Effect of acetylation and permethylation on the conformation and candidacidal activity of salivary histatin-5

Summary Salivary histatins provide the non-immune defense against oral pathogens such as Candida albicans. The structural requirements of histatin-5 for anticandida activity were examined with respect to its ability to adopt helical structures, its electrostatic interactions and the hydrogen-bonding potency of its basic residues. For this purpose, the lysine and/or histidine residues of histatin-5 were chemically modified by acetylation and permethylation. Acetylated histatin-5 retained its ability to adopt helical structures in 2,2,2-trifluoroethanol, but completely lost its ability to kill yeast cells. In contrast, permethylated histatin-5 shows very little tendency to adopt a helical structure, but retained significant anticandida activity. The results suggest that the candidacidal activity can arise even when the histatin does not have the ability to adopt helical structures. The candidacidal activity of the derivatives is discussed in terms of electrostatic interactions and hydrogen-bonding potency.     

Biochemical mechanisms of realization of antiviral and interferon-inducing activity of amixine and its analogs

Antiviral and interferon inducing activity of the amixine and its some derivatives, as well as their influence on the proteolytic enzymes activity, monooxygenase activity of the microsomal fraction, level of the lipids peroxidation were studied. Lack of correlation between antiviral and interferon inducing activity in the investigated series of compounds was found. Vice versa, the good correlation between interferon inducing activity and the elastase-like activities inhibition ability of the compounds was observed. It allows to state the assumption, that only one ability of compounds to induce of an interferon doesn't suffice for obtaining of high titres of interferon, and while their rather high antiproteolitic activity is necessary. It's shown, that except for one compound the influence of amixine and its derivatives on the red-ox enzymes activity well correlates with their ability to the interferon-inducing. All presented above allows to attribute amixine and its derivatives to polymodal antiviral agents.     

Dok-R mediates attenuation of epidermal growth factor-dependent mitogen-activated protein kinase and Akt activation through processive recruitment of c-Src and Csk

Dok-R has previously been shown to associate with the epidermal growth factor receptor (EGFR) and become tyrosine phosphorylated in response to EGF stimulation. The recruitment of Dok-R to the EGFR, which is mediated through its phosphotyrosine binding (PTB) domain, results in attenuation of mitogen-activated protein kinase (MAPK) activation. Dok-R's ability to attenuate EGF-driven MAPK activation is independent of its ability to recruit rasGAP, a known attenuator of MAPK activity, suggesting an alternate Dok-R-mediated pathway. Herein, we have determined the structural determinants within Dok-R that are required for its ability to attenuate EGF signaling and to associate with c-Src and with the Src family kinase (SFK)-inhibitory kinase, Csk. We demonstrate that Dok-R associates constitutively with c-Src through an SH3-dependent interaction and that this association is essential to Dok-R's ability to attenuate c-Src activity and diminish MAPK and Akt/PKB activity. We further illustrate that EGF-dependent phosphorylation of Dok-R requires SFK activity and, more specifically, that SFK-dependent phosphorylation of tyrosine 402 on Dok-R facilitates the inducible recruitment of Csk. We propose that recruitment of Csk to Dok-R serves to bring Csk to c-Src and down-regulate its activity, resulting in a concomitant attenuation of MAPK and Akt/PKB activity. Furthermore, we demonstrate that Dok-R can abrogate c-Src's ability to protect the breast cancer cell line SKBR3 from anoikis and that an association with c-Src and Csk is required for this activity. Collectively these results demonstrate that Dok-R acts as an EGFR-recruited scaffolding molecule that processively assembles c-Src and Csk to attenuate signaling from the EGFR.     

Modulation of complement activityin Vitro andin Vivo byYersinia wild and mutant strains

The ability of released proteins (Yops) and surface lipopolysaccharides (LPS) from the wild-type strain Yersinia enterocolitica 8081-L2, serotype 0:8 to influence the complement activity was determined. Yops and LPS from wild-type and mutant strains showed different ability to affect the classical pathway (CP) functional complement activity in vitro. The serum CP activity was inhibited during the infection induced with six Y. enterocolitica and three Y. pseudotuberculosis strains in rabbits. The changed complement activity might be of importance for the course of Yersinia infections.     

Relationship between chewing ability and high-level functional capacity in an 80-year-old population in Japan

Objectives: To evaluate the association between high‐level functional capacity and chewing in a middle‐old community‐based population. Background: Although basic and instrumental activities of daily living are known to be associated with chewing ability in the elderly, an association between higher levels of competence and chewing ability has not been evaluated in the elderly. Materials and methods: The association between chewing ability using a number of different foods and high‐level functional capacity by the Tokyo Metropolitan Institute of Gerontology was evaluated in 694, 80‐year‐old people residing in Fukuoka Prefecture, Japan. Results: A significant correlation was found, using multiple regression or logistic regression analyses adjusted for various confounding factors, between the number of total chewable foods, hard foods or moderately hard foods, and total functional capacity, instrumental activity, intellectual activity or social role ability. In contrast, the number of slightly hard foods, easily chewable foods and remaining teeth were only partly related to total functional capacity and intellectual activity. Conclusion: High‐level functional capacity including intellectual activity and social role in middle‐old elderly was associated with the ability to chew hard foods than to chew easily chewable foods. Maintenance of chewing ability in elderly might result in better intellectual activity and social role.     

Matrix metalloproteinases and collagen catabolism

The matrix metalloproteinase (MMP)/matrixin family has been implicated in both normal tissue remodeling and a variety of diseases associated with abnormal turnover of extracellular matrix components. The mechanism by which MMPs catabolize collagen (collagenolysis) is still largely unknown. Substrate flexibility, MMP active sites, and MMP exosites all contribute to collagen degradation. It has recently been demonstrated that the ability to cleave a triple helix (triple-helical peptidase activity) can be distinguished from the ability to cleave collagen (collagenolytic activity). This suggests that the ability to cleave a triple helix is not the limiting factor for collagenolytic activity-the ability to properly orient and potentially destabilize collagen is. For the MMP family, the catalytic domain can unwind and cleave a triple-helical structure, while the C-terminal hemopexin-like domain appears to be responsible for properly orienting collagen and destabilizing it to some degree. It is also possible that exosites within the catalytic and/or C-terminal hemopexin-like domain may exclude some MMPs from cleaving collagen. Overall, it appears that many proteases of distinct mechanisms possess triple-helical peptidase activity, and that convergent evolution led to a few proteases possessing collagenolytic activity. Proper orientation and distortion of the triple helix may be the key factor for collagenolysis.     

Phospholipase D couples survival and migration signals in stress response of human cancer cells

MDA-MB-231 human breast cancer cells belong to a highly invasive metastatic cell line that depends on phospholipase D (PLD) activity for survival when deprived of serum growth factors. In response to the stress of serum withdrawal, there is a rapid and dramatic increase in PLD activity. Concomitant with increased PLD activity, there was an increase in the ability of MDA-MB-231 cells to both migrate and invade Matrigel. The ability of MDA-MB-231 cells to both migrate and invade Matrigel was dependent on both PLD and mTOR, a downstream target of PLD signals. Serum withdrawal also led to a PLD-dependent increase in the expression of the stress factor, hypoxia-inducible factor-1alpha. These data reveal that PLD survival signals not only prevent apoptosis but also stimulate cell migration and invasion, linking the ability to suppress apoptosis with the ability to metastasize.     

Effect of modification on physico-chemical and biological properties of haptoglobin. Acetylation, iodination and nitration.

Human haptoglobin (Hp) type 2-1 was modified with N-acetylimidazole, iodine or tetranitromethane (TNM), and the ability of the obtained derivatives to form with haemoglobin (Hb) complexes with peroxidase activity, was estimated. At low reagent to protein molar ratios, 11 tyrosine residues were nitrated, 12 acetylated and 13 iodinated. The biological activity of NO2-Hp and I-Hp amounted to 40% of the activity of native Hp whereas the activity of Ac-Hp only to 16%. The derivatives modified at high ratios of N-acetylimidazole or iodine lost the ability to bind with Hb. Deacylation. of tyrosines and partial liberation of acetylated xi-amino groups resulted in partial recovery of the activity. As demonstrated by polyacrylamide-gel electrophoresis, the modification of Hp with high excess of TNM or iodine induced polymer formation     

Correlated variability in motor activity and emotionality in selecting rats for high and low values of active avoidance conditioned reflexes

The open-field behaviour of rats selected for high and low active avoidance level was studied. The data obtained showed the correlated response of two selected lines for avoidance ability with the locomotor activity and emotional reactivity in the open-field situation. The line of a low avoidance value had the low activity level and high defecation score in comparison to the alternative one and to progenitory Krushinsky--Molodkina strain. The correspondence of learning ability and emotionality-motivation performance has been discussed.     

黄河鲤水产细菌的分离及其毒力因子和群体感应信号分子的检测

【背景】水产细菌病害制约水产养殖业健康发展,群体感应与细菌毒力因子的产生密切相关,群体感应调控细菌的毒力因子特性值得进一步研究。【目的】探究群体感应与黄河鲤细菌病害的关系,明确群体感应对细菌毒力因子特性的影响。【方法】通过16S rRNA基因测序并构建系统进化树确定筛选菌株的进化地位,通过脱脂牛奶平板法和偶氮酪蛋白法检测菌株胞外蛋白酶活力,采用结晶紫染色法对菌株的生物膜形成能力进行测定,通过报告菌株BB170和CV026分别测定菌株产信号分子AI-2和高丝氨酸内酯的能力,外源添加高丝氨酸内酯检测信号分子对菌株胞外蛋白酶活力和生物膜形成能力的影响。【结果】哈夫尼亚菌(Hafnia sp.) Z11和气单胞菌(Aeromonas sp.) Z12具有高水平的胞外蛋白酶活力和生物膜形成能力,能够分泌AHLs信号分子且具有菌体密度依赖性。外源添加HSL对菌株毒力因子特性有不同程度的影响,外源添加高浓度的N-丁酰基高丝氨酸内酯(C4-HSL)和N-己酰基高丝氨酸内酯(C6-HSL)能够分别提高菌株Z11和Z12的胞外蛋白酶活力和生物膜形成能力。【结论】高浓度群体感应信号分子AHLs对哈夫尼亚菌和气单胞菌胞外蛋白酶活性有促进作用,说明该2种菌的群体感应现象可能会影响其毒力。     

Comparative studies on biological activities of subcomponents C1q of the first component of human, bovine, mouse and guinea-pig complement

Both the haemolytic activity and the binding ability to immunoglobulin G(IgG) (Fc-binding ability) were comparatively assayed among human, bovine, mouse and guinea-pig C1q. The haemolytic activity was measured by using the sensitized sheep erythrocytes with rabbit immunoglobulin M(IgM)- or IgG-haemolysin. The Fc-binding ability was assayed by using immune complexes made of rabbit IgG-antibody against human serum albumin as well as agglutination of latex particles coated with human, bovine or rabbit IgG (IgG-latex). The specific haemolytic activity was comparable with between bovine and mouse C1q, while those of guinea pig and human C1q were significantly lower than those of the others. Only the human and mouse C1q showed significantly positive agglutinating activity of human or bovine IgG-latex. In the case of the use of rabbit IgG-latex, each of these C1q gave much weaker agglutination. On the other hand, the ability of all these C1q to bind to Fc of immune complexes specifically was almost comparable. The discrepancy in specific activities between the haemolysis and the Fc-binding ability may suggest that these two biological activities are not always correlative and that these are independent biological phenomena.     

Infectivity and Arthritis Induction of Borrelia japonica on SCID Mice and Immune Competent Mice: Possible Role of Galactosylceramide Binding Activity on Initiation of Infection

We investigated the relationship between the binding activity to galactosylceramide (GalCer) and the arthritis induction activity of Borrelia japonica. The B. japonica strains maintained the ability to induce arthritis in inbred C3H/HeN and immunodeficient SCID mice, but the ability was lower than that of Borrelia burgdorferi sensu stricto virulent strain 297. Histopathological changes were restricted to the joints, and a marked effusion of polymorphonuclear neutrophils into the joint space was found. The binding activity of B. japonica strains to GalCer was lower than that of the virulent strain 297 but higher than that of the high-passage strain 297. The lower infectivity and virulence of B. japonica may explain its lower binding ability to GalCer.     

Changes in the functional activity and kinetics of macrophages and lymphocytes as affected by retinoic acid

The influence of water-soluble compound of retinoic acid on the ability of human blood cell precursors to transform into macrophages, phagocytic macrophage activity, proliferation and marker properties of lymphocytes and the condition of erythrocytes was studied. It was found that retinoic acid caused erythrocyte damages, induced the ability of precursor cells to transforms into macrophages, did not increase significantly their functional activity and facilitated the formation of granulocyte-lymphocyte-macrophage aggregates. In the absence of macrophages, the action of retinoic acid was followed by the decrease in the expressivity of precursor cells to transform into macrophages, did by the suppression of their proliferative activity.     

Synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their β1,4-GalT inhibitory and anti-cancer properties

8-Hydroxyquinoline scaffold is a privileged structure used in designing a new active agents with therapeutic potential. Its connections with the sugar unit is formed to improve the pharmacokinetic properties. The broad spectrum of activity of quinoline derivatives, especially glycoconjugates, is often associated with the ability to chelate metal ions or with the ability to intercalate into DNA. Simple and effective methods of synthesis glycoconjugates of 8-hydroxyquinoline and 8-hydroxyquinaldine derivatives, containing an O-glycosidic bond or a 1,2,3-triazole linker in their structure, have been developed. The obtained glycoconjugates were tested for their ability to inhibit β-1,4-Galactosyltransferase, as well as inhibit cancer cell proliferation. It was found that used glycoconjugation strategy influenced both improvement of activity and improvement of the bioavailability of 8-HQ derivatives. Their activity depends on type of attached sugar, presence of protecting groups in sugar moiety and presence of a linker between sugar and quinolone aglycone.     

Effect of trifluoperazine and colchicine on smooth muscle cellular proliferative and secretory activity induced by hypercholesterolemic medium in vitro

The present study, addressed to understand the mechanism behind the cholesterol-induced proliferative and collagen secretory activity of smooth muscle cells, revealed that cholesterol-induced smooth muscle cellular DNA synthesis and collagen secretion was mediated through its ability to amplify the intracellular cGMP signal because of the fact that Trifluoperazine (an anticalmodulin and blocker of phospholipase A2) and colchicine (an antitubulin and inhibitor of guanyl cyclase) inhibited DNA synthesis and collagen-secretory activity of smooth muscle cells by their ability to decrease the cGMP levels within smooth muscle cells. From these results we suggest that membrane cholesterol modulated phospholipase 'A2' activity may be the basic mechanism involved in cholesterol-induced proliferative and collagen-secretory activity of smooth muscle cells in vitro.     

Development of selective tolerance to the serotonin behavioral syndrome and suppression of locomotor activity after repeated administration of either 5-MeODMT or mCPP

Repeated administration to rats of the 5-HT -selective agonist 5-methoxy-N, N-dimethyltryptamine (5-MeODMT)^1A produced tolerance to the ability of a test dose of 5-MeODMT to produce the serotonin behavioral syndrome, but not to the ability of a test dose of the 5-HT_1B -selective agonist m-chlorophenylpiperazine (mCPP) to decrease locomotor activity. Conversely, repeated administration of mCPP produced tolerance to the ability of a test dose of mCPP to decrease locomotor activity, but not to the ability of a test dose of 5-MeODMT to elicit the serotonin behavioral syndrome. The lack of cross-tolerance between these two selective agonists is consistent with the idea that the serotonin behavioral syndrome and suppression of locomotor activity are mediated by different subtypes of the 5-HT₁ receptor.     

Nonspecific double-stranded DNA binding activity of simian virus 40 large T antigen is involved in melting and unwinding of the origin

Helicase activity is required for T antigen to unwind the simian virus 40 origin. We previously mapped this activity to residues 131 and 616. In this study, we generated a series of mutants with single-point substitutions in the helicase domain to discover other potential activities required for helicase function. A number of DNA unwinding-defective mutants were generated. Four of these mutants (456RA, 460ED, 462GA, and 499DA) were normal in their ability to hydrolyze ATP and were capable of associating into double hexamers in the presence of origin DNA. Furthermore, they possessed normal ability to bind to single-stranded DNA. However, they were severely impaired in unwinding origin-containing DNA fragments and in carrying out a helicase reaction with an M13 partial duplex DNA substrate. Interestingly, these mutants retained some ability to perform a helicase reaction with artificial replication forks, indicating that their intrinsic helicase activity was functional. Intriguingly, these mutants had almost completely lost their ability to bind to double-stranded DNA nonspecifically. The mutants also failed to melt the early palindrome region of the origin. Taken together, these results indicate that the mutations have destroyed a novel activity required for unwinding of the origin. This activity depends on the ability to bind to DNA nonspecifically, and in its absence, T antigen is unable to structurally distort and subsequently unwind the origin.     

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.