Interaction of lactoferrin and lysozyme with casein micelles

On addition of lactoferrin (LF) to skim milk, the turbidity decreases. The basic protein binds to the caseins in the casein micelles, which is then followed by a (partial) disintegration of the casein micelles. The amount of LF initially binding to casein micelles follows a Langmuir adsorption isotherm. The kinetics of the binding of LF could be described by first-order kinetics and similarly the disintegration kinetics. The disintegration was, however, about 10 times slower than the initial adsorption, which allowed investigating both phenomena. Kinetic data were also obtained from turbidity measurements, and all data could be described with one equation. The disintegration of the casein micelles was further characterized by an activation energy of 52 kJ/mol. The initial increase in hydrodynamic size of the casein micelles could be accounted for by assuming that it would go as the cube root of the mass using the adsorption and disintegration kinetics as determined from gel electrophoresis. The results show that LF binds to casein micelles and that subsequently the casein micelles partly disintegrate. All micelles behave in a similar manner as average particle size decreases. Lysozyme also bound to the casein micelles, and this binding followed a Langmuir adsorption isotherm. However, lysozyme did not cause the disintegration of the casein micelles.     

Psychophysiological mechanisms of cognition disorders and optimization of their correction: I. Psychophysiological effects of transcranial micropolarization used as a corrective therapy for different disturbances in the control of wakefulness,emotions, and cognition in schoolchildren aged 10–12 years with learning difficulties

We have summarized the results of psychophysiological studies in schoolchildren aged 10–12 years with cerebral functional disturbances of hypoxic-ischemic or traumatic origin with learning difficulties. We have differentiated three types of disintegration of ultraslow information-controlling systems that reflect specific disturbances in the interaction of cortical-brainstem, limbic-reticular, and suprasegmental mechanisms of regulation of the wakefulness level and are characterized by emotional and cognitive disturbances typical of each type of disintegration. Adolescents with moderately and strongly decreased resistance to transitory hypoxia differed in psychological and psychophysiological effects of corrective and recovery therapy, which included transcranial micropolarization (TCMP) and psychological-pedagogical sessions during and between TCMP sessions and during the intervals between repeated TCMP courses. We discuss the psychophysiological mechanisms of the corrective effect of low-intensity direct currents with parameters close to the native ultraslow processes of the brain and the body systems regulated by these parameters.     

Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates.

The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.     

The Effects of X-rays on the Embryonal Growth and Development of Hordeum vulgare L.

The effects of softer X-rays as an external agent on the developingembryos were manifested by a number of abnormalities such asenlargement and vacuolation of cells, disturbances in cell walls,disintegration of cells leading to the formation of cavities,disturbances in distribution of meristematic tissues, a numberof proliferations in different parts of the embryo, and retardedgrowth of the embryo during the early post-radiation period.In addition to these, deeply stained globular cytoplasmic bodieswere observed in radiated embryonic cells. Very similar kindsof abnormalities were produced by softer X-rays starting from400 r. and including 800 r. The frequency of occurrence anddegree of proliferations in embryos irradiated with 800 r.,however, were relatively higher than those produced by 400 r.and 500 r. Treatments with 300 r. units of irradiation inducedonly the appearance of deeply stained globular bodies withincytoplasm and no effects were visible with dosages below 300r. Comparative measurements of cell- lengths of the treatedand control embryos at undifferentiated as well as at differentiatedstages showed an increase in cell-lengths within radiated embryos.Radiated embryos showed defective pattern of germination. HarderX-rays were found to be less effective in inducing embryonicabnormalities than softer rays when equal calculated dosagesof the two were applied to the flowering spikes of Hordeum vulgareL.     

A multistage model for the action of cytotoxic T lymphocytes in multicellular conjugates

We propose a multistage stochastic model to explain data on the kinetics of target cell lysis by cytotoxic T lymphocytes in multicellular conjugates. A novel feature of our model is that we explicitly consider both the lethal hitting stage and the target cell disintegration stage of the cytolytic process. Further, we allow for the possibility that target cell disintegration is itself a complex process composed of many events. The comparison of our model with the data of other investigators suggests that cytotoxic T cells deliver lethal hits at random to undamaged target cells. Having received a lethal hit, the target cell disintegrates over a variable length of time. The disintegration times of target cells from different conjugates appear to be randomly distributed and to be consistent with a model in which disintegration occurs by at least two major, sequential, rate-limiting events. For conjugates containing one lymphocyte and multiple target cells, the mean rate at which a lethally hit target cell disintegrates is found to be independent of the total number of target cells in the conjugate. Our model predicts that in such multicellular conjugates, individual target cells lyse one by one, on average at approximately 30-min intervals, thus agreeing closely with previously reported experimental observations.     

The histochemical detection of acetylcholine esterase--an indicator of the disintegration of membranes

The presence of ACHE in erythrocytes membrane in vitro but not detectable ultrahistochemically has been explored as a sign of membranes disintegration. The final results of the investigations have shown, that the increase in ACHE activity of the erythrocyte membrane is a qualitative sign and a semiquantitative measure of serious disturbances in the membrane structure.     

The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.     

Disturbance of action potential generation in the mollusk giant neuron after preliminary membrane hyperpolarization

A disturbance of action potential generation in the mollusk giant neuron was investigated by intracellular recording. The anodal-breaking effect resulting from cessation of intracellular hyperpolarization was chosen as the factor evoking splitting of the action potential into its separate components. This effect was shown to be accompanied by splitting of the high-amplitude action potential into low-amplitude components. This splitting arises at a certain critical strength of the hyperpolarizing current (the disintegration threshold) and it increases with an increase in the intensity and duration of the hyperpolarizing current. Splitting of the action potential into components is regarded as disturbance of the connections between individual areas of the excitable membrane of the neuron soma. Frequent repetition of hyperpolarization leads to accumulation of the disturbances and intensifies the disintegration of the action potential into its components.     

The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding.

The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.     

Lectin binding patterns in normal canine endometrium and in bitches with pyometra and cystic endometrial hyperplasia

Cystic endometrial hyperplasia (CEH) and pyometra in the bitch are dioestral syndromes, supposed to be caused by hormonal disturbances and changes in endometrial steroid hormone receptor levels. Histologically, the endometria show cystic dilated glands and, if bacteria succeed in invading the uterus, pyometra may develop in the following metoestrus. In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of uterine glands and surface epithelium in healthy dogs and in dogs with CEH and pyometra. Lectin binding is a useful tool to identify glycoconjugates, especially of the glycocalyx, which has essential functions in the endometrium during reproduction. Uterine tissue was obtained from 18 healthy bitches in metoestrus or anoestrus and 18 bitches with a clinical diagnosis of CEH or pyometra. Normal endometria showed cycle-dependent changes in SBA, PNA, HPA and UEA binding during metoestrus and anoestrus. LCA did not show cycle-dependent changes and WGA bound to Golgi regions in the apical parts of surface epithelial cells only in metoestrous. Endometria with inflammatory alterations lost cycle-specific lectin binding patterns and, with increasing severity of pathological changes, showed a marked decrease in binding intensity to the glandular and surface epithelial glycocalyx and secretions. In dogs with CEH, unaltered glands with generally strong lectin binding to the glycocoalyx and Golgi regions were found adjacent to altered glands. The decrease of lectin binding in pyometra cases is supposed to be a result of glandular exhaustion after cystic hyperplasia. In addition, bacterial adhesion to sugar residues on the uterine surface epithelium might impede lectin binding.     

CNPase activity in myelin from adult rat brains after prolonged lead exposure in vivo

The present study investigated the sensitivity of rats cerebral myelin to prolonged toxicity of lead (Pb) that imitates environmental exposure to this metal. The results indicated that 90 days exposure of young adult rats to lead in drinking water affects the morphology of myelin sheaths, expressed in disintegration of its multilamellar structure. Both, the protein content and the activity of the myelin-specific enzyme CNPase (2',3'-cyclic nucleotide 3-phosphodiesterase), were lowered. The Michaelis-Menten kinetic for CNPase in myelin obtained from control and Pb-treated rats was different. Km increased and Vmax decreased when compared to controls. Observed disturbances in enzyme activity may be one of the potential reasons of the ultrastructural changes. It is thus tempting to speculate that Pb may be considered as a one of the factors contributing to demyelinating diseases.     

Effects of plantain and corn starches on the mechanical and disintegration properties of paracetamol tablets

The effects of plantain starch obtained from the unripe fruit of the plantMusa paradisiaca L. (Musaceae) on the mechanical and disintegration properties of paracetamol tablets have been investigated in comparison with the effects of corn starch BP using a 2³ factorial experimental design. The individual and combined effects of nature of starch binder (N), concentration of starch binder (C), and the relative density of tablet (RD) on the tensile strength (TS), brittle fracture index (BFI), and disintegration time (DT) of the tablets were investigated. The ranking of the individual effects on TS was RD>C≫N, on BFI was C≫RD>N and on DT was N>C>RD. The ranking for the interaction effects on TS and DT was N-C≫N-RD>C-RD, while that on BFI was N-C≫C-RD>N-RD. Changing nature of starch from a “low” (plantain starch) to a “high” (corn starch) level, increasing the concentration of starch binding agent from 2.5% to 10.0% wt/wt, and increasing relative density of the tablet from 0.80 to 0.90, led to increase in the values of TS and DT, but a decrease in BFI. Thus, tablets containing plantain starch had lower tensile strength and disintegration time values than those containing corn starch, but showed better ability to reduce the lamination and capping tendency in paracetamol tablet formulation. The interaction between N and C was significantly (P<.001) higher than those between N and RD and between C and RD. There is therefore the need to carefully choose the nature (N) and concentration (C) of starch used as binding agent in tablet formulations to obtain tablets of desired bond strength and disintegration properties. Furthermore, plantain starch could be useful as an alternative binding agent to cornstarch, especially where faster disintegration is required and the problems of lamination and capping are of particular concern. Published: October 22, 2005     

Levels of consciousness and levels of activation

In the paper Pavlov's idea is used about "the bright spot of consciousness" as a zone of increased excitability which moves over the cerebral cortex. Zone of increased activation is revealed migrating from the frontal parts of the left hemisphere to the occipital parts of the right hemisphere. By means of various methods (record of amplitude and latency of the visual evoked responses, frequency and amplitude of the dominant rhythm, spectral-coherent analysis of the electroencephalogram) the activation of focus in the cerebral cortex was singled out, which moved, depending on the novelty, complication and degree of automatization of the task, from the frontal parts of the left hemisphere to the occipital parts of the right hemisphere. A parallel is drawn between Pavlov's hypothesis on the focus of consciousness as foci of increased brain activity, and revealed foci of activation. The conclusion is made about fluctuations of consciousness level as a factor which is in the basis of activation focus migration.     

Ethylene-induced changes of chloroplast structure in Satsuma mandarin (Citrus unshiu Marc.)

Ethylene-induced changes of chloroplast structure in Satsumamandarin (Citrus unshiu Marc.) were examined using light andelectron microscopy. In ethylene-treated fruits, the numberof chloroplasts decreased; this was especially remarkable incells distant from the epidermis. Rapid reduction in chloroplastsize was a characteristic feature. The inner membrane system of the chloroplasts of ethylene-treatedfruits disintegrated prior to the disintegration of other cellstructures. The disintegration of the membranes within the chloroplastswas expressed by the word "melt". The double-layered structureof lamellar and granal membranes was degraded and the membranelayers became separated. Another interesting feature was the appearance of finger-likeprotuberances and peripheral reticula in the chloroplasts ofthe ethylene-treated fruits. (Received June 13, 1977; )     

Monoclonal antibodies against human immunodeficiency virus type 1 integrase: epitope mapping and differential effects on integrase activities in vitro.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the integration of viral DNA into the host chromosome, an essential step in retroviral replication. As a tool to study the structure and function of this enzyme, monoclonal antibodies (MAbs) against HIV-1 IN were produced. Epitope mapping demonstrated that the 17 MAbs obtained could be divided into seven different groups, and the selection of MAbs representing these groups were tested for their effect on in vitro activities of IN. Four groups of MAbs recognized epitopes within the region of amino acids (aa) 1 to 16, 17 to 38, or 42 to 55 in and around the conserved HHCC motif near the N terminus of IN. MAbs binding to these epitopes inhibited end processing and DNA joining and either stimulated or had little effect on disintegration and reintegration activities of IN. Two MAbs binding to epitopes within the region of aa 56 to 102 in the central core or aa 186 to 250 in the C-terminal half of the protein showed only minor effects on the in vitro activities of IN. Three Mabs which recognized on epitope within the region of aa262 to 271 of HIV-1 IN cross-reacted with HIV-2 IN. MAbs binding to this epitope clearly inhibited end processing and DNA joining and stimulated or had little effect on disintegration. In contrast to the N-terminal-specific MAbs, these C-terminal-specific MAbs abolished reintegration activity of IN.     

The combined activities of the temporal cortical area and hippocampus in man during the localization of a moving sound image

In patients with epileptic lesions in the cortex and mediobasal structures of the brain, studies have been made on the perception of spatial position of sound images during dichotic stimulation. It was established that the extreme interval which is necessary for formation of sensation of the moving sound image increases during right-side lesions of the temporal cortex. During left-side lesion of the temporal lobe, more diffuse disturbances in the trajectory of image movement (from the right and left) are observed, whereas right-side lesions result in disturbances of movement only at the opposite side of the latter. Cortical lesions and those in the mediobasal parts of the temporal lobe are accompanied by identical gradient of disturbances in the trajectory of sound image movement and short-term imprinting of succession of signals which differ with respect to their spatial position. Maximum disturbances are observed during lesions in the cortical and mediobasal parts of the temporal lobe, whereas only cortical lesions or only hippocampal lesions result in less significant disturbances. It is suggested that combined activity of the auditory cortex and hippocamp is necessary for localization of a sound source.     

Dynamic Change of Global and Local Information Processing in Propofol-Induced Loss and Recovery of Consciousness

Whether unique to humans or not, consciousness is a central aspect of our experience of the world. The neural fingerprint of this experience, however, remains one of the least understood aspects of the human brain. In this paper we employ graph-theoretic measures and support vector machine classification to assess, in 12 healthy volunteers, the dynamic reconfiguration of functional connectivity during wakefulness, propofol-induced sedation and loss of consciousness, and the recovery of wakefulness. Our main findings, based on resting-state fMRI, are three-fold. First, we find that propofol-induced anesthesia does not bear differently on long-range versus short-range connections. Second, our multi-stage design dissociated an initial phase of thalamo-cortical and cortico-cortical hyperconnectivity, present during sedation, from a phase of cortico-cortical hypoconnectivity, apparent during loss of consciousness. Finally, we show that while clustering is increased during loss of consciousness, as recently suggested, it also remains significantly elevated during wakefulness recovery. Conversely, the characteristic path length of brain networks (i.e., the average functional distance between any two regions of the brain) appears significantly increased only during loss of consciousness, marking a decrease of global information-processing efficiency uniquely associated with unconsciousness. These findings suggest that propofol-induced loss of consciousness is mainly tied to cortico-cortical and not thalamo-cortical mechanisms, and that decreased efficiency of information flow is the main feature differentiating the conscious from the unconscious brain.     

Mechanisms of transforming growth factor β induced cell cycle arrest in palate development

Immaculate and complete palatal seam disintegration, which takes place at the last phase of palate development, is essential for normal palate development. And in absence of palatal midline epithelial seam (MES) disintegration, cleft palate may arise. It has been established that transforming growth factor (TGF) β induces both epithelial mesenchymal transition (EMT) and/or apoptosis during MES disintegration. It is likely that MES might cease cell cycle to facilitate cellular changes prior to undergoing transformation or apoptosis, which has never been studied before. This study was designed to explore whether TGFβ, which is crucial for palatal MES disintegration, is capable of inducing cell cycle arrest. We studied the effects of TGFβ1 and TGFβ3, potent negative regulators of the cell cycle, on p15ink4b activity in MES cells. We surprisingly found that TGFβ1, but not TGFβ3, plays a major role in activation of the p15ink4b gene. In contrast, following successful cell cycle arrest by TGFβ1, it is TGFβ3 but not TGFβ1 that causes later cellular morphogenesis, such as EMT and apoptosis. Since TGFβ signaling activates Smads, we analyzed the roles of three Smad binding elements (SBEs) on the p15ink4b mouse promoter by site specific mutagenesis and found that these binding sites are functional. The ChIP assay demonstrated that TGFβ1, not TGFβ3, promotes Smad4 binding to two 5' terminal SBEs but not the 3' terminal site. Thus, TGFβ1 and TGFβ3 play separate yet complimentary roles in achieving cell cycle arrest and EMT/apoptosis and cell cycle arrest is a prerequisite for later cellular changes.     

Real-time monitoring of disintegration activity of catalytic core domain of HIV-1 integrase using molecular beacon

HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN–CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN–CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN–CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity.     

Degradation of glucagon receptors by dispase treatment of isolated intact rat hepatocytes

Collagenase-isolated rat hepatocytes were treated with dispase II, a neutral proteolytic enzyme which is often used for the disintegration of neonatal cells. The treatment of hepatocytes with dispase II caused a significant reduction of glucagon binding to the intact cells. The deleterious effect of this enzyme on the specific glucagon binding sites is accompanied by a reduction of the maximum intracellular cyclic AMP production.