment of gastric corpus. Total flow measured by a venous effluent technique demonstrated close correlation with microsphere-measured flow (r = 0.98, slope = 0.95) in 12 dogs given histamine, gastrin, or isoproterenol. In 12 histamine-stimulated dogs, mucosal flow measured by aminopyrine clearance and by microspheres also showed good agreement (r = 0.96, slope = 0.83). No evidence was found to indicate that microspheres altered hemodynamic or gastric function. In all experiments less than 1% of the total gastric radioactivity passed through arteriovenous shunts. The mucosa always contained a statistically adequate number of spheres (greater than 400), but the submucosa and muscularis frequently did not. Microspheres of all sizes mixed adequately in large arteries, but a significant difference was found in the distribution of 16- and 26 mum spheres between mucosa and submucosa, presumably because of streaming of the larger spheres past mucosal arteries. It was concluded that, with the techniques developed in our laboratory, microspheres could be a highly useful tool for quantitating gastric regional blood flow under a variety of experimental conditions.     

Structure of Escherichia coli After Freeze-Etching

Survival of Escherichia coli, quick-frozen under conditions similar to those employed for freeze-etching, is close to 100%. For determination of cell shrinkage, the diameters of freeze-etched E. coli cells (average, 0.99 mum) were compared with those of preparations after negative staining and after ultrathin sectioning. Negatively stained cells measured from 0.65 to 1.0 mum in diameter, and ultrathin sections showed average cell diameters of 0.70 mum. Freeze-etched replicas of logarithmically growing, as well as stationary, E. coli B cells revealed a smooth, finely pitted cell surface in contrast to cell surfaces seen with other preparative methods. The frozen cell wall may cleave in two planes, exposing (i) a smooth fracture face within the lipid layer and (ii) in rare instances an ill-defined particulate layer. Most frequently, however, cleavage of the envelope occurred between wall and protoplasmic membrane; large areas of the membrane were then exposed and showed a surface studded with predominantly spherical particles, an appearance which did not significantly change when the cells were fixed in formaldehyde and osmium tetroxide before freeze-etching. The distribution of these particles differed between logarithmically growing cells and stationary cells.     

Effect of diameter on membrane capacity and conductance of sheep cardiac Purkinje fibers

Membrane electrical properties were measured in sheep cardiac Purkinje fibers, having diameters ranging from 50 to 300 mum. Both membrane capacitance and conductance per unit area of apparent fiber surface varied fourfold over this range. Membrane time constant, and capacitance per unit apparent surface area calculated from the foot of the action potential were independent of fiber diameter, having average values of 18.8 +/- 0.7 ms, and 3.4 +/- 0.25 muF/cm2, respectively (mean +/- SEM). The conduction velocity and time constant of the foot of the action potential also appeared independent of diameter, having values of 3.0 +/- 0.1 m/s and 0.10 +/- 0.007 ms. These findings are consistent with earlier suggestions that in addition to membrane on the surface of the fiber, there exists a large fraction of membrane in continuity with the extracellular space but not directly on the surface of the fiber. Combining the electrical and morphological information, it was possible to predict a passive length constant for the internal membranes of about 100 mum and a time constant for chaning these membranes in a passive 100-mum fiber of 1.7 ms.     

Alleles of the major histocompatibility complex play a role in the pathogenesis of pancreatic acinar atrophy in dogs

Exocrine pancreatic insufficiency (EPI) is a disease wherein pancreatic acinar cells fail to synthesize and secrete sufficient amounts of digestive enzymes for normal digestion of food. EPI affects many dog breeds, with a dramatically higher prevalence in the German shepherd dog (GSD) population. In this breed and perhaps others, EPI most often results from degeneration of the acinar cells of the pancreas, a hereditary disorder termed pancreatic acinar atrophy (PAA). Evidence of lymphocytic infiltration indicates that PAA is an autoimmune disease, but the genetic etiology remains unclear. Data from global gene expression and single nucleotide polymorphism profiles in the GSD suggest the involvement of the major histocompatibility complex [MHC; dog leukocyte antigen (DLA)]. To determine if alleles of the MHC influence development of EPI, genotyping of polymorphic class I (DLA-88) and II loci (DLA-DRB1, DLA-DQA1, and DLA-DQB1) was carried out for 70 affected and 63 control GSDs, and four-locus haplotypes were determined. One haplotype containing a novel allele of DLA-88 is very highly associated with EPI (OR?>?17; P?=?0.000125), while two haplotypes were found to confer protection from EPI (P?=?0.00087 and 0.0115). Described herein is the genotyping of MHC class I and II loci in a GSD cohort, establishment of four-locus haplotypes, and association of alleles/haplotypes with EPI.     

DNA of a Human Hepatitis B Virus Candidate

Particles containing DNA polymerase (Dane particles) were purified from the plasma of chronic carriers of hepatitis B antigen. After a DNA polymerase reaction with purified Dane particle preparations treated with Nonidet P-40 detergent, Dane particle core structures containing radioactive DNA product were isolated by sedimentation in a sucrose density gradient. The radioactive DNA was extracted with sodium dodecyl sulfate and isolated by band sedimentation in a preformed CsCl gradient. Examination of the radioactive DNA band by electron microscopy revealed exclusively circular double-stranded DNA molecules approximately 0.78 mum in length. Identical circular molecules were observed when DNA was isolated by a similar procedure from particles that had not undergone a DNA polymerase reaction. The molecules were completely degraded by DNase 1. When Dane particle core structures were treated with DNase 1 before DNA extraction, only 0.78-mum circular DNA molecules were detected. Without DNase treatment of core structures, linear molecules with lengths between 0.5 and 12 mum, in addition to the 0.78-mum circles were found. These results suggest that the 0.78-mum circular molecules were in a protected position within Dane particle cores and the linear molecules were not within core structures. Length measurements on 225 circular molecules revealed a mean length of 0.78 +/- 0.09 mum which would correspond to a molecular weight of around 1.6 x 10(6). The circular molecules probably serve as primer-template for the DNA polymerase reaction carried out by Dane particle cores. Thermal denaturation and buoyant density measurements on the Dane particle DNA polymerase reaction product revealed a guanosine plus cytosine content of 48 to 49%.     

Reculture of Electrofocused Foetal Rabbit Skin Cells

Cells from a primary culture of 30 day-old foetal rabbit skin were electrofocused and recultured. Certain fractions (pH 10-3) grew best on reculture. Electrofocused cells formed monolayers in six weeks. The focused cells were dormant for a period preceding growth. Increased cell density and cell contact of recultured cells occurred when they were exposed to millipore filtered media from unfocused cells. Primary culture cells exposed to 1 mw L -aspartic acid, L -ornithine, or L -lysine in culture media, when treated with trypsin and electrofocused indicated different profiles of pH maxima. These recultured cells differed from the primary culture cells in morphology and growth rate.     

Cultivation of virus antigen in fibroblast cells using a glass fiber bed reactor

The anchorage-dependent cell line, MRC-5, was cultivated successfully on glass fibers with diameters ranging from 24 to 120 mum, despite vast differences in substrate curvature. Multilayer cell growth was observed, particularly for fiber diameters 30 mum and below, which differed from the typical monolayer growth observed in T-flask cultivations. Cells were maintainable at a reduced incubation temperature and were demonstrated to support virus replication for the 21-day antigen production period. Direct microscopic observation, along with indirect calculations, indicated that only a small fraction (about 10%) of the total available fiber surface area was occupied by cells. Thus, productivity per unit surface area was replaced by productivity per unit medium volume when evaluating fiber bed performance. Antigen and protein yields, as well as nutrient uptakes, were 1.5- to 2.5-fold greater than parallel T-flask cultures when compared on this basis. Corresponding available surface area-based values were 10- to 15-fold lower for the fiber bed reactor. The multilayer cell morphology obtained in the fiber bed was attractive for antigen production when immobilized in a column reactor system. (c) 1993 John Wiley & Sons, Inc.     

Blood lymphocyte volumes and diameters in patients with chronic lymphocytic leukemia and normal controls

The electronic modal lymphocyte volumes of 151 patients with chronic lymphocytic leukemia (CLL) and 305 normal controls were determined by the hydrodynamically focused multi-channel Coulter TF analyser. The mean volumes of the normally distributed groups were 166 +/- 19.3 (range 126-216) fl in patients with CLL and 206 +/- 14.4 (range 126 +/- 246) fl in normal controls. The calculated cell diameters were 6.8 (6.2-7.4) micron and 7.3 (6.8-7.8) micron respectively. Our data do not support previous reports about relations between cell size and clinical stages of the Rai and Binet classifications.     

Denaturation mapping of R factor deoxyribonucleic acid.

The R factor NR1 consists of two components: a resistance transfer factor which harbors the tetracycline resistance genes (RTF-TC) and the r-determinants component which harbors the other drug resistance genes. Using partial denaturation mapping it is possible to distinguish the RTF-TC region from the r-determinants region of the composite R factor NR1 DNA which has a contour length of 37 mum and a density of 1.712 g/ml. The r-determinants region was a relatively undenatured 8.5-mum segment of the molecule when the deoxyribonucleic acid was partially denatured at pH 10.7. An RTF-TC genetic segregant of NR1 which had lost the r-determinants component had a contour length of 28.7 mum and a density of 1.710 g/ml. Characterization of an RTF-TC using partial denaturation mapping at pH 10.7 confirmed that the relatively undenatured 8.5-mum r-determinants segment of the composite R factor had been deleted. Circular, transitioned NR1 DNA molecules (1.716 to 1.718 g/ml), whose contour lengths were consistent with an RTF-TC plus an integral number of tandem copies of r-determinants, were also characterized by denaturation mapping. The relatively undenatured region in these molecules had a length equal to an integral number of copies of r-determinants and was located at the same site in the partially denatured RTF-TC as the single copy of r-determinants in the 37-mum composite NR1. This indicates that there is a unique integration site for r-determinants in the RTF-TC component. The R factor UCR122, a TC deletion mutant of NR1, was also characterized by denaturation mapping. The translocation of the TC resistance gene(s) on the denaturation map permitted the alignment of the denaturation map with the heteroduplex map of Sharp et al. (u073). Linear and circular monomeric and presumed multimeric r-determinants DNA molecules (p = 1.718 g/ml) were partially denatured at a higher pH (11.10). The r-determinants multimers showed a repeating 8.3-mum (monomeric) partial denaturation pattern indicating a head-to-tail arrangement of monomers in these poly-r-determinant molecules.     

Fluorescence detection for the XLI analytical ultracentrifuge

Analytical ultracentrifugation (AUC) provides first-principle hydrodynamic and thermodynamic information concerning the size, shape and interactions of macromolecules. The fundamental measurement needed in AUC is the macromolecular concentration as a function of radial position and time. Currently, the Beckman Coulter XLI analytical ultracentrifuge may be equipped with absorbance and refractive detectors, which provide complementary concentration determinations. For detecting trace quantities of materials, fluorescence detection offers unique advantages over either absorbance or interference detection. A prototype fluorescence detector for the XLI analytical ultracentrifuge has been developed and its characteristics determined. An Ar(+) laser provides a continuous 488-nm excitation beam. Radial resolution is achieved by scanning the focused beam along a radial axis. Detection of the fluorescence signal uses a co-axial, front-face optical configuration to reduce inaccuracies in the concentration caused by inner filter effects. A high-speed A/D data acquisition system allows the fluorescence intensity to be monitored continuously and at a sufficiently high angular resolution so that at any radial position the intensities from all of the samples may be acquired at each revolution. The fluorescence detector is capable of detecting concentrations as low as 300 pM for fluorescein-like labels. The radial resolution of the fluorescence detector is comparable to that of the absorbance system. Both sedimentation velocity and sedimentation equilibrium measurements may be made with the fluorescence detector. Results are presented comparing data acquired using the fluorescence with those acquired using the absorbance detector.     

Size measurements on isolated rat heart cells using coulter analysis and light scatter flow cytometry

Isolated ventricular muscle cells from the adult rat heart have been examined by both Coulter analysis and light scatter flow cytometry. The dispersed cell preparations contain two main cell types: viable, rod-shaped cells and damaged, round cells. Coulter analytical techniques provided statistical data on cell volume for both cell types. The contribution of each population to the Coulter pulse height distributions were separated by a subtraction method using data obtained from digitonin-treated preparations that contain only round cells. A shape factor for cells aligned with the flow direction was computed from light microscope measurements and the effects of cell orientation within the Coulter aperture were approximately assessed. The estimated volumes for intact myocytes compare favourably with those reported in the literature. No significant size difference was observed between fresh and fixed cells.Narrow angle, forward light scatter measurements were made on individual cells flowing across a focused laser beam. Both scatter pulse height and pulse width (pulse duration) distributions were collected. Values for myocyte length calculated from pulse width information agree well with published data and confirm that the hydrodynamic forces in the flow system produced alignment of the cells with the flow direction. Scatter pulse width distributions reveal two distinct peaks assignable to either rod or round cells. Preliminary electronic gating experiments, using pulse height signals, suggest that signals derived from round cells could be eliminated entirely using a gating regime based on pulse width. This would enable flow cytometric measurements to be made on only the intact myocytes present in heterogeneous preparations.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.     

Interaction of urokinase with alpha2-macroglobulin investigated by isoelectric focusing. Evidence for non-specific dissociable binding.

The binding of urokinase to human alpha2M (alpha2-macroglobulin) was investigated in comparison with the formation of the equimolar trypsin-alpha2M complex. Experiments were performed by molecular-sieving on Sephadex G-200, subunit conversion by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis after reduction and isoelectric focusing in linear sucrose gradients with ampholytes pH 3.5-10.0. Urokinase activity was determined with alpha-N-acetyl-L-lysine methyl ester and by activation of plasminogen on unheated fibrin plates. alpha2M was determined by single radial immunodiffusion. alpha2M was capable of binding some urokinase by a non-specific type of attachment that could be disrupted by isoelectric focusing but not by gel filtration. The pI of the undissociated trypsin-alpha2M complex was 6.0, and differed from that of the pure alpha2M (5.2-5.4). Likewise the pI of the immunoreactive alpha2M was 5.2 after exposure to urokinase, whereas the dissociated urokinase focused at pI 10.2. This indicated lack of true inhibitor-complex formation, which was also sustained by total absence of subunit conversion. The results are in agreement with our previous findings with pancreatic and urinary kallikreins.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10³–10⁴ V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strenght it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yields a mean value of about 1.6 V for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 °C as shown by incorporation of ¹³¹I-labeled albumin and repeated dielectric breakdown.     

Effect of altered G levels on deposition of particulates in the human respiratory tract.

The primary mode of depositon of particles in the respiratory tract in the size range 0.5-10 mum diam (unit density) is sedimentation. The rate of sedimentation is directly proportional to the velocity of settling of the particle. Therefore, the total deposition of particles in the respiratory tract as well as the region of deposition is affected by changes in gravity. Human subjects were exposed to aerosols of 2.02-mum-diam polyvinyltoluene particles at 0, 0.5, 1.0, and 2.0 G. Total deposition was measured at each G level. Results indicate an almost linear increase in total deposition with increasing G levels over the range studied. The deposition measured at 1 G was less than reported in earlier experiments and the deposition at levels less than 1 G was less than had been calculated by Muir and Beeckmans. These data show that although sedimentation plays the major role in depostion of 2.02 mum particles, it is less than previously described.     

Genome-Wide Analysis in German Shepherd Dogs Reveals Association of a Locus on CFA 27 with Atopic Dermatitis

Humans and dogs are both affected by the allergic skin disease atopic dermatitis (AD), caused by an interaction between genetic and environmental factors. The German shepherd dog (GSD) is a high-risk breed for canine AD (CAD). In this study, we used a Swedish cohort of GSDs as a model for human AD. Serum IgA levels are known to be lower in GSDs compared to other breeds. We detected significantly lower IgA levels in the CAD cases compared to controls (p = 1.1×10⁻⁵) in our study population. We also detected a separation within the GSD cohort, where dogs could be grouped into two different subpopulations. Disease prevalence differed significantly between the subpopulations contributing to population stratification (λ = 1.3), which was successfully corrected for using a mixed model approach. A genome-wide association analysis of CAD was performed (n _cases = 91, n _controls = 88). IgA levels were included in the model, due to the high correlation between CAD and low IgA levels. In addition, we detected a correlation between IgA levels and the age at the time of sampling (corr = 0.42, p = 3.0×10⁻⁹), thus age was included in the model. A genome-wide significant association was detected on chromosome 27 (p_raw = 3.1×10⁻⁷, p_genome = 0.03). The total associated region was defined as a ∼1.5-Mb-long haplotype including eight genes. Through targeted re-sequencing and additional genotyping of a subset of identified SNPs, we defined 11 smaller haplotype blocks within the associated region. Two blocks showed the strongest association to CAD. The ∼209-kb region, defined by the two blocks, harbors only the PKP2 gene, encoding Plakophilin 2 expressed in the desmosomes and important for skin structure. Our results may yield further insight into the genetics behind both canine and human AD.     

Identities of four low-molecular-mass subunits of the photosystem I complex from Anabaena variabilis ATCC 29413. Evidence for the presence of the psaI gene product in a cyanobacterial complex

Photosystem I (PSI) complex of Anabaena variabilis ATCC 29413 consists of at least 11 subunits, 9 of which are resolved by high resolution gel electrophoresis. N-terminal amino acid sequences of the four subunits with molecular masses of 6.8, 5.2, 4.8 and 3.5 kDa were determined. Based on the sequence homology, the 3.5 kDa subunit was revealed to correspond to PSI-I (the gene product of psaI), which had so far been detected only in higher plant PSI complexes. The 6.8 kDa protein and 4.8 kDa protein were identified as gene products of psaK and psaJ, respectively. The 5.2 kDa protein was homologous to a 4.8 kDa subunit of PSI of the thermophilic cyanobacterium Synechococcus vulcanus, suggesting that this protein is a component of PSI in cyanobacteria.