A study of the interaction between Helicobacter pylori and components of the human fibrinolytic system

The interaction of plasminogen, tissue plasminogen activator (t-PA) and urokinase with a clinical strain of Helicobacter pylori was studied. Plasminogen bound to the surface of H. pylori cells in a concentration-dependent manner and could be activated to the enzymatic form, plasmin, by t-PA. Affinity chromatography assays revealed a plasminogen-binding protein of 58.9 kDa in water extracts of surface proteins. Surface-associated plasmin activity, detected with the chromogenic substrate CBS 00.65, was observed only when plasminogen and an exogenous activator were added to the cell suspension. The two physiologic plasminogen activators, t-PA and urokinase, were also shown to bind to and remain active on the surface of bacterial cells. epsilon-Aminocaproic acid caused partial inhibition of t-PA binding, suggesting that the kringle 2 structure of this activator is involved in the interaction with surface receptors. The activation of plasminogen by t-PA, but not urokinase, strongly depended on the presence of cells and a 25-fold enhancer effect on the initial velocity of activation by t-PA compared to urokinase was established. Furthermore, a relationship between cell concentration and the initial velocity of activation was demonstrated. These findings support the concept that plasminogen activation by t-PA on the bacterial surface is a surface-dependent reaction which offers catalytic advantages.

Serum cytokines in patients with Brazilian pemphigus foliaceus (fogo selvagem)

Pemphigus foliaceus (PF) is characterized by acantholysis determined by IgG4 binding to desmoglein I, a 160-kDa desmosomal glycoprotein. To investigate the immunopathological aspects of Brazilian PF, we determined levels of serum cytokines in patients with PF. Twenty-five patients with PF and a control group consisting of 10 healthy individuals were studied. Serum IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-gamma were measured in the two groups by ELISA. The median concentration of IL-2 was lower in PF patients compared to the control group (0.45 and 9.50 pg/ml, respectively), as also was the concentration of IL-4 (0.26 and 10.16 pg/ml, respectively). The same was observed for IL-5 (7.94 and 15.74 pg/ml, respectively) and for IFN-gamma (5.90 and 8.58 pg/ml, respectively). For IL-10 and IL-12, higher concentrations were observed in PF compared to the control group (IL-10: 24.76 and 20.92; IL-12: 2.92 and 1.17 pg/ml, respectively). Considering the Th1/Th2 paradigm, it seems that a Th2 profile, mainly represented by IL-10, predominates in PF.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Two related thrombin-like enzymes present in Bothrops atrox venom

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1) interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP) have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5%) followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

Aggrecan structure in amphibian cartilage

The structure of the large proteoglycan present in the bullfrog epiphyseal cartilage was studied by immunochemical and biochemical methods. The isolated monomer showed a polydisperse behavior on Sepharose CL2B, with a peak at Kav = 0.14. Chondroitin sulfate chains were identified by HPLC analysis of the products formed by chondroitinase digestion and mercuric acetate treatment. These chains have approximately 38 disaccharides, a Di45:Di68 ratio of 1.6 and GalNAc4S + GalNAc4,6S are the main non-reducing terminals. Keratan sulfate was identified by the use of two monoclonal antibodies in Western blots after chondroitinase ABC treatment. A keratan sulfate-rich region (~110 kDa) was isolated by sequential treatment with chondroitinase ABC and proteases. We also employed antibodies in Western blotting experiments and showed that the full length deglycosylated core protein is about 300 kDa after SDS-PAGE. Domain-specific antibodies revealed the presence of immunoreactive sites corresponding to G1/G2 and G3 globular domains and the characterization of this large proteoglycan as aggrecan. The results indicate the high conservation of the aggrecan domain structure in this lower vertebrate.

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

Autoimmunity and molecular mimicry in tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy

Viruses share antigenic sites with normal host cell components, a phenomenon known as molecular mimicry. It has long been suggested that viral infections might trigger an autoimmune response by several mechanisms including molecular mimicry. More than 600 antiviral monoclonal antibodies generated against 11 different viruses have been reported to react with 3.5% of cells specific for uninfected mouse organs. The main pathological feature of tropical spastic paraparesis/human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (TSP/HAM) is a chronic inflammation of the spinal cord characterized by perivascular cuffing of mononuclear cells accompanied by parenchymal lymphocytic infiltration. We detected the presence of autoantibodies against a 98- to 100-kDa protein of in vitro cultured human astrocytes and a 33- to 35-kDa protein from normal human brain in the serum of HTLV-I-seropositive individuals. The two cell proteins exhibited molecular mimicry with HTLV-I gag and tax proteins in TSP/HAM patients, respectively. Furthermore, the location of 33- to 35-kDa protein cross-reaction correlated with the anatomical spinal cord areas (in the rat model) in which axonal damage has been reported in several cases of TSP/HAM patients. Our experimental evidence strongly suggests that the demyelinating process occurring in TSP/HAM may be mediated by molecular mimicry between domains of some viral proteins and normal cellular targets of the spinal cord sections involved in the neurodegeneration.

Early remodeling of rat cardiac muscle induced by swimming training

The aim of the present investigation was to study the effect of acute swimming training with an anaerobic component on matrix metallopeptidase (MMP) activity and myosin heavy chain gene expression in the rat myocardium. Animals (male Wistar rats, weighing approximately 180 g) were trained for 6 h/day in 3 sessions of 2 h each for 1 to 5 consecutive days (N = 5 rats per group). Rats swam in basins 47 cm in diameter and 60 cm deep filled with water at 33 to 35ºC. After the training period a significant increase (P < 0.05) was observed in the heart weight normalized to body weight by about 22 and 35% in the groups that trained for 96 and 120 h, respectively. Blood lactate levels were significantly increased (P < 0.05) in all groups after all training sessions, confirming an anaerobic component. However, lactate levels decreased (P < 0.05) with days of training, suggesting that the animals became adapted to this protocol. Myosin heavy chain-ß gene expression, analyzed by real time PCR and normalized with GAPDH gene expression, showed a significant two-fold increase (P < 0.01) after 5 days of training. Zymography analysis of myocardium extracts indicated a single ~60-kDa activity band that was significantly increased (P < 0.05) after 72, 96, and 120 h, indicating an increased expression of MMP-2 and suggesting precocious remodeling. Furthermore, the presence of MMP-2 was confirmed by Western blot analysis, but not the presence of MMP-1 and MMP-3. Taken together, our results indicate that in these training conditions, the rat heart undergoes early biochemical and functional changes required for the adaptation to the new physiological condition by tissue remodeling.

Association of urinary 90 kDa angiotensin- converting enzyme with family history of hypertension and endothelial function in normotensive individuals

We described angiotensin-I-converting enzyme (ACE) isoforms with molecular masses of 190, 90, and 65 kDa in the urine of normotensive offspring of hypertensive subjects. Since they did not appear in equal amounts, we suggested that 90 kDa ACE might be a marker for hypertension. We evaluated the endothelial response in normotensive offspring with or without family history of hypertension and its association with the 90 kDa ACE in urine. Thirty-five normotensive subjects with a known family history of hypertension and 20 subjects without a family history of hypertension, matched for age, sex, body weight, and blood pressure, were included in the study. Endothelial function was assessed by ultrasound and a sample of urine was collected for determination of ACE isoforms. In the presence of a family history of hypertension and detection of 90 kDa ACE, we noted a maximal flow mediated dilation of 12.1 ± 5.0 vs 16.1 ± 6.0% in those without a previous history of hypertension and lacking urinary 90 kDa ACE (P < 0.05). In subjects with a family history of hypertension and presenting 90 kDa ACE, there were lower levels of HDL-cholesterol (P < 0.05) and higher levels of triglycerides (P < 0.05). Subjects with 90 kDa ACE irrespective of hypertensive history presented a trend for higher levels of triglycerides and HDL-cholesterol (P = 0.06) compared to subjects without 90 kDa ACE. Our data suggest that the 90 kDa ACE may be a marker for hypertension which may be related to the development of early atherosclerotic changes.

Meditationes sancti evangelii 33

Invokaatio: Deo duce.

Meditationes sanctarum epistolarum 33

Arkit: A-B4.

Chronicon episcoporum Finlandensium 33

Arkit: 1 arkintunnukseton lehti, 3T-3U4.

An interleukin-33/ST2 signaling deficiency reduces overt pain-like behaviors in mice

Interleukin (IL)-33, the most recent member of the IL family of cytokines, signals through the ST2 receptor. IL-33/ST2 signaling mediates antigen challenge-induced mechanical hyperalgesia in the joints and cutaneous tissues of immunized mice. The present study asked whether IL-33/ST2 signaling is relevant to overt pain-like behaviors in mice. Acetic acid and phenyl-p-benzoquinone induced significant writhing responses in wild-type (WT) mice; this overt nociceptive behavior was reduced in ST2-deficient mice. In an antigen-challenge model, ST2-deficient immunized mice had reduced induced flinch and licking overt pain-like behaviors. In the formalin test, ST2-deficient mice also presented reduced flinch and licking responses, compared with WT mice. Naive WT and ST2-deficient mice presented similar responses in the rota-rod, hot plate, and electronic von Frey tests, indicating no impairment of motor function or alteration in basal nociceptive responses. The results demonstrate that IL-33/ST2 signaling is important in the development of overt pain-like behaviors.

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.