A new haemolysin from Staphylococcus aureus which lyses horse erythrocytes

A new haemolysin from Staphylococcus aureus produced opaque zones of haemolysis on horse blood agar but did not lyse equine erythrocytes suspended in phosphate-buffered saline. The haemolysin was not neutralized by normal rabbit serum and was distinct from alpha-, beta- and delta-haemolysins as well as human leucocidin. Partially purified preparations produced erythema when injected intradermally into rabbit skin.     

Inhibition of sickle beta-chain (betaS)-dependent polymerization by nonhuman alpha-chains. A superinhibitory mouse-horse chimeric alpha-chain

Horse alpha-chain inhibits sickle beta-chain-dependent polymerization; however, its inhibitory potential is not as high as that of mouse alpha-chain. Horse alpha-(1-30) and alpha-(31-141) segments make, respectively, minor and major contributions to the inhibitory potential of horse alpha-chain. The sum of the inhibitory potential of the two segments does not account for the inhibitory potential of the full-length horse alpha-chain. Although the polymerization inhibitory potential of horse alpha-chain is lower than mouse alpha-chain, the inhibitory potential of horse alpha-(31-141) is comparable to that of mouse alpha-(31-141). When mouse alpha-(1-30) is stitched to horse alpha-(31-141), the product is a chimeric alpha-chain with an inhibitory potential greater than mouse alpha-chain. In contrast, the stitching of horse alpha-(1-30) with mouse alpha-(31-141) had no additional inhibitory potential. Molecular modeling studies of HbS containing the mouse-horse chimeric alpha-chain indicate altered side-chain interactions at the alpha1beta1 interface when compared with HbS. In addition, the AB/GH corner perturbations facilitate a different stereochemistry for the interaction of the epsilon-amino group of Lys-16(alpha) with the beta-carboxyl group of Asp-116(alpha), resulting in a decrease in the accessibility of the side chain of Lys-16(alpha) to the solvent. Based on molecular modeling, we speculate that these perturbations by themselves, or in synergy with the altered conformational aspects of the alpha1beta1 interactions, represent the molecular basis of the superinhibitory potential of the mouse-horse chimeric alpha-chains.     

Kinetic measurements of molecular interactions by spectrofluorometry

The kinetics of antibody-antigen interactions are reviewed in terms of general trends observed in both polyclonal and monoclonal antibody populations. Anti-fluorescein antibodies are featured in the review as model proteins to explore fluorescence-based kinetic measurements. Since the fluorescence of the fluorescein ligand is significantly quenched upon interaction with both polyclonal and monoclonal anti-fluorescein antibodies, the quenching parameter can be advantageously employed in measuring the rates of association (k1) and dissociation (k2). The near diffusion-limited k1 rates and the prolonged k2 rates are discussed in terms of antibody affinity and mechanisms involved in ligand binding. Specific prolongation effects of reagents, such as anti-metatype antibodies, on the dissociation rate are discussed in terms of antibody dynamics and conformational substates.     

beta 2-Microglobulin and the kidney: an overview

beta 2 microglobulin is a potentially amyloidogenic low molecular weight protein. Increased serum levels are seen in renal diseases that decrease glomerular filtration and/or tubular reabsorption, dialysis patients, chronic inflammatory diseases, and certain malignancies. Various aspects of beta 2 microglobulin metabolism and its accumulation in the kidney are addressed.     

The non-specific binding of immunoglobulins to silicone implant materials: the lack of a detectable silicone specific antibody

Recent studies have suggested that anti-silicone antibodies develop in patients implanted with silicone materials. The majority of these studies have utilized enzyme-linked immunosorbent assay (ELISA) methodology with a silicone material substrate as a means to detect the presence of the anti-silicone antibody. The current studies were undertaken to determine whether the binding of IgG to a silicone substrate was consistent with an antigen-specific antibody interaction or the result of non-specific hydrophobic interactions. While significant differences were detected in serum from silicone antibody "positive" and "negative" patients when the ELISA was conducted using a phosphate buffered saline (PBS)-0.05% Tween 20 (Tween) blocking system, the difference in the responses was attenuated when protein blocking systems were used or when incubation times were decreased. Furthermore, ELISA studies, using purified mouse and human IgG, demonstrated a concentration-dependent binding of IgG to silicone elastomer substrate which was also attenuated when a protein blocking system was used in lieu of Tween. In controlled animals studies in which female B6C3F1 mice were implanted with silicone gel or silicone elastomer for 180 days, no difference was observed between the implanted animals and the PBS control animals with respect to binding of IgG to the silicone substrate. Similar studies in female Fischer 344 rats implanted with silicone gel for 84 days also failed to demonstrate the presence of anti-silicone antibody. Collectively, the results suggest that the binding of IgG to silicone implant materials is non-specific in nature, consistent with the well-recognized interactions between hydrophobic molecules (IgGs) and hydrophobic surfaces (silicones) in an aqueous-based system.     

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize beta1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion

Cell migration may depend on integrin-mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three-dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three-dimensional collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co-clustering of beta1, beta2, or beta3 integrins with F-actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high-affinity beta1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion-perturbing anti-beta1, -beta2, -beta3, and alpha v integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, beta1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A-activated cells. Hence, T lymphocytes migrating in three-dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion-dependent migration strategies employed by other cells.     

The use of MAB 1977 monoclonal antibody for the immunohistochemical localization of beta 1 integrins in paraffin-embedded human kidney

AIMS AND BACKGROUND: Integrins are widely known cell membrane receptors for extracellular matrix molecules. The beta 1 integrin subgroup is mainly expressed by kidney cells; immunolocalization of these molecules is usually carried out on cryostatic sections. A commercial monoclonal antibody directed against the human beta 1 integrin was tested in order to design a method for the detection of this antigen in formalin-fixed, paraffin-embedded human kidney tissue. METHODS: Specimens obtained from nephrectomies were fixed with 10% formalin and embedded in paraffin. Three different detection protocols were applied after incubation with the anti-human beta 1 integrin monoclonal antibody (MAB 1977): 1) immunoperoxidase with labeled streptavidin biotin (LSAB), using biotinylated secondary antibodies, peroxidase-labeled biotin-streptavidin, and 3,3'-diaminobenzidine tetra-hydrochloride (DAB) as the revealing system; 2) immunoperoxidase with tyramide signal amplification (TSA), using biotinylated secondary antibodies, streptavidin-peroxidase, tyramide, streptavidin-peroxidase again and DAB; 3) indirect immunofluorescence with fluorescein-labeled anti-mouse immunoglobulins. RESULTS: The beat results were obtained with the LSAB detection protocol preceded by a predetection step with proteinase k. Proteinase k pretreatment did not significantly damage the tissue morphology and successfully unmasked beta 1 integrin antigens. Nonspecific background staining was reduced by a blocking step with swine serum. Similar results were obtained with the TSA detection method; however, although lower concentrations of anti-beta 1 integrin immunoglobulins and of secondary biotinylated antibody were employed, there was more undesired background staining than with the LSAB protocol. CONCLUSIONS: The method reported and discussed here may represent a valid tool for research and diagnostic applications based upon detection of beta 1 integrin in paraffin-embedded human tissues.     

The characterization of composite agarose/hydroxyethylcellulose matrices for the separation of DNA fragments using capillary electrophoresis

Mixtures of the polysaccharide derivatives, 19% hydroxyethylated SeaPrep agarose (SP-AG) and hydroxyethylcellulose (HEC), in aqueous buffer solutions are applied for the first time to the separation of DNA fragments using capillary electrophoresis (CE). These matrices form unique size-sieving networks that allow the separation of a wide size range of DNA fragments in a single analysis. Relative to their homogeneous counterparts, the composite separation matrices provide enhanced selectivity properties of DNA fragments, especially for fragments greater than 1000 base pairs (bp) in length. Additionally, the effects on separation performance of capillary temperature, the incorporation of a DNA intercalator, and applied field strength are demonstrated. Solution viscosity measurements of the homogeneous and composite matrix solutions were made in order to establish the entanglement threshold concentrations for the unique size-sieving solutions. The relatively low solution viscosities of the composite separation matrices allow reproducible replacement of the separation matrix between analyses. The mechanism of separation of DNA fragments for the composite matrices is proposed.     

Decreased severity of ethanol withdrawal behaviors in kainic acid-treated rats

The involvement of kainate (KA)-sensitive regions in ethanol withdrawal behaviors was investigated in male Wistar rats given three intraperitoneal (IP) injections of KA (12 mg/kg) or saline each followed by recovery at 4 degrees C for 5 h and room temperature for 3 days and a final KA or saline injection at room temperature. Some animals received MK-801 (1 mg/kg, IP) 30 min after each injection and one group received saline only. The saline/saline, saline/MK-801, and KA/MK-801 groups displayed typical ethanol withdrawal behaviors 8-12 h after ethanol withdrawal. These behaviors were attenuated in the KA/saline group. Audiogenic seizures could be induced in all treatment groups 12 h after withdrawal. There was severe neuronal degeneration in the hippocampal CA region and the piriform cortex of the KA/saline-treated animals that was reduced by MK-801 treatment. The inferior colliculus remained intact. These results suggest that the N-methyl-D-aspartate receptor mediates KA-induced damage in limbic structures and that these regions may play an important role in typical, but not audiogenically induced ethanol-withdrawal behaviors.     

Fate of antibodies bound to lymphocyte surface. II.--Degradation and release of immune complexes

The fate of horse anti-human lymphocyte globulins, rabbit anti-human beta2 microglobulin antibodies and anti-HL-A alloantibodies radiolabelled with 125I and complexed to their corresponding lymphocyte surface antigens, was investigated. During prolonged incubation at 37 degrees C, part of these antibodies were actively released into the surrounding medium after enzymatic degradation occuring either intracellularly or at the cell surface. Metabolism of the antibodies was temperature-dependent and involved only antibodies able to react with antigens on the cell surface. Sephadex G200 filtration of the small percentage of non dialysable radioactive products in the supernatant revealed that all the 125I-anti-human lymphocyte globulin was recovered as a macromolecular structure in the void volume. Similar experiments were performed with 51Cr labelled lymphocytes coated with anti-lymphocyte globulins: treatment with anti-horse immunoglobulin precipitated part of 51Cr activity in the void volume which was therefore associated with anti-human lymphocyte globulin as an immune complex. The same protocol applied to the other two antibodies studied showed that anti-beta2 microglobulin antibodies were recovered both as macromolecular and 7 S immune complexes, whereas anti-HL-A antibodies were recovered as macromolecular, 7 S and still smaller immune complexes.     

Tractional force generation by porcine Müller cells. Development and differential stimulation by growth factors

PURPOSE: To assess the ability of retinal Müller cells to generate tractional forces during dedifferentiation in culture and to assess their responsiveness to contraction-stimulating growth factors. METHODS: Müller cells were isolated from papain-DNase-digested porcine retina. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and alpha smooth muscle actin (alpha SMA). Tractional force generation was assessed as a function of Müller cell contraction of collagenous extracellular matrices in vitro. The effects of potential promoters were assessed by addition directly to culture medium. The contributions of specific promoting to the contraction-promoting activity in serum were assessed by adding neutralizing antibodies and measuring loss of stimulatory activity. RESULTS: Freshly isolated Müller cells did not generate substantial matrix contraction. However, this activity increased 150-fold within 12 days in culture and continued to increase during the next 21 days. Development of the capacity for extracellular matrix contraction coincided with the acquisition of immunodetectable alpha SMA and loss of GFAP. Matrix contraction by Müller cells was stimulated in a dose-dependent fashion by human serum, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). Müller cells were not stimulated by transforming growth factor beta 1 (TGF beta 1), transforming growth factor beta 2 (TGF beta 2), or endothelin-1 (E1). Neutralizing antibodies against PDGF and IGF-I reduced the activity in human serum by 37% and 58%, respectively, and 87% when added together. CONCLUSIONS: Porcine Müller cells in culture acquire the ability to contract extracellular matrices and thus generate tractional forces. Acquisition of this activity coincides with alpha SMA expression and loss of GFAP. Further, this activity is dependent on the presence of exogenous promoters, including PDGF or IGF-I.     

Immunolocalization of transforming growth factor-beta 1 and transforming growth factor-beta 2 in the mouse ovary during gonadotrophin-induced follicular maturation

An immunohistochemical approach was utilized to evaluate the cellular distribution of transforming growth factor-beta 1 (TGF beta 1) and transforming growth factor beta 2 (TGF beta 2) at different stages of follicle development in the prepubertal mouse ovary under the following conditions: (i) after pregnant mare's serum gonadotrophin (PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin (HCG) treatment; (iii) after PMSG and HCG treatment plus mating. In the immature ovary, TGF beta 1 and TGF beta 2 immunoreactivities are localized in theca and granulosa cells and in oocytes. After PMSG treatment, TGF beta 1 and TGF beta 2 immunoreactivities are localized in granulosa cells; in addition, TGF beta 2 staining is noted in the matrix surrounding antral cells. Staining for both TGR beta 1 and TGF beta 2 drops in the theca but persists in the oocyte. PMSG plus HCG treatment results in a significant increase in TGF beta 1 and TGF beta 2 immunoreactivity in the theca and in the maintenance of TGF beta 1 staining in both basal granulosa cells and cumulus cells whereas TGF beta 2 immunoreactivity is essentially localized in the matrix surrounding cumulus cells. Staining for TGF beta 1 and TGF beta 2 persists in the oocyte. Following PMSG plus HCG treatment and mating, TGF beta 1 immunoreactivity is localized in the luteal cells of corpora lutea and TGF beta 2 shows a similar localization pattern. This study provides evidence that TGF beta 1 and TGF beta 2 peptides are expressed in specific cell types during induced follicular maturation in the mouse ovary.     

Immunoglobulin G, F(AB')2, and fab fragment uptake kinetics in isolated perfused rat liver and rat hepatic cells

The interaction of 125I-radiolabeled immunoglobulin G (IgG), F(ab')2, and Fab fragments with different modes of production (polyclonal or monoclonal), belonging to different subclasses (IgG1 and IgGT) and derived from different sources (mouse, rat, and horse) with liver, was investigated by using isolated perfused rat liver and isolated rat hepatic parenchymal cells (PCs) and non-parenchymal cells (NPCs) in suspension. Lactosaminated-bovine serum albumin (Lac-BSA) and formaldehyde-bovine serum albumin were used as markers of specific binding to PCs and NPCs, respectively. Using the isolated perfused rat liver model, data clearly indicated a very weak hepatic extraction ratio (< 0.003) for IgGs and fragments in comparison with Lac-BSA (extraction ratio = 0.398) over the 3 hr of the experiments. No breakdown or higher molecular weight compounds were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Biliary excretion of IgGs and fragments ranged from 0.07 to 0.3%, mainly as free iodine-125. In contrast, 7% of Lac-BSA was excreted unchanged in bile, and 10% of free iodine was excreted at 3 hr. In vitro binding studies showed no specific binding of any antibody and fragment proteins at 4 degrees C or 37 degrees C. In contrast, saturable uptake was observed for Lac-BSA with PCs and formaldehyde-bovine serum albumin with NPCs. Both models demonstrated that nonspecific antibody/fragment interactions occurred with rat liver. Several hypotheses can be formulated to explain why liver-antibody interactions depend on more complex antibody molecular states (aggregated structure and immune complex) rather than the monomeric structure investigated in the present study.     

Stimulation of G-CSF gene expression in the macrophage cell line by contact with extracellular matrix proteins and a pre-B leukaemia cell line

Freshly prepared dentine specimens of human teeth were perfused with either horse serum or physiologic saline. After application of AllBond2, ART Bond, Syntac or an experimental dentine bonding agent called P-Bond, a composite cylinder was added and cured at the same time. After 1500 thermal cycles with constant imitation of intrapulpal pressure, the shear bond strengths were measured. Resulting shear bond strength values were analysed with Students t-test or Mann-Whitney Test. The values for AllBond2 were not significantly different. The values for ART Bond (P < 0.05) and for Syntac (P < 0.05) were significantly higher if the dentine was perfused with horse serum. For P-Bond (P < 0.001) the values were significantly higher if the dentine was perfused with physiologic saline. According to these results it does not seem to be appropriate to take a clear decision as to which of the two perfusing media investigated might be more suitable to imitate in vivo conditions.     

Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica)

This paper presents the results of viscosity determinations on aqueous solutions of bovine serum albumin (BSA) at a wide range of concentrations and at temperatures ranging from 5 degrees C to 45 degrees C. On the basis of these measurements a general formula connecting the relative viscosity eta r with temperature T and concentration c of the dissolved proteins was established: [formula: see text] The quantities alpha, beta, B, D and delta E are described in the text below. A simple substantiation of the formula was also given. This relation gives immediately the Mooney approximation and allows the prediction of the values of the parameter S and a self-crowding factor K in this approximation. By applying an asymptotic form of the formula such rheological quantities as the intrinsic viscosity and Huggins coefficient were calculated.     

Enhancement of cell interactions with collagen/glycosaminoglycan matrices by RGD derivatization

The interaction of three cell types important to the wound repair process with collagen/glycosaminoglycan (GAG) dermal regeneration matrices covalently modified with an Arg-Gly-Asp (RGD)-containing peptide was characterized. Function-blocking monoclonal antibodies directed against various integrin subunits were used to demonstrate that human fibroblasts attached to the unmodified matrix through the integrin, alpha2beta1. Human endothelial cells and human keratinocytes, however, attached minimally to the unmodified matrix. After modification of the collagen/GAG matrix with RGD-containing peptide, endothelial cells and keratinocytes attached and spread well on the matrix. This attachment was RGD dependent as evidenced by essentially complete inhibition with competing soluble peptide. In terms of overall cell number, fibroblast cell attachment remained unchanged on the RGD peptide-modified matrix compared to the unmodified material. Antibody and peptide inhibition studies demonstrate, however, that attachment to the modified matrix was mediated by both alpha2beta1 and RGD-binding integrins. We have successfully introduced a specific RGD receptor-mediated attachment site on collagen/GAG dermal regeneration matrices, resulting in enhanced cell interaction of important wound healing cell types. This modification could have important implications for the performance of these matrices in promoting dermal regeneration.     

Immunization of cows with ferric enterobactin receptor from coliform bacteria

The serum and milk immunoglobulin (Ig) G responses of lactating dairy cows were determined following immunization with ferric enterobactin receptor FepA. Escherichia coli 471 was cultured in iron-depleted medium, and outer membrane proteins were extracted by 2% N-lauroylsarcosine sodium salt and 2% Triton X-100. The FepA was isolated from the outer membrane proteins by ion-exchange chromatography. Twenty cows were assigned to four treatment groups of 5 cows blocked by breed and days in milk. Treatment groups were vaccinated with 100 micrograms of FepA, 500 micrograms of FepA, Escherichia coli J5 bacterin, or sterile phosphate-buffered saline. Primary immunization was at approximately 200 d in milk, and booster immunizations were given 14 and 28 d later. Serum and whey IgG titers to FepA in cows vaccinated with FepA were significantly higher than those from cows vaccinated with either E. coli J5 bacterin or phosphate-buffered saline. Serum and whey IgG titers to FepA were elevated by 14 d in cows vaccinated with FepA. Significant differences were not observed between doses of FepA. The degree of cross-reactivity of purified IgG from cows vaccinated with FepA to E. coli and Klebsiella pneumoniae isolates was significantly higher than that to a control isolate that lacked FepA production. Immunization with FepA elicited an immunological response in serum and milk.     

Synergism between NMDA and domoic acid in a murine model of behavioural neurotoxicity

We have examined the behavioural neurotoxicity of domoic acid (DOM) and kainic acid (KA) in mice following administration of ligands active at the N-methyl-d-aspartate (NMDA) receptor. Groups of female CD-1 mice (n=4) were injected i.p. with saline or one of three doses of either DOM or KA. Doses of DOM and KA were selected from the steep portion of the respective dose response curves and were equitoxic when compared between the two ligands. Toxicity was recorded as both total cumulative toxicity over 60 min according to a previously validated 7 point rating scale, and as the latency to the onset of tremors and/or convulsions. Five minutes prior to administration of either agonist mice were injected with either saline, NMDA (40 mg/kg) or a combination of NMDA and 15 mg/kg CPP (3-[2-carboxypiperazine-4-yl]propyl-1-phosphonic acid). Neither NMDA nor CPP at these doses produced significant changes from baseline responding when injected prior to saline. Injection of NMDA prior to DOM, however, resulted in significantly increased cumulative toxicity and significantly reduced latencies to seizures at the two highest doses of DOM (3.75 and 5.0 mg/kg). NMDA-induced potentiation of DOM toxicity was completely antagonized by co-administration of CPP. In contrast, injection of NMDA prior to KA did not result in significant changes in KA toxicity at any of the doses tested using either index of behavioural toxicity. These results confirm previous reports of synergism between DOM and ligands acting at the NMDA receptor in isolated neurons, and provide further evidence of pharmacological dissociation of the actions of DOM and KA in vivo.     

Electrofusion of zona-free mouse embryonic cells in electrolytes and their development in vitro

The influence of increasing the physical electrofusion parameters, direct current (DC) pulse strength, pulse duration, pulse number, alternating current (AC) voltage and alignment time, in electrolytes on the rates of fusion, degeneration and development of zona-free mouse 2-cell embryos were examined. Furthermore, the effects of physiological saline and mannitol as fusion media and various mouse strains were also evaluated. Dulbecco's phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum was used as the main fusion solution. A significant increase in the rate of fusion (P < 0.05) was obtained by increasing pulse strength from 30 to 300 V/mm. The embryos fused at the pulse strengths of 30 to 70 V/mm had significantly higher development rates to blastocysts compared with those fused at 100 to 300 V/mm (P < 0.05). There were no significant differences in the rates of fusion, degeneration and development to blastocysts when the pulse duration was increased from 30 to 90 microseconds. Although fusion rates were increased (P < 0.05) by increasing the pulse number up to 4, a significant decrease (P < 0.05) in development to blastocysts was observed when the pulse number was 5. Application of AC voltage prior to the DC pulse tended to increase the fusion rate (89.2-93.8%), compared with fusion with the DC pulse only (75.0%). Prolongation of alignment time from 5 to 15 sec had no effect on the fusion rate. Under the optimum conditions (2 pulses of DC of 70 V/mm, 70 microseconds pulse duration and AC of 5 V/mm for 5 sec), no significant difference was obtained in the fusion and development rates in different mouse strains, nor were fusion and development rates significantly different among PBS, physiological saline and mannitol solutions (P > 0.05).