Molecular Motions and Interactions in Aqueous Solutions of Thymosin-ß4 , Stabilin CTD and Their 1 : 1 Complex, Studied by 1 H-NMR Spectroscopy.

Wide-line 1 H NMR measurements were extended and all results were interpreted in a thermodynamics-based new approach on aqueous solutions of thymosin-ß4 (Tß4 ), stabilin cytoplasmic domain (CTD), and their 1 : 1 complex. Energy distributions of potential barriers controlling the motion of protein-bound water molecules were determined. Heterogeneous and homogeneous regions were found in the protein-water interface. The measure of heterogeneity of this interface gives quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to ∼20 % of the individual whole proteins. About 40 % of the binding sites of free Tß4 get involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tß4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

Distinct hydration properties of wild-type and familial point mutant A53T of α-synuclein associated with Parkinson's disease.

The propensity of α-synuclein to form amyloid plays an important role in Parkinson's disease. Three familial mutations, A30P, E46K, and A53T, correlate with Parkinson's disease. Therefore, unraveling the structural effects of these mutations has basic implications in understanding the molecular basis of the disease. Here, we address this issue through comparing details of the hydration of wild-type α-synuclein and its A53T mutant by a combination of wide-line NMR, differential scanning calorimetry, and molecular dynamics simulations. All three approaches suggest a hydrate shell compatible with a largely disordered state of both proteins. Its fine details, however, are different, with the mutant displaying a somewhat higher level of hydration, suggesting a bias to more open structures, favorable for protein-protein interactions leading to amyloid formation. These differences disappear in the amyloid state, suggesting basically the same surface topology, irrespective of the initial monomeric state.

Hydration water/interfacial water in crystalline lens.

Wide-line (1)H NMR signal intensity, spin-lattice and spin-spin relaxation rates and differential scanning calorimetry (DSC) measurements were done on avian (chicken and turkey) crystalline lenses between -70 degrees C and +45 degrees C to provide quantitative measures of protein hydration characteristic of the protein-water interfacial region. These measures are of paramount importance in understanding both the physiology of crystalline lens and its transitions to the cataractous pathological state characterized by the formation of opaque protein aggregates. Water mobility shows a characteristic transition at about -60 degrees C, which is identified as the melting of the interfacial/hydrate water. The amount of water in the low-temperature mobile fraction is about h = 0.4 g water/g protein, which equals the hydration required for protein activity. The amount of mobile water is temperature-independent up to about -10 degrees C, with a significant increase at higher temperatures below 0 degrees C. Above 0 degrees C, the relaxation processes can be described by a single (for spin-lattice) and by a triple (for spin-spin relaxation) exponential function. The spin-spin relaxation rate component of R(2) = 10-20 s(-1) and its dynamical parameters characterize the interfacial water at ambient or physiological temperatures. When considered an independent phase, the specific heat of the hydrate water obtained by a combination of DSC and NMR data in the temperature range -43 degrees C to -28 degrees C is higher than that of pure/bulk water. This discrepancy can only be resolved by assuming that the hydrate water is in strong thermodynamic coupling with the protein matrix. The specific heat for the system composed of the protein molecule and its hydration water is 4.6 +/- 0.3 J g(-1) K(-1). Thus, in a thermodynamic sense, crystalline protein and its hydrate layer behave as a highly-interconnected single phase.

Effects of cholecystokinin octapeptide and its fragments on brain monoamines and plasma corticosterone.

The effects of intracerebroventricular administration of an 80 pmole dose of cholecystokinin octapeptide sulphate ester, unsulphated cholecystokinin octapeptide and their fragments were tested on the dopamine, norepinephrine and serotonin contents of the rat hypothalamus, mesencephalon, amygdala, septum, cerebral cortex and striatum, as well as on the plasma corticosterone level. Cholecystokinin octapeptide sulphate ester and the tyrosine-sulphate-methionine and tyrosine-sulphate-methionine-glycine fragments increased the dopamine and norepinephrine contents of the hypothalamus and mesencephalon. The same compounds increased the dopamine content of the amygdala, while they decreased the dopamine and norepinephrine concentrations in the striatum. The plasma corticosterone level was also increased. The unsulphated cholecystokinin octapeptide and its fragments had no effects on the brain monoamine contents and slight but not significant effect on the plasma corticosterone level. The data suggest that the presence of the tyrosine-sulphate-methionine dipeptide is essential in the effects of cholecystokinin octapeptide sulphate ester on the monoamine contents of different brain areas, as well as on the plasma corticosterone level.

Antibody-dependent cell-mediated cytotoxicity (ADCC) in Parkinson's disease.

The possible role of a non-specific cell-mediated immune reaction in the pathogenesis of Parkinson's disease (PD) is discussed. It was found that the killer cell activity of PD patients below 60 years of age was significantly lower than in the older age groups or in the age-matched control group. On the other hand, it was also found that the killer cell activity of PD patients with severe symptoms (Hoehn-Yahr's IV, V stage) was significantly higher than that of the milder cases. These results support the hypothesis that an ADCC reaction--mediated by the killer cells--may play a role in the pathogenesis of PD.

[Serum aminoterminal type III procollagen peptide level and killer cell activity in patients with alcoholic liver diseases]. / Alkoholos májbetegek aminoterminális procollagén-III-peptid szérum szintje és killer-sejt aktivitása.

The authors examined the aminoterminal type III procollagen peptide level of serums and killer-cell activity peripheric blood lymphocytes with 75 patients suffering from ethanol originated liver diseases as well as control samples from 40 healthy volunteers. Determination of type III procollagen peptide (Fab) took place by the RIA method. The cytotoxic activity of killer-cells was tested against human red blood cells. Both in fatty liver and chronic alcoholic hepatitis the level of type III procollagen peptide increased, while in liver cirrhosis the same level reached a value three times of the normal. At the same time in cirrhosis hepatitis an increased killer-cell activity could be observed. Type III procollagen peptide values were also analysed in view of the cytotoxic capacity of killer-cells. At first ill, then healthy control individuals were divided into three groups according killer-cell activity values. Results have shown that in the group with a high level killer-cell activity average type III procollagen peptide values were significantly greater as compared to those of the medium or low level activity groups. These results might indicate a relation between a conditional antibody-dependent cellular cytotoxicity reaction and increasing collagen synthesis.

Wide-line NMR and protein hydration.

In this chapter, the reader is introduced to the basics of wide-line NMR, with particular focus on the following: (1) basic theoretical and experimental NMR elements, necessary before switching the spectrometer and designing the experiment, (2) models/theories for the interpretation of measured data, (3) definition of wide-line NMR spectrometry, the description of the measurement and evaluation variants, useful hints for the novice, (4) advice on selecting the solvent, which is not a trivial task, (5) a note of warning that not all data are acceptable in spite of the statistical confidence. Finally, we wrap up the chapter with the results on two proteins (a globular and an intrinsically disordered).

Protein-water and protein-buffer interactions in the aqueous solution of an intrinsically unstructured plant dehydrin: NMR intensity and DSC aspects.

Proton NMR intensity and differential scanning calorimetry measurements were carried out on an intrinsically unstructured late embryogenesis abundant protein, ERD10, the globular BSA, and various buffer solutions to characterize water and ion binding of proteins by this novel combination of experimental approaches. By quantifying the number of hydration water molecules, the results demonstrate the interaction between the protein and NaCl and between buffer and NaCl on a microscopic level. The findings overall provide direct evidence that the intrinsically unstructured ERD10 not only has a high hydration capacity but can also bind a large amount of charged solute ions. In accord, the dehydration stress function of this protein probably results from its simultaneous action of retaining water in the drying cells and preventing an adverse increase in ionic strength, thus countering deleterious effects such as protein denaturation.

[Destruction of the alveolar bone during development of periodontal disease]. / Destrukcija alveolarne kosti u toku razvoja parodontopatije.

The papers surveys the mechanisms of alveolar bone destruction in the course of parodontopathy development. The results obtained in the studies carried out so far have shown that human inflamed gingiva contains biologically active substance Interleukin-1 (IL-1), tumor necrosis factor and prostaglandin E2 in concentration which are high above the minimal values that can induce in vitro the bone resorption. It has been generally understood that alveolar bone destruction is a result of the increased osteoclastic activity caused by the synchronous effect of mentioned mediators released from the structural cells and cells of inflamed infiltrate into the bone tissue encircling.

[Immunoglobulin A levels in the saliva in patients with periodontal disease]. / Koncentracija imunoglobulina A u pljuvacki osoba s parodontopatijom.

The objective of this study was to establish the concentration of immunoglobulin A in saliva and to determine a possible connection between this concentration and the intensity of gingival inflammation. The study comprised 30 healthy persons with a mean age of 37.5 years in whom periodontitis was diagnosed after clinical examination and x-ray analysis. The control group consisted of 30 healthy persons with a mean age of 23.0 years with healthy periodontium. The intensity of clinical gingival inflammation was determined by gingival index after Loben. On the basis of the gingival index values the examinees were divided into three groups. Immunoglobulin A concentration was determined in mixed, nonstimulated saliva by method of nephelometry. Average value of immunoglobulin A in saliva of periodontitis patients was 0.30 g/l and it is significantly lower than in the control group (p < 0.001). It has been established that in persons with increased intensity of gingival inflammation values of immunoglobulin A in the saliva are decreased.

The effect of hexetidine spray on dental plaque following periodontal surgery.

The aim of the present study was to determine the effects of a 0.2% hexetidine spray, used as a supplement to regular oral hygiene measures, on dental plaque and gingival condition following periodontal surgery. This study was carried out on 38 patients who required 2 episodes of periodontal surgery. Examinations regarding dental plaque were performed at 0, 7, 14, 21 and 28 days, while the condition of the gingiva were examined at 0 and 28 days. Dental plaque was assessed by the Turesky modification of Quigley-Hein index; the gingival condition was evaluated using the gingival index of Löe-Silness and the papilla bleeding index. In a double-blind cross-over study of 28 days duration, significant reduction in plaque accumulation and an improvement in wound healing were demonstrated for the test spray compared to the placebo.

[New findings on pathogenic mechanisms of collagen fiber damage during development of periodontal diseases]. / Novija saznanja o patogenetskim mehanizmima ostecenja kolagenih vlakana u toku razvoja parodontopatije.

This paper reviews new knowledge on collagen destruction of the periodontium during development of periodontal diseases. Numerous clinical and experimental studies reveal important data in regard to recognition of these pathogenetic mechanisms. Two of them are described: intracellular-not depending on collagenase and extracellular-depending on collagenase. Which of these mechanisms is more important or what their concrete relationship is like, is still unknown.

[Effect of doxycycline in the treatment of progressive periodontal disease]. / Efekti doksiciklina u lecenju uznapredovale parodontopatije.

The aim of this paper was to establish effects of doxycycline on gingival inflammation and depth of periodontal sacculi in individuals with a form of paradontopathy in progress. The investigation was performed in two parallel groups of examinees from 38 to 56 years of age. The same kind of parodontal therapy was carried out in all patients and after that by a method of random sampling, where the examiner has not taken part, 30 patients were chosen and doxycycline was applied per os (200 mg on the first day and then 100 mg a day during a period of 13 days). Analyzing gathered results a significant decrease of gingival inflammation and of periodontal sacculi was established in persons treated with doxycycline. These results show that a 2 week application of doxycycline per os in combination with parodontal therapy is effective in treating paradontopathy in progress.

[Diagnosis of precancerous conditions of the oral cavity]. / Dijagnoza prekanceroznih stanja usne duplje.

Malignant neoplasms take the second place among the leading causes of death. The investigation was aimed at the determination of the incidence of precancerous states of the oral cavity according to the sex, localization and etiological factors. The diagnosis of the disease was based on anamnesis, clinical examination and pathohistological data. In 1987 chronic oral changes were detected in 53 patients--40 men and 13 women. The mean age of the subjects was 50.3 years. The results reveal that of 53 patients 17 had oral changes of whom 5 were diagnosed to have cancer, while in 2 patients with leukoplakia the presence of atypical cells was detected pathohistologically. The changes were most frequently localized on the left and right cheek, left lateral side of the tongue and the lower lip. Smokers comprised 71%, alcohol consumption and caries made up for 15.3%, while inappropriate dentures and the presence of galvanic current was found in 9.6% of the cases.

[Stereometric characteristics of inflammatory gingival infiltrates]. / Stereometrijska karakteristika zapaljenjskog infiltrata gingive.

The aim of this paper was to assess stereometric characteristics of gingival cell infiltrate in regard to intensity of clinical inflammation. Materials used for assessment were 70 specimen of human gingiva removed during periodontal surgery therapy. Just before the surgery in the area where specimen was to be taken, intensity of clinical gingival inflammation was determined by gingival index after Loben. Considering stereometric parameters numerical density of lymphocytes and plasma cells were established. Analyzing results it was established that by increase of intensity of clinical gingival inflammation and increase of cell infiltrate occurs, whereas an increase of numerical plasma cells' density is statistically highly important (p < 0.001), while the numerical density of lymphocytes does not changes significantly (p > 0.05).

Carbenicillin half-life in children with early diabetes mellitus.

It has been shown earlier by the authors that the glomerular filtration rate (GFR) and the penicillin clearance are increased in short-term childhood diabetes. Recently, GFR and carbenicillin half-life (T/2) were determined in insulin-treated children with a diabetes duration of 1-5 years, and in a healthy control group. An inverse relationship, between carbenicillin T/2 and GFR was found in both groups. The GFR was significantly higher, and the carbeniciliin T/2 significantly lower in diabetic children than in healthy controls. There was no correlation to blood sugar levels. It is suggested that the accelerated elimination of penicillin and carbenicillin in early diabetes necessitates higher dosages of these drugs for an adequate therapeutic effect.

Penicillin clearance in diabetic children.

The clearance of crystalline penicillin G was found to be significantly higher in children in the early period of diabetes than in age-matched healthy controls. The therapeutic importance of the finding is emphasized.

Killer-cell activity in alcohol-originated diseases of the liver, and the effect of alcohol on the K-cell functions under in vitro conditions.

Non-specific cell immunity and, within it, the change in K-cell activity, can be relevant in alcohol-induced diseases of the liver. It was examined for this reason how alcohol in its different concentrations influences the activity of K-cells under in vitro conditions. Furthermore, the cytotoxic capacity of K-cells was defined in 22 chronic alcoholics and 112 patients with alcohol induced hepatopathies. The latter were divided into subgroups. Cytotoxic capacity of lymphocytes in the peripheral blood was determined in a test against human red blood cells. 123 healthy volunteers made up the control group. A high concentration of alcohol was needed to impede K-cell capacity under in vitro conditions. It is supposed that the gradual growth in K-cell activity registered in cases of alcohol-induced hepatopathy may point--though only indirectly--to the development of an antibody-dependent cellular cytotoxic reaction.

Relationship between the immune system and the diseases of the central nervous system.

Considering the eventual role of non-specific cell-mediated immune reactions in the pathogenesis of Parkinson's syndrome based on the destruction of dopaminergic cells of the substantia nigra the killer cell activity of patients suffering from this disease has been examined. According to the results the killer cell activity of Parkinson patients is significantly lower in the age group below 60 years as compared to the higher age groups. When comparing the age groups below 60 years, significantly lower activity was measured in the patients than in the controls. Killer cell activity is significantly higher in patients suffering from more severe conditions (Hoehn-Yahr's stage IV-V) when compared to the milder cases. These results suggest the possibility that killer cell-mediated ADCC reaction may play a role in the pathogenesis of the disease. The results of these examinations open new therapeutic perspectives. It may be hoped that, as a result of our increasing knowledge and technical progress in immunology, the damaged immune system could be selectively influenced and target specific immune therapy could be used in the near future by means of for instance inactivations of cytotoxic cells, elimination of antibodies or other immunological methods.