Selective accumulation of monoclonal antibodies against neurospecific enolase in brain tissue of rats with middle cerebral artery occlusion

Preparations of I¹²⁵-labeled monoclonal antibodies against neurospecific enolase and mouse plasma IgG1 were injected intravenously to rats immediately after unilateral occlusion of the middle cerebral artery. Radioactivity of I¹²⁵-labeled monoclonal antibodies against neurospecific enolase in the brain tissue progressively increased, reached a maximum by the 48th hour, and remained practically unchanged after 72 h. At the same time radioactivity of labeled IgG1 in the brain tissue and radioactivity of both preparations in the blood, liver, spleen, kidneys, heart, and lungs decreased over 72 h. Selective accumulation of I¹²⁵-labeled monoclonal antibodies against neurospecific enolase was less significant in the brain tissue of the contralateral hemisphere and cerebellum not exposed to ischemia.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 10, pp. 388–392, October, 2004     

Detection of IgG antibodies againstBordetella pertussis with125I-protein A

A method for the detection of IgG antibodies againstBordetella pertussis is described, based on the principle of sandwich radioimmunoassay.¹²⁵I protein A is used as radioactive tracer. The influence of amounts of antigen, antibody, radioactive tracer, incubation time and temperature were tested and the optimal conditions for the assay are described. The procedure offers a simple, quick, and sensitive method for detecting antibodies againstB. pertussis. Application and limitation of the test are discussed.     

INK4a-ARF alterations in Barrett’s epithelium,intraepithelial neoplasia and Barrett’s adenocarcinoma

Introduction The INK4a-ARF [CDKN2A]- locus on chromosome 9p21 encodes for two tumour suppressor proteins, p16^INK4a and p14^ARF, which act as upstream regulators of the Rb-CDK4 and p53 pathways. To study the contribution of each pathway to the carcinogenesis of Barretts adenocarcinoma, we analysed the alterations of p14^ARFand p16^INK4a in preneoplastic and neoplastic lesions of this disease.Materials and methods After microdissection, DNA of 15 Barretts adenocarcinomas, 40 Barretts intraepithelial neoplasms (n=20 low- and n=20 high-grade) and 15 Barretts mucosa without neoplasia was analysed for INK4-ARF inactivation using DNA sequence and loss of heterozygosity (LOH) analysis, methylation-specific polymerase chain reaction, restriction-enzyme-related polymerase chain reaction and immunohistochemistry.Results We detected 9p21 LOH, p16^INK4a methylation and p16^INK4a mutations in Barretts adenocarcinomas in 5 of 15 (33%), 8 of 15 (53%) and 1 of 15 (7%) patients, respectively. P14^ARF was methylated in 3 of 15 (20%) adenocarcinomas. In Barretts intraepithelial neoplasia, p16^INK4a was altered in 12 of 20 (60%) high-grade and in 4 of 20 (20%) low-grade intraepithelial neoplasms. In Barretts mucosa without intraepithelial neoplasia p16^INK4a was methylated in one case (7%). P14^ARF was intact in Barretts mucosa without intraepithelial neoplasia.Conclusions We conclude that most Barretts intraepithelial neoplasms contain genetic and/or epigenetic INK4a-ARF alterations. Methylation of p16^INK4a appears to be the most frequent epigenetic defect in the neoplastic progression of Barretts tumourigenesis.     

Block of sodium currents by the calcium antagonist D600 in human heart cell segments

The effects of micromolar concentrations of racemic D600 on the transmembrane inward sodium current (I_Na) were investigated in voltage clamped, intracellularly perfused, human heart cell segments. Extracellular D600 blocked I_Na in a voltage- and ratedependent manner as shown by the enhanced I_Na depression with a reduction of the resting transmembrane polarization (Vm) and stimulation interval (SI). D600 action was manifested as a voltage-dependent slowing down of the Na⁺ channels' recovery kinetics after a pulsed excitation, with greater recovery times during longer depolarized states, excited or non-excited. This phenomenon seems linked to the improved control of the intracellular environment normally influencing channel activity, and to the increased ratio of non-sarcolemmal to sarcolemmal cell membranes for this preparation.     

An autoradiographic study of uptake of exogenous glycine by vertebrate spinal cord splices in vitro

Summary Spinal cord slices from rat and goldfish were incubated with³H-glycine and³H-leucine. After fixation, the slices were examined by both light and electron microscopic autoradiography. Light microscopic autoradiograms showed, in slices incubated with³H-glycine, a high level of uptake at discretely localized sites in the ventral horn grey matter with particular concentrations around the perikarya of motor neurons. Electron microscopic autoradiograms revealed that glycine had been taken up by axon terminals containing flat synaptic vesicles. There was no uptake into terminals containing round vesicles. The spinal cord slices incubated with³H-leucine showed very low levels of radioactivity randomly scattered throughout the tissue.     

Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle myosin

Summary Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeletal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin—27 kDa, 50 kDa and 20 kDa—aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1–23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the anti-27 K and anti-50 K epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the anti-50 K to S-1 decreased upon actin binding, while that of the anti-27 K was not affected by binding of S-1 to F-actin. The anti-50 K antibody inhibited the K⁺ (EDTA) and the actin-activated ATPase activity of S-1, while the anti-27 K had no effect. The results indicate that either the epitope of the anti-50 K is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.     

Whole exon 5 and intron 5 replaced by RHCE in DVa(Hus)

The D^Va(Hus) was previously investigated through cDNA analysis, which revealed an RHD-CE(5)-D hybrid allele. However, the 5 and 3 breakpoints remain unknown. In this article, gene recombinations between the RHD and RHCE alleles were investigated by a combination approach of a sequence-specific primer PCR (PCR-SSP) and an RHD full-length coding region sequencing method on two Chinese subjects with weak D phenotypes. The hybrid Rhesus box of each individual was also investigated through an established PCR-based method. As a result, two partial D phenotypes, D^Va(Hus) and D^VI type III, were identified, each carrying one hybrid RHD-CE-D allele. The two samples were also serotyped with Rh phontypes of DccEe and DCcee, respectively. Other sequencing analyses of the D^Va(Hus) sample showed that the sequence of intron 4 is identical with RHD, whereas the whole sequence of exon 5 and intron 5 is identical with RHCE except for seven polymorphisms in the intron 5. We may concluded that in the case of this Chinese D^Va(Hus), the whole exon 5 and complete intron 5 of a total segment of 1801 nucleotides were replaced by RHCE suggesting that the breakpoints of the replaced region are the 5 end of the exon 5 and the 3 end of the intron 5.     

Assessment of Safety and the Immune Response to the CD4 “Internal Antigen” Mouse Anti-Idiotypic MAb 16D7 in Four Patients with SLE

We have analyzed the immune response elicited with the human CD4 internal antigen anti-idiotypic monoclonal antibody 16D7 in four patients with active systemic lupus erythematosus and assessed the safety of the treatment. Patients 1 and 2 received three 2-mg 16D7 subcutaneous (SC) injections at 3-week intervals and mainly developed IgG1, whereas IgG1, IgG3, and IgG4 were detected in the sera of the other two patients (3 and 4) who received the same amount of 16D7 on days 0, 28, and 70. 16D7-F(ab`)<sub>2</sub> isotypic determinant-specific antibodies levels, evaluated by sera reactivity with the 16D7-isotype matched anti-idiotypic monoclonal antibody 14D6-F(ab`)₂, were low or undetectable in patient 1 and became detectable following the first and second boosters in patient 3 and patients 2 and 4, respectively. The highest level was seen in patients 3 and 4. The focusing pattern (spectrotype) of 16D7 idiotypic-specific antibodies suggested that multiple V genes are involved or many somatic mutations occur. Once established, each patients spectrotype remained stable. Although spectrotype were individually distinct, all four patients produced CD4-specific antibodies, indicating that this response crosses the genetic barrier. Disease relapsed after 11 (patient 2), 40 (patients 3 and 4), and 125 (patient 1) weeks. The lack of side effects and the prolonged periods of disease control (33 and 103 weeks after the last booster) warrants an extension of this study.     

Effect of Ultralow Doses of Antibodies to S-100 Antigen (Proproten-100) on Spatial Learning in Rats

In experiments on rats we studied the effect of potentiated antibodies against S-100 antigen on training a step-down passive avoidance task and choice between drinking bowls with sucrose solution. Peroral treatment with antibodies accelerated inhibition of ineffective and punished locomotor reactions in animals.__________Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 139, No. 2, pp. 163–165, February, 2005     

Immunochemical Properties of Anti-Galα1–3Gal Antibodies After Sensitization with Xenogeneic Tissues

In antigen-driven immune responses to proteins, antibodies of low avidity and limited complement fixing capacity undergo affinity maturation to yield antibodies of higher avidity which fix complement to a greater extent. The products of antigen-driven responses to carbohydrates are less defined. To investigate the evolution of natural antibodies against carbohydrates following sensitization, we studied natural antibodies specific for Gal1-3Gal in patients sensitized to that antigen as a result of perfusion of their blood through porcine livers for the treatment of hepatic failure. The natural antibodies against Gal1-3Gal, which occur in all unsensitized individuals, were predominantly IgM and IgG2, with average functional avidities of 5 × 10^–9 and 2 × 10^–8 M, respectively. After sensitization, the classes of anti-Gal1-3Gal included IgM, IgG2, and IgG1, and the average functional avidities of IgM and IgG were 3 × 10^–9 and 2 × 10^–9 M, respectively. The activation of complement by anti-Gal1-3Gal per microgram of Ab, measured by the fixation of C3bi on porcine cells, increased after sensitization owing to changes in subclass and avidity. Deposition of C3bi correlated with the concentrations of IgG1 and IgM but not IgG2 against Gal1-3Gal. Consistent with this finding, purified IgG1, but not IgG2, anti-Gal1-3Gal fixed complement on porcine cells. These results demonstrate that the properties of anticarbohydrate antibodies evolve after sensitization to increase complement fixation on potential targets. These properties may result from the altered costimulation of the humoral response to Gal1-3Gal due to sensitization.     

Mechanisms of Hyperpolarizing Effect of GABA on Resting Potential of the <Emphasis Type="Italic">Lumbricus Terrestris</Emphasis> Muscular Wall Somatic Cells

GABA, baclofen, isoguvacine increase, and cis-4-aminocrotonic acid does not modify resting membrane potential of muscle cells. Bicuculline, phaclofen, N-ethylmaleimide, chlorpromazine, verapamil, and removal of Ca²⁺ from bathing solution abolished the effect of baclofen, while U73122 and D609 were ineffective in this respect. The authors conclude that the Lumbricus terrestris muscle cells contain GABAergic structures similar to a- and b-receptors. Activation of GABA receptors induced Cl^- inward current and Ca²⁺ entry with subsequent activation of calmodulin-like proteins, which causes membrane hyperpolarization by increasing the effect of pumping potential on resting membrane potential.__________Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 139, No. 2, pp. 219–222, February, 2005     

Developmental transitions in the myosin patterns of two fast muscles

Summary Transitions in myosin patterns were examined in situ by immunofluorescence in two fast muscles of the developing chicken, the pectoralis and the posterior latissimus dorsi. Myosin isoforms were localized using stage-specific monoclonal antibodies against the heavy chain of pectoralis myosin. Two antibodies (12C5 and 10H10) recognize adult and late embryonic myosin. They reacted weakly with both the pectoralis and posterior latissimus dorsi at 10 days in ovo, but intensely at 18 days in ovo. Both muscles were completely unreactive with an adult-specific antibody (5C3), indicating that the staining with 12C5 and 10H10 at 18 days in ovo reflects embryonic myosin. Thus two different embryonic isoforms are expressed sequentially in each muscle. Both 12C5 and 10H10 reacted weakly again with these muscles after hatching. The reappearance of a strong positive response to both antibodies, at 28 days in the pectoralis and after 60 days in the posterior latissimus dorsi, correlated well with the first appearance of a response to the adult-specific antibody, 5C3, signalling the beginning of the adult pattern. Both muscles reacted strongly with an antibody (5B4) specific for neonatal myosin between 18 days in ovo and 60 days after hatching. In the pectoralis, embryonic was replaced by neonatal myosin in most fibres by 14 days after hatching; by 28 days, both adult and neonatal myosin were expressed in most fibres; and in the adult, neonatal myosin was replaced entirely by the adult isoform. In contrast, many fibres in the posterior latissimus dorsi still expressed both embryonic and neonatal myosins up to at least 60 days post-hatch, and the remaining fibres expressed the neonatal isoform; the neonatal isoform was present in some fibres even in the adult posterior latissimus dorsi. We have therefore demonstrated in situ four different heavy chain isoforms in two different fast muscles. Early embryonic, late embryonic, neonatal and eventually adult isoforms are expressed in each muscle and more than one isoform often coexists in the same fibre.     

Chemotherapy of a patient because of spuriously elevated alpha-fetoprotein levels

Summary Because of spuriously elevated alpha-fetoprotein levels, a course of polychemotherapy was given to a patient. We purified and identified the serum factor responsible for falsely high AFP levels as an IgG directed against mouse IgG. On gel filtration it behaves differently from true AFP, exhibiting a molecular weight of around 150,000. Chromatography on Protein A-Sepharose revealed properties indistinguishable from those of IgG. The patient had never been treated with mouse antibodies, neither for diagnostic nor therapeutic purposes and he had had no contact with animals professionally.Abbreviations AFP Alpha-fetoprotein - TSH Thyrotropin - HCG Human choriogonadotropin - OPD O-Phenylendiamine     

Calcium-dependent action potentials in guinea-pig olfactory cortex neurones

Ca²⁺-dependent action potentials were recorded in guinea pig olfactory neurones in vitro (23°–25°C). In most cells (in the presence of tetrodotoxin: TTX) the current-voltage relationship displayed anomalous rectification (apparent high slope resistance) at potentials 20 mV depolarized to the resting membrane potential (–80 mV) and strong outward rectification at more positive potentials. Intracellular Cs⁺-loading blocked outward rectification and increased action potential duration. Such spikes were TTX-insensitive and were further prolonged by external addition of tetraethylammonium (TEA) or Ba²⁺. Spikes recorded from Cs⁺-loaded, TTX/TEA-treated neurones displayed a prolonged plateau and an after-depolarization. They persisted when Ba²⁺ or Sr²⁺ were substituted for external Ca²⁺, but not when Mg²⁺ was the sole extracellular divalent cation. The spikes were blocked in the presence of Cd²⁺ but persisted when 82% of the extracellular Na⁺ was substituted by choline. A TTX-insensitive, slowly inactivating inward current at depolarized potentials is believed to account for the subthreshold anomalous rectification and prolonged spike plateau.     

Effects of antibodies against S-100 antigen in ultralow doses (Proproten-100) on acquisition of avoidance response in rats

We studied the effect of potentiated antibodies against S-100 antigen on learning of avoidance responses of 2 types. Peroral administration of antibodies promoted inhibition of locomotor activity and feeding behavior, which was associated with electrical pain stimulation. Our results indicate that the preparation in ultralow doses modulates the mechanism of memory formation.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 12, pp. 629–631, December, 2004This revised version was published online in April 2005 with a corrected cover date.     

Development of pump electrogenesis in hypokalaemic rat muscle

In hypokalaemic rats maintained on a potassium deficient diets for 10–50 days, the isolated Na-loaded and K-depleted (Na-rich) muscle fibers showed the membrane potential less than –115 mV in fresh muscles of normal rats in K⁺-free Krebs solution. Upon adding 5 mM K⁺ to the K⁺-free medium bathing the soleus muscles, the measured potentials of Na-rich muscles always exceeded the membrane potentials of fresh muscles in 5 mM K⁺. The hyperpolarization was dependent on the amount of intracellular Na⁺ concentration ([Na]_i) accumulated during the potassium deficiency. The electrogenic Na-pump was activated by an increase of [Na]_i of less than 5 mM. Further increases in [Na]_i resulted in increases in membrane potential which appeared to approach a limit at [Na]_i levels higher than 65 mM.     

Effect of Active Fraction of Cerebral on Expression of Caspase-3 and β-Amyloid Precursor Protein during Therapy of Hemorrhagic Stroke in the Acute and Delayed Periods

Active anti-stroke fraction of Cerebral preparation (extract of water-soluble molecules from brain tissue of animals with hemorrhagic stroke) decreased caspase-3 expression and improved survival of experimental animals in the acute period after hemorrhagic stroke.__________Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 139, No. 2, pp. 175–177, February, 2005     

Chromaticity of synaptic inputs to H1 horizontal cells in carp retina: analysis by voltage-clamp and spectral adaptation

Summary Cone photoreceptor inputs to H1 horizontal cells (H1 HCs) in carp retina were studied by measuring light-modulated currents (I_L) to monochromatic stimuli (460, 533, 688 nm) under a voltage-clamp condition. By using double-barrelled micro-electrodes H1 HCs were voltage-clamped whilst perfusing with dopamine to uncouple the cells. The I_L of the H1 HCs driven by each cone input was segregated by selective chromatic adaptation, and differences in the kinetics of the I_L of the H1 HCs were revealed. Thus, all together, three types of I_L were observed: (1) a fast outward current to the long-wavelength stimulus; (2) a slow outward current to the middle-wavelength stimulus; and (3) a delayed inward current that followed the peak of slow outward current to the short-wavelength stimulus. The reversal potentials of the three currents were estimated to be at least 20 mV more positive than the dark resting potential by extrapolation of the I_L-V curve. These observations are consistent with the idea that the H1 HCs receive sign-inverting, conductance decreasing synaptic input(s) from at least one other cone mechanism, in addition to the main conventional EPSP type synaptic input from red-sensitive cones.     

Absence of calcium channels in neonatal rat aortic myocytes

We have investigated whole-cell Ba²⁺ currents through Ca²⁺ channels (I_Ba) in single myocytes freshly isolated from the aortic media of neonatal (1-day-old) and adult (12-week-old) rats. In neonatal myocytes, I_Ba was undetectable even in presence of the dihydropyridine (DHP) agonist Bay K 8644. Binding of [³H]Nitrendipine on crude plasma membrane preparation of media confirmed the absence of DHP-receptors. By contrast, a robust DHP-sensitive L-type I_Ba was recorded in adults which was consistent with the presence of specific [³H]Nitrendipine binding sites. In conclusion, neonatal aortic myocytes do not express any Ca²⁺ channels. The acquisition of L-type Ca²⁺ channels may be related to cell differentiation and acquisition of contractility during postnatal development.     

The contractile behaviour of EGTA- and detergent-treated heart muscle

Summary Tension responses of rat ventricular trabeculae subjected to successive treatment with EGTA and Triton X-100 are described in order to investigate the effects of chemical skinning techniques. In some preparations the alkaloid saponin was also used before Triton. Ultrastructural evidence is cited that the EGTA-treatment fails to render cells hyperpermeable, i.e. freely permeable to small ions, whereas both saponin and Triton do so. In this paper we show that contractile responses like those described previously for the EGTA-treated tissue can be obtained. However, more detailed examination shows that such behaviour is quantitatively distinct from that of conventionally skinned fibres in a way that is incompatible with the notion of hyperpermeability. The Ca-sensitivity after treatment with either EGTA, saponin or Triton is identical in our hands. However, this is not explained by free access of Ca (and EGTA) to the intracellular space in the EGTA-treated preparation: contractures develop with very different time courses, being fastest after Triton and only marginally slower when first exposed to saponin but a factor of five times slower after EGTA-treatment alone. This applies to contractures evoked direct from Ca²⁺ concentration 10^–9 m to the test Ca²⁺ concentration at constant total buffer concentration.EGTA-treated fibres develop tension when ATP or creatine phosphate (CrP) are removed from the bath. However, responses to ADP and to CrP changes persist with millimolar levels of ATP present, quite unlike the Triton-skinned muscle. Exposure to each of a variety of solutions for 24h produce preparations showing similar behaviour: whatever the explanation for the EGTA-skinning phenomenon it is not dependent upon low bathing Ca²⁺ concentration. On the basis of the functional characteristics described here, and the structural results cited, we conclude that the cell membrane continues to function as a selective permeability barrier after EGTA-treatment: this treatment does not produce a model of a selectively skinned cardiac cell.