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It has been well known that Vibrio cholerae inhabit in environmental water. As many patients infected with cholera toxin-producing V. cholerae O1 (toxigenic V. cholerae O1) emerge in Kolkata, India, it has been thought that toxigenic V. cholerae O1 is easily detected in environmental water in Kolkata. However, we could not isolate toxigenic V. cholerae O1 from environmental water in Kolkata, though NAG Vibrio (generic name of V. cholerae non-O1/non-O139) is constantly detected. To clear the reason for the non-isolation of toxigenic V. cholerae O1, we examined the viability of V. cholera O1 and NAG Vibrios in the artificial low ionic strength aquatic solution. We found that the viability of toxigenic V. cholerae O1 in the solution is low, but that of NAG Vibrios is high. Subsequently, we examined the viability of NAG Vibrios possessing cholera toxin gene (ctx) in the same condition and found that the viability of these NAG Vibrios is low. These results indicate that the existence of ctx in V. cholerae affects the viability of V. cholerae in the aquatic solution used in this experiment. We thought that there was closely relation between the low viability of toxigenic V. cholerae O1 in the artificial low ionic strength aquatic solution and the low frequency of isolation of the strain from environmental water.

The use of monoclonal antibodies (mAbs) for the treatment of a variety of diseases is rapidly growing each year. Many mAbs are administered intravenously using i.v. bags containing 0.9% NaCl (normal saline). We studied the aggregation propensity of these antibody solutions in saline and compared it with a low ionic strength formulation buffer. The mAb studied in this work is prone to aggregate, and is known to form a viscoelastic network at the air-solution interface. We observed that this interfacial elasticity increased when formulated in saline. In the bulk, the mAbs exhibited a tendency to self-associate that was higher in saline. We also studied the aggregation of the mAbs in the presence of polysorbate-20, typically added to formulations to mitigate interfacial aggregation. We observed that with surfactants, the presence of salt in the buffer led to a greater mAb adsorption at the interface and resulted in the formation of more particulate aggregates. Our results show that the addition of salt to the buffer led to differences in the interfacial aggregation in mAb formulations, showing that stress studies used to screen for mAb aggregation intended for i.v. administration should be performed in conditions representative of their intended route of administration.

Liquid-liquid phase separation (LLPS) of a monoclonal antibody solution was investigated at low ionic strength in the presence of oligovalent anions, such as citrate, trimellitate, pyromellitate and mellitate. Phase separation was observed at the isoelectric point of the antibody at pH8.7 as well as in more acidic pH regions in the presence of the tested oligovalent ions. This can be attributed to charge neutralization via binding of the oligovalent anions to the positively charged antibody. The influence of the anion concentration on liquid-liquid phase separation with respect to the net charge of the antibody was examined. Similarities to the formation of a complex coacervate were shown to apply. These findings enable us to understand the usage of excipients to rationally induce or avoid liquid-liquid phase separation at low ionic strength. Furthermore we present a method to directly examine the competition of different ions for the solvation shell, called buffer equilibration.

It has been regarded that Vibrio cholerae O1 inhabit in environmental water. As many cholera patients emerge in Kolkata, it has been thought that V. cholerae O1 is easily detected in environmental water in Kolkata. However, the detection of V. cholerae O1 is rare, though other V. cholerae (NAG Vibrio) is constantly detected. To clear the reason for the difference of the detection rate of two Vibrios, we examined the viability of V. cholera O1 and NAG Vibrios in low ionic strength aquatic medium. We observed greater declining viability of V. cholerae O1 possessing cholera toxin gene (ctx) in low ionic strength solution, but the decline of NAG Vibrios non-possessing ctx is small. To evaluate the concerning of ctx in the viability, we examined the viabilities of V. cholerae O1which do not possess ctx and NAG Vibrios possessing ctx under the same condition. The result indicated that the existence of the ctx induces the decrease the viability of the host in low ionic strength solution. The decrease observed in this experiment might relate with the low detection of V. cholerae O1 possessing ctx in environmental water, though NAG Vibrio is constantly detected.

Lipobeads are spherical vesicles of lipid membrane supported by a hydrogel core that hold promise as next-generation biosensors. However, previous methods for generating lipobeads had limitations due to the use of organic solvents and low ionic strength solutions. To overcome these limitations, we developed a method for forming lipobeads by using a gel-assisted swelling approach. Our results show that lipobeads form in osmotic strength solution and supposed to contain no residual organic solvent.

It has been regarded that Vibrio cholerae O1 inhabit in environmental water. As many cholera patients emerge in Kolkata, it has been thought that V. cholerae O1 is easily detected in environmental water in Kolkata. However, the detection of V. cholerae O1 is rare, though other V. cholerae (NAG Vibrio) is constantly detected. To clear the reason for the difference of the detection rate of two Vibrios, we examined the viability of V. cholera O1 and NAG Vibrios in low ionic strength aquatic medium. We observed greater declining viability of V. cholerae O1 possessing cholera toxin gene (ctx) in low ionic strength solution, but the decline of NAG Vibrios non-possessing ctx is small. To evaluate the concerning of ctx in the viability, we examined the viabilities of V. cholerae O1which do not possess ctx and NAG Vibrios possessing ctx under the same condition. The result indicated that the existence of the ctx induces the decrease the viability of the host in low ionic strength solution. The decrease observed in this experiment might relate with the low detection of V. cholerae O1 possessing ctx in environmental water, though NAG Vibrio is constantly detected.

Impurities in indicator salts can significantly bias spectrophotometric pH determinations. In this work, two purified sulfonephthalein indicators, meta-cresol purple (mCP) and phenol red (PR), were tested for analysis of freshwater pH on the free hydrogen ion concentration scale. These two purified indicators were characterized for the first time under low ionic strength conditions, providing their molar absorption coefficients and dissociation constants along with their temperature dependence from 8 °C to 30 °C. At 25 °C, the infinite dilution constants ( pKIo) were determined to be 8.6606 and 8.0642 for mCP and PR, respectively. The accuracy and precision of the method, evaluated with a variety of buffers with known pH, were found to be +0.0014 pH units and ±0.0022 pH units, respectively (n = 30). The pH values of different freshwater samples were also determined using both indicators. The mCP and PR results were all within ± 0.01 pH units of each other with three out of seven pH differences within ± 0.001 pH units, indicating the high consistency between these two indicator methods. The work presented here is the first parallel comparison with two purified indicators used to determine pH of the same freshwater samples.

Summary The effects of 5 mm l-histidine (l-His) on water-binding capacity, gel strength, thermal gelling properties of chicken breast myofibrillar proteins (MPs) in 1 mm NaCl or 0.6 m NaCl solutions (pH 7.0) were investigated. l-His could significantly increase the solubility and thermal gelling ability of MPs in 1 mm NaCl. l-His at 1 mm NaCl shortened the water relaxation time and decreased the water mobility of MPs gel. l-His promoted the formation of MPs gel structure with small pores and thin strands at 1 mm NaCl. These resulted in the enhanced water retention and weak gel strength of MPs in low ionic strength solution. The water-binding capacity of MPs gels formed in 1 mm NaCl containing 5 mm l-His was equivalent to that with 0.6 m NaCl. The information could offer certain theoretical foundation to apply l-His as sodium salt substitute for developing low-salt meat gelling product with high yield.

Permeability tests of the compacted bentonite-sand mixture using salt solutions were carried out. Exposure of the Ca type-bentonite to NaCl or KCl solutions after fully saturation with deionized water caused ion exchange of the montmorillonite. After Ca ion exchanged to Na ion, the swelling pressure of the sample increased and the permeability decreased when the low ionic strength condition, comparing with the initial value before Na ion exchange. While, after Ca ion exchanged to K ion, increase in the swelling pressure and reduction of the permeability were insignificant. K and Na ions showed the different effect of ion exchange on the engineering properties of the bentonite.

Background/Aims: In a variety of investigations described in the literature it was not clear to what extent the transmembrane potential red blood cells (RBCs) was changed after the cells have been transferred into low ionic strength (LIS) solutions. Another open question was to find out how fast the transmembrane potential of RBCs in LIS solution will change and which final new equilibrium value will be reached. Methods: The transmembrane potential of human and bovine RBCs was investigated using the potential-sensitive fluorescent dye DIBAC4(3) (bis(1,3-dibutylbarbituric acid) trimethine oxonol) as well as the CCCP (carbonylcyanide-m-chlorophenylhydrazone) method. Results: Under physiological conditions the transmembrane potential was about -10 mV in agreement with literature data. However, when the RBCs were transferred into an isosmotic low ionic strength medium containing sucrose the transmembrane potential increased to +73 mV and +81 mV for human and bovine RBCs, respectively. In case of human RBCs it continuously decreased reaching finally an equilibrium state of -10 mV again after 30 - 60 min. For bovine RBCs the transmembrane potential declined more slowly reaching a value of +72 mV after 30 min. Conclusions: Investigations of parameters of RBCs depending on transmembrane potential cannot be performed with human RBCs in LIS media.

Various colloidal phases of Wyoming type Ca/Na-montmorillonite have been investigated experimentally by sedimentation and swelling tests of originally pure Ca- or Na-montmorillonite in different CaCl2/NaCl solutions of low ionic strength. Forces contributing to colloid (de)stability are discussed, and the experimental findings are compared with a theoretical sol formation zone in the [Ca2+]–[Na+] diagram derived from simple assumptions regarding the forces. It is found that the sol formation ability drastically lowers when calcium ions are present in the system. This effect could be due to an explicit influence of these ions on edge face interactions.

The structure of single filaments of horny cells of normal and psoriatic epidermis was studied in the electron microscopy in situ and in filaments reconstituted from urea extracts by dialysis, against low ionic strength buffer. It was found that both in situ and reconstituted filaments consist of 20 A protofibrils. In filaments of normal horny cells the protofibrils seem to form a rope-like structure while in those of psoriatic horny cells protofibrils appear randomly arranged. The filaments reconstituted from extracts of psoriatic scales range in width from 90 to 500 A, indicating a defect in lateral assembly of protofibrils. Numerous small particles are detectable at high magnification in extracts of both normal and psoriatic horny cells, which are about 20 A wide and 200 A long. These particles are consisted to be the basic structural units from which the protofibrils are formed, by end-to-end junctions.

Insulin glargine (Lantus®, Aventis Pharma, Deutschland, GmbH) is a new long-acting human insulin analog. Structural modification of the insulin molecule at two sites alters its pH, causing insulin glargine to precipitate in the neutral environment of subcutaneous tissue and to form a depot that is slowly absorbed into the bloodstream. In this paper insulin glargine aggregation is investigated by light scattering. This study shows that, in a physiologic-like pH (even at low ionic strength) conditions, aggregation phenomena occur, giving rise to compact structures with radius of hundreds of nanometers. The aggregation of insulin glargine can be responsible for its slow in situ absorption allowing for a more controlled release.

Within the UK blood transfusion services, there is currently no recommendation for the use of either clotted or anticoagulated samples for antibody identification testing. This report describes three cases in which the detection of IgM antibodies was impeded by the use of anticoagulated samples. Two patient samples, referred for compatibility testing, were both identified as having IgM complement-activating anti-S and the remaining case involved an antenatal patient with IgM complement-activating anti-Vel. In all three cases, the coincidental referral and investigation of both clotted and anticoagulated samples led to the discrepancy in serum and plasma test results becoming apparent. Potential errors in selection of suitable blood for transfusion and appropriate antenatal management were avoided by correct identification of the antibodies present using the clotted samples. Immunohematology 2006;22:72–77.

Iontophoretic transdermal delivery of sumatriptan was investigated in vitro . Among the conditions tested, 0.25 mA/cm 2 and low ionic strength (NaCl 25 mM) was the best experimental condition to increase its transport across the skin. The flux increased 385-fold respective to passive diffusion, thus resulting in a transdermal flux of sumatriptan of 1273 ± 83 nmol/cm 2 h. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association

The interaction between human serum albumin (HSA) and the short-chain phospholipid dioctanoylphosphatidylcholine (diC8PC) in aqueous solutions at pH 3.2, 7.4, and 10.0 at high and low ionic strength has been investigated. Static and dynamic light scattering methods have shown that the HSA is partially associated in these aqueous solutions with an association number of 1.67 ± 0.02. On the addition of phospholipid up to a concentration equal to its critical micellar concentration (cmc) the association increases, and at high ionic strength (0.188 M) at pH 3.2 and 7.4 the HSA is largely dimeric with a small proportion of trimeric and possibly more associated species. The interaction can be followed using laser Doppler velocimetry to measure the ζ potentials of HSA in the presence of diC8PC. At pH 3.2 where both HSA and diC8PC are positively charged, the interaction results in a marked decrease in ζ potential as the cmc of the PC is approached. At pH 10.0 where HSA is negatively charged and diC8PC is zwitterio...

Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.

Most ADAMTS13 assays use non-physiological conditions (low ionic strength, low pH, barium chloride), are subject to interference from plasma proteins, hemoglobin and bilirubin, and have limited sensitivity, especially for inhibitors.We addressed these constraints by designing a substrate that can be used in undiluted plasma.A polypeptide was expressed in E. coli that corresponds to von Willebrand factor Gln(1599) -Arg(1668) , with mutations N1610C and K1617R and an N-terminal Gly. Substrate FRETS-rVWF71 was prepared by modifying Cys(1610) with DyLight 633 (abs 638 nm, em 658 nm) and the N-terminus with IRDye QC-1 (abs 500-800 nm). Assays were performed at pH 7.4 in 150 mm NaCl, 10 mm CaCl2 .Serum and plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity with FRETS-rVWF71. Neither bilirubin (≤ 20 mg dL(-1) ) nor hemoglobin (≤ 20 g L(-1) ) interfered with product detection. Assays with FRETS-rVWF71 and FRETS-VWF73 gave similar results (R(2 ) = 0.95) for plasma from 80 subjects with thrombotic microangiopathy, 22 subjects with other causes of thrombocytopenia, and 20 healthy controls. The limit of detection with FRETS-rVWF71 for ADAMTS13 activity was ≤ 0.3%. Inhibitor assays with FRETS-rVWF71 gave titers ~2.5-fold higher than with FRETS-VWF73 and clearly distinguished patients with and without inhibitors.FRETS-rVWF71 is suitable for ADAMTS13 assays in minimally diluted plasma or serum without interference from proteins, bilirubin or free hemoglobin in plasma. Optimized detection of ADAMTS13 inhibitors will facilitate the monitoring of antibody responses during the treatment of thrombotic thrombocytopenic purpura.

International audience; Fat loss during cooking of duck "foie gras" is the main problem for both manufacturers and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yields remains high and uncontrolled. To understand the biochemical effects of postslaughter processing on fat loss during cooking, this study characterizes for the first time the protein expression of fatty liver during chilling using a proteomic approach. For this purpose the proteins were separated according to their solubility: the protein fraction soluble in a buffer of low ionic strength (S) and the protein fraction insoluble in the same buffer (IS). Two-dimensional electrophoresis was used to analyze the S fraction and mass spectrometry for the identification of spots of interest. This analysis revealed 36 (21 identified proteins) and 34 (26 identified proteins) spots of interests in the low-fat-loss and high-fat-loss groups, respectively. The expression of proteins was lower after chilling, which revealed a suppressive effect of chilling on biological processes. The shot-gun strategy was used to analyze the IS fraction, with the identification of all the proteins by mass spectrometry. This allowed identification of 554 and 562 proteins in the low-fat-loss and high-fat-loss groups, respectively. Among these proteins, only the proteins that were up-regulated in the high-fat-loss group were significant (p value = 3.17 X 10(-3)) and corresponded to protein from the cytoskeleton and its associated proteins. Taken together, these results suggest that the variability of technological yield observed in processing plants could be explained by different aging states of fatty livers during chilling, most likely associated with different proteolytic patterns.

The sensitivity of the low ionic strength solution antiglobulin test (LISS-AGT), polyethylene glycol antiglobulin test (PEG-AGT), low ionic strength solution solid-phase anti-globulin test (LISS-SPAT), gel low ionic strength solution antiglobulin test (GEL-LISS), and gel papain test (GEL-PAP) was compared in titration studies of 460 sera containing identified IgG alloantibodies. The GEL-PAP was 100% sensitive to detect Rh antibodies, whereas the PEG-AGT was the most sensitive to detect Kell, Duffy, Kidd, Ss, and rare blood group antibodies. The better performance of PEG-AGT was especially obvious with Kell, Duffy, and Ss antibodies (S = 100%). When the sensitivity of the LISS-AGT, PEG-AGT, GEL-LISS, and GEL-PAP was evaluated in different routines, the GEL-LISS showed to be more sensitive than PEG-AGT in the detection of clinically significant antibodies. These discrepant results showed that the performance of a technique may change when it is applied as a routine. © 1996 Wiley-Liss, Inc.

The mechanisms by which oral lactobacilli, one of the three major genera of cariogenic bacteria, attach to tooth surfaces are unknown. We hypothesize that recognition of collagen, the major component of dentin, may be a mechanism which localizes these bacteria to exposed root surfaces as well as to carious lesions which have penetrated the dentin. We found that the majority of oral Lactobacillus spp. strains recognize and bind collagen type I. Binding of 125I-labeled collagen type I to two strains of L. casei rhamnosus has been characterized in some detail. These strains were previously characterized with respect to their attachment to dentin (Switalski and Butcher, 1994). The process of 125I-collagen binding was mediated via specific as well as charge interactions. The putative adhesin-mediated (specific) interaction involved a limited number of bacterial surface components (2 x 103/cell). Under conditions conducive for non-specific interactions (low ionic strength), the binding was higher by an order of magnitude. Collagen binding strains were found to adhere to collagen-coated surfaces, while strains unable to bind collagen adhered to a much lesser extent. Adherence of bacteria to collagen-coated surfaces could be competitively inhibited with collagen. These interactions may target collagen-binding strains of lactobacilli to dentin collagen in the oral cavity and thus play a role in the pathogenesis of root surface and/or coronal caries. Interference with this collagen-mediated attachment of lactobacilli may provide effective means of caries control, particularly in view of the fact that other oral acidogenic microbiota also interact with collagen.

Phosphorylation of glial fibrillary acidic protein (GFAP) induces disassembly of the filaments. An amino-terminal fragment of bovine GFAP (G-Hf) was produced by lysylendopeptidase digestion. G-Hf formed ribbon-like filaments in the presence of GFAP even in low ionic strength, whereas the fragment itself did not form any structures. Only one (PK3) of the five V8 protease fragments of G-Hf accelerated GFAP assembly to the same degree as G-Hf did, whereas the other fragments did not. When PK3 was cleaved into two fragments, it lost the assembly-accelerating property. The sequence of PK3 was determined as RRRVTSATRRSYVSSSE, which corresponded to residues 3-19 of porcine GFAP. It was concluded that PK3 contains a sequence indispensable for GFAP assembly and that neither PK1 (RRRVTS) nor PK2 (ATRRSYVSSSE) included all of the sequence. A single phosphorylation of PK3 by cyclic AMP-dependent protein kinase diminished its assembly-accelerating property. The phosphorylation site was determined as Ser-12 of porcine GFAP. It was shown that single phosphorylation of the amino-terminal head domain, which contains an indispensable sequence for GFAP assembly, might be sufficient for GFAP disassembly.

International audience; Hyaluronan (HA) is the substrate of hyaluronidase (HAase). In addition, HA is able to form electrostatic complexes with many proteins, including HAase. Experiments have shown the strong inhibition of the HA hydrolysis catalysed by HAase when performed at low HAase over HA concentration ratio and under low ionic strength conditions. Non-catalytic P proteins are able to compete with HAase to form electrostatic complexes with HA and thus to modulate HAase activity. We have modelled the HA-HAase-P system by considering the competition between the two complex equilibria HA-P and HA-HAase, the Michaelis-Menten type behaviour of HAase, and the non-activity of the electrostatically complexed HAase. Simulations performed by introducing experimental data produce a theoretical behaviour similar to the experimental one, including all the atypical phenomena observed : substrate-dependence, enzyme-dependence and protein-dependence of HAase. This shows that our assumptions are sufficient to explain the behaviour of the system and allows us to estimate unknown parameters and suggest new developments.

Gelation properties were evaluated on high ionic strength soluble (HIS) proteins alone and in combination with low ionic strength soluble (LIS) or insoluble (IN) proteins from beef skeletal, heart, lung and spleen tissues. Apparent stress and strain at failure were greatest in skeletal HIS 6% (w/w) protein gels, followed by heart, lung and spleen. Expressible moisture was greatest in lung HIS protein gels. Microstructures of 6% (w/w) HIS protein gels from skeletal, cardiac and lung tissues were fibrous, while lung HIS protein gels had a globular matrix. Substitution of LIS or IN proteins into HIS protein gels altered expressible moisture, gel microstructure and apparent stress and strain at failure.

Low potassium-type (LK) sheep red blood cells show a significant increase of the residual (i.e., ouabain-insensitive) K + influx when the ionic strength of the solution is decreased. This effect is absent from high potassium-type (HK) sheep red blood cells. The KCl cotransport system is not involved since three different manoeuvres to suppress the KCl cotransport (replacement of Cl − by NO 3 − , volume-decrease, inhibition by anti-L 1 antibodies) have no effect on the low ionic strength-stimulated K + influx.

The present report describes the case of an elderly woman who was admitted to the hospital with sepsis and subsequently developed a transient 2:1 atrioventricular block following administration of a low-ionic strength contrast agent for computed tomography. The block resolved after approximately 24 h and did not require a temporary pacemaker. The case identifies an unusual consequence following an anaphylactic reaction to a low-ionic strength computed tomography contrast agent and suggests the potential role of histamine release as a mechanism underlying this reaction.

Microwaves offer the prospect of rapid and uniform heating of frozen organs. This is significant in the context of cryopreservation, and particularly of vitrification, because microwave heating may help to avoid crystallization or recrystallization of ice during warming, minimize any effects of high cell density, and reduce thermal-mechanical stresses. Previous work has established a rationale for reducing the ionic strength of solutions used to prepare tissues for microwave heating, since this permits the use of lower frequencies, which makes heating more uniform, without increasing the risk of thermal runaway (T, P. Marsland, S. Evans, and D. E. Pegg, Cryobiology 24 , 311–323, 1987). In this paper we report a study of two possible low ionic strength perfusates, in rabbit kidneys, using 48 hr of hypothermic storage and autotransplantation as the test system. This model was chosen because there is a great deal of basic information about it. Both a single-pass “flush” preservation solution and a solution designed for continuous perfusion gave excellent results. The continuous perfusion system, which would be the more suitable for introducing cryoprotectants, gave five of five surviving animals with peak serum creatinine levels of 353–555 μmol/liter normal histology in three cases, and only very minor damage in the other two. There would therefore seem to be no obstacle to the use of perfusates having a low ionic strength in renal cryopreservation studies.

The poor agreement between different workers regarding the exact mechanism involved in the adsorption of aurocyanide onto activated carbon is shown to be due to differences in the experimental conditions they employed. The effects of oxygen and ionic strength are shown to be interrelated. Adsorption under conditions of high ionic strength, as found in gold plants, involves an ion-pair mechanism whereas, under conditions of low ionic strength, a portion of the gold is adsorbed by electrostatic interacyion with ion-exchange sites formed through oxidation of the carbon surface by molecular oxygen.

The AhR was identified and characterized in cytosol from human tonsils obtained at surgery from children 2–6 years of age. The receptor was detectable with [3H]TCDD in tonsils from all donors studied, and sedimented at ≈ 9 S on sucrose gradients in low ionic strength. Scatchard plot analysis revealed that, similar to AhR from other human tissues, the apparent binding affinity of the AhR in human tonsils (≈ 8 nM) was in all cases lower than that observed in rat liver cytosol (1–2 nM).

The pelvic skeleton of threespine stickleback fish contributes to defence against predatory vertebrates, but rare populations exhibit vestigial pelvic phenotypes. Low ionic strength water and absence of predatory fishes are associated with reduction of the pelvic skeleton, and lack of Pitx1 expression in the pelvic region is evidently the genetic basis for pelvic reduction in several populations. Pelvic vestiges in most populations are larger on the left (left-biased), apparently because Pitx2 is expressed only on that side. We used whole-mount in situ hybridization to study Pitx1 expression in 19 populations of Gasterosteus aculeatus from lakes around Cook Inlet, Alaska, USA. As expected, specimens from six populations with full pelvic structures usually expressed Pitx1 in the limb bud; those from eight populations with left-biased pelvic reduction usually did not express it. Specimens from one of three populations with right-biased or unbiased pelvic reduction sometimes expressed Pitx1. One of two populations in which the pelvic spines (but not the girdle) are usually absent often expressed Pitx1. In terms of Jacob's 1977 'tinkering' metaphor, Pitx1 was the spare part with which natural selection usually tinkered for stickleback pelvic reduction, but it also tinkered with other genes that have smaller effects.

The resistance to association of circulating multiply-modified low-density lipoproteins (LDL) isolated from human blood and characterized by a decreased content of sialic acids in comparison with native LDL was studied by analysing light transmission fluctuations. LDL association was stimulated by decreasing environmental ionic strength. It is established that circulating modified LDL are less resistant to association than native LDL. Association of LDL in a medium with low ionic strength was irreversible. Probably, increased capacity to irreversible association determines the atherogenic properties of circulating modified LDL subfraction.

Thin filaments were prepared from rabbit and beef skeletal muscle with three different procedures, both at high and low ionic strength. Nebulin was always found to be associated with the myosin fraction and was always absent from the thin filament fraction.

A technique was developed, whereby proteins from animal muscle tissue were solubilized under conditions of low ionic strength and low pH, that retained high yields of proteins, which when precipitated and neutralized were capable of producing gels with good functional properties. Gels made from acid-solubilized proteins commenced gelation at a substantially lower temperature when compared to washed muscle. Fluorescence data suggests extensive protein unfolding at pH values between 5.0 and 3.0, whereupon further acid addition in the range 3.0-2.0 resulted in a re-folding of the proteins. Extensive unfolding of the protein appears to be crucial to the development of a gel upon precipitation and neutralization.

Data reported in the literature suggest that crossbridges in rapid equilibrium between attached and detached states (weakly binding bridges), demonstrated in relaxed skinned fibres at low ionic strength, could be present also in intact fibres under physiological conditions. In addition, it was suggested that the well known leading of stiffness over force during the tension development in stimulated muscle fibres could be due to an increased number of weakly binding bridges induced by the stimulation. The experiments reviewed in this paper were made to investigate these possibilities. Fast ramp length changes were applied to single frog muscle fibres at rest and during the early phases of activation. The corresponding force changes were analysed, searching for the components expected from the presence of weakly binding bridges. The results showed no mechanical indication for the presence of weakly binding bridges in both skinned and intact fibres, either at rest or during activation. It was also found that a portion of the fibre stiffness increase induced by stimulation leads the formation of crossbridges.

Abstract 2276 Is has long been known that RBC antibody detection is enhanced following a decrease in the ionic concentration. In the past we added into our routine pre-transfusion antibody screen a new test, which employs ionic strength conditions between 15 to 17% of the ionic strength of 0.9% NaCl (vLIS/IAT): 2 drops of patient's plasma and one drop of 3% RBC are incubated for 15 minutes in 0.5 mL of 5% mannitol, pH 6.0 with phosphate buffer, containing 8mM EDTA. The RBCs are washed with normal saline and tested for agglutination by an anti-IgG reagent. Other published low ionic strength methods decrease the ionic concentration less, up to about 50%. In the process of pre-transfusion testing we found 711 patients with one, two or more blood group antibodies. The following table shows the specificities and quantities of clinically significant antibodies detected by these two methods. The following antibody specificities were not included: those that were present only in small quantities, directly agglutinating IgM antibodies and anti-Lea and Lebantibodies which are usually considered to be clinically insignificant. Antibody specificity Total Number detected % Detected by IAT % Detected by vLIS/IAT C 71 71.8% 93.0% c 36 80.5% 92.0% D 147 68.7% 97.3% E 220 40.3% 95.5% Fya 44 70.5% 95.5% Jka 60 60.0% 96.7% K 154 89.0% 76.0% M 23 34.8% 87.0% S 18 50.0% 100% Total 773 Avg. 62.8% Avg. 92.6% Overall, vLIS/IAT detected an increase in clinically relevant antibodies compared to IAT. This is clearly seen in the case of anti-E, anti-D, anti-Jka, anti-M and anti-S antibodies. Anti-K antibodies, however, were detected preferably by IAT. It should be noted that more anti-Le antibodies, although not shown here, were detected either by IAT or by direct agglutination (IgM antibodies). In summary, these data show that vLIS/IAT improved antibody detection when incorporated into routine pre-transfusion tests. They also indicate that one method does not detect all clinically significant antibodies; therefore it is beneficial to use two methods which in combination cover the varying spectra of RBC antibodies. Disclosures: No relevant conflicts of interest to declare.

Upon absorption of large oppositely charged electrolytes such as proteins, polyionic hydrogels are frequently observed to separate into dense shell–swollen core states. We have developed a theory that in a detailed way takes into account the inhomogeneous swelling and distribution of the protein within such a core–shell separated gel. With this we investigated whether the core–shell separation can be an equilibrium state or if it must be understood as a dynamical phenomenon. Restricting ourselves to spherical gels with an unlimited supply of protein, we found that as an intermediate between a swollen and a collapsed gel the core–shell state can indeed be the one of lowest free energy but this state is not stable. In such cases where formation of a shell could occur spontaneously there was no further thermodynamic barrier to complete collapse of the gel (but possibly dynamical ones). The core–shell separation was favourable in systems of high charge and low ionic strength and was explained, within our theory, by the fact that the energy gain in packing proteins and polyions closely together outweighs the entropy loss of the uneven distribution.

BACKGROUND: Polyethylene glycol (PEG) has been shown to potentiate antigen-antibody reactions. STUDY DESIGN AND METHODS: To investigate the utility of PEG in pretransfusion testing, a blinded comparison study of PEG and a low-ionic-strength additive solution (LISS) was conducted. A total of 500 patient samples were tested in parallel with reagent antibody-detection cells using blind-coded PEG and LISS potentiators. RESULTS: In 34 (34%) of 100 samples with known antibodies in the Rh, Kell, Duffy, Kidd, and MNS systems, PEG antiglobulin reactions were stronger (total score, 382) than LISS antiglobulin reactions (total score, 216), and in 66 cases (66%), they were equal to those of LISS. Of 400 samples without detectable antibodies, 384 were negative with PEG and LISS, and 16 were positive in PEG tests and negative in LISS. Seven of the 16 were clinically important antibodies (D, 1; E, 3; Fya, 1; Jka; 1; Jkb, 1), and four were clinically benign antibodies (Le(a), 2; McCc, 1; Sda, 1). Five of the 16 demonstrated inconclusive PEG reactions, for a false-positive rate of 5 in 400 (1.3%). Of the 500 samples, none was negative in PEG tests and positive in LISS (0% false-negative rate). CONCLUSION: Although PEG demonstrates a relatively high false-positive rate, PEG is more sensitive than LISS in detecting clinically significant antibodies.

Cyanomet human hemoglobin has been crystallized at a chloride ion concentration and pH similar to physiological conditions. Molecular replacement calculations definitively show that the hemoglobin subunits are arranged in the Y quaternary form recently discovered in carbon monoxy hemoglobin Ypsilanti (99 Asp–Tyr), and subsequently observed in carbon monoxy normal human hemoglobin crystallized at low ionic strength and low pH. The structure has been refined at 2.09 A resolution to an R-value of 0.232, and further refinement is currently underway. Although the refinement is not yet complete, our results are the first indication that the Y structure may represent an important quaternary form of liganded hemoglobin under physiological buffer conditions. These results suggest the need for a reexamination of structure–function correlations in the hemoglobin system. © 1994 John Wiley & Sons, Inc.

The two-domain structure of streptokinase (Sk) was demonstrated by scanning calorimetric investigations at neutral pH and low ionic strength. The melting pattern of the protein is composed of two two-state transitions at TtrS1 = 45.9 +/- 0.4 degrees C with delta H1 = 431 +/- 18 kJ/mol, and TtrS2 = 60.1 +/- 1.3 degrees C with delta H2 = 306 +/- 16 kJ/mol. The partial specific heat capacity of native Sk was determined to be Cp = 1.42 +/- 0.17 J/K/g and the denaturational heat capacity change associated with the two transitions, delta Cp1 = 0.21 J/K/g and delta Cp2 = 0.38 J/K/g, respectively. The overall melting pattern of Sk remains almost unchanged at a variety of tested solvent compositions, except at pH 4 (and below) and in the presence of denaturants. The two domains show different susceptibility to urea. It is proposed that the less thermostable domain is located within the N-terminal part (residues 1-230), and the more thermostable one, within the C-terminal region.

Maximal calcium-activated tension (Tmax) was measured from saponin-and alpha-toxin permemabilized strips of rat anococcygeus muscle stimulated repetitively. 50 mins after saponin treatment, Tmax had declined to 20% of the initial value. Calmodulin (1µM) only partially reduced this decline. Lowering the ionic strength of the bathing solution from 0.2M to 0.07M markedly reduced the rate of decline of tension. Addition of calmodulin to the bathing medium of low ionic strength prevented the decline of force still further; 50 mins after permeabilization, Tmax declined to 80% of the original level. Loss of endogenous calmodulin should be prevented by permeabilization with alpha-toxin from Staphylococcus aureus. Nevertheless, Tmax still declined to approximately 50% of the original value after 50mins. However, negligible force reduction was observed if the bathing solution had an ionic strength of 0.07M.

1. 1. In contrast to rabbit skeletal muscle α-actinin, which is extracted only by low ionic strength solutions, scallop α-actinin has been extracted in a wide range of ionic strength and its relative content is maximal in physiological ionic strength solutions. 2. 2. The yield of α-actinin from scallop myofibrils sharply increases in the presence of NaHCO 3 which may be used for isolation of α-actinin from molluscs muscles. 3. 3. NaHCO 3 -solution completely removes α-actinin from scallop myofibrils, although α-actinin from rabbit skeletal muscles has not been extracted by NaHCO 3 . 4. 4. It is suggested that these differences are connected with structural differences in the Z-line of cross-striated muscles of scallop and rabbit.

Resume La technique du test de Coombs a basse force ionique a ete decrite par Low , Garratty et coll. , Moore et Mollison a la suite des travaux de Hugues-Jones et coll. sur le role de l'abaissement de la force ionique sur la fixation des anticorps. Le but de cet article est de preciser la place reelle de ce test dans les recherches d'agglutinines irregulieres et les tests de compatibilite. Cette etude comparative a porte sur les 9 tests rapportes dans le tableau II. La premiere etape de ce travail a consiste a etudier 53 anticorps irreguliers, nous permettant ainsi de selectionner les tests les plus performants : le test de Coombs a basse force ionique (15 min.), le test de Coombs a basse force ionique utilisant des hematies traitees par les enzymes (15 min.), le test a la papaine (40 min.) et le test manuel au polybrene. La seconde etape a consiste en l'etude de 68 autres anticorps dans ces 4 techniques. Elle nous a permis de conclure que l'association du test de Coombs a basse force ionique (15 min.) et du test a la papaine conduisait a un depistage complet de tous les anticorps testes. L'interet du test de Coombs a basse force ionique est double : une plus grande sensibilite et un gain de temps. L'utilisation de la trypsine n'est pas necessaire. Le nombre des fausses reactions positives n'est pas augmente par l'utilisation de ce test. Le test manuel au polybrene, bien que performant, ne peut etre preconise du fait de certaines insuffisances. En matiere de recherches d'agglutinines irregulieres et de tests de compatibilite, l'association test de Coombs a basse force ionique et test a la papaine apparait comme la plus satisfaisante pour assurer la securite immunologique des transfusions de globules rouges.

Two low ionic strength-dependent autoagglutinins were studied and found to have anti-Pr1 specificity. This specificity was determined by studies with enzyme-treated and neuraminidase-treated human red blood cells (RBCs), animal RBCs and chemically-modified sialoglycoproteins, all suspended in a low ionic strength solution (LISS). Both IgM complement-binding cold agglutinins had a wide thermal range and caused in vitro hemolysis of some LISS-suspended RBCs at 37 degrees C. Compatible blood was found for these patients by using techniques that did not employ LISS.

The results of the detection of the Rh antigens (D, C, E, c) and the S and K antigens by an elution technique comparing the AutoAnalyser and a manual method for the detection of eluted antibody are presented. Investigation of a series of anti-D sera demonstrates that while it is not too difficult to select antisera for the detection of the D antigen in fresh stains ( i.e. up to 4 weeks old) some of these sera will not be satisfactory for typing older stains. The AutoAnalyser system used is low ionic strength/polybrene which has a capacity for detecting 0.002 μg/ml of anti-D, but in spite of this degree of sensitivity the manual technique for the detection of eluted antibody compared very favourably and tended, where older stains were being tested, to give more conclusive results than the AutoAnalyser. The superiority of the manual technique was most pronounced when detecting the S and K antigens.

SUMMARY: A mutant (dev-1515), which forms either spores or stalk cells depending on the cultural conditions, was isolated from dev-1510, a mutant of Dictyostelium discoideum NC-4. When grown with Escherichia coli on a nutrient-rich medium, dev-515 formed a hemispherical cell mass in which most cells differentiated into spores. Culture with E. coli on a nutrient-intermediate medium inhibited amoebae from aggregating normally and they differentiated into stalk cells only. Development of washed vegetative cells of the mutant proceeded further without bacteria on either medium. Cells cultured in a roller tube were profoundly affected by the ionic strength of the medium. Almost all the cells in an agglutinate differentiated into stalk cells in a medium of low ionic strength, while most cells differentiated into spores in a medium of high ionic strength.

In measuring total galactosyltransferase activity in the pleural effusions from patients with benign or malignant diseases, we found no significant difference between the two groups (p greater than 0.05). However, a small amount of a galactosyltransferase variant, GT(l), could be separated from other galactosyltransferase enzymes in malignant pleural effusions by anion-exchange chromatography (DEAE-cellulose) with a buffer of low ionic strength. Other galactosyltransferases were eluted from the column with buffer of higher ionic strength. Using a mini-column method, we detected GT(l) enzyme in 19 of 26 specimens fro cancer patients, as compared with eight of 25 specimens from patients with benign disorders. The appearance of GT(l) enzyme in pleural effusion may be a tumor-associated phenomenon.

The investigation presents evidence for the value of suspending red cells in a low ionic strength medium for elution experiments since this has the effect of increasing the uptake of antibodies by the red cells. In addition it has been shown that for the titration of low affinity antibodies, and also of eluted antibodies, the combining capacity of which may be impaired through the elution process, it is an advantage to use a LISS-enzyme (papain) technique.