Expression and regulation of αB-crystallin in the kidney in vivo and in vitro

αB-crystallin, a major component of the mammalian eye lens, is a small heat shock protein and molecular chaperone that is also abundant in the mammalian kidney. The present study aimed to characterize more closely the intrarenal expression and regulation of αB-crystallin in vivo and in vitro. In normal rat kidney, the expression of αB-crystallin mRNA and protein were both close to the detection limit in cortex, but increased steeply from the outer to the inner medulla where αB-crystallin constitutes approximately 2% of total tissue protein. Immunohistochemistry disclosed papillary collecting duct cells and thin limbs as the major sites for intrapapillary αB-crystallin immunoreactivity. In rats subjected to sucrose diuresis for 3 days, αB-crystallin mRNA expression was reduced by 27 and 46% in outer and inner medulla, respectively. In agreement with the results obtained in vivo, in Madine–Darby canine kidney cells, αB-crystallin mRNA and protein were induced significantly by elevating the medium osmolality to 500 mosm/kg H₂O by the addition of NaCl and raffinose, and also by urea. The NaCl-induced increase in αB-crystallin expression was concentration-dependently blunted by SP600125, a specific JNK inhibitor. Overexpression of αB-crystallin in 293 cells resulted in increased tolerance to acute osmotic stress. These results indicate that αB-crystallin may be regulated by papillary interstitial tonicity in a JNK-dependent process. Moreover, the high abundance of αB-crystallin in the renal medulla may be important for cell survival in an environment characterized by extreme interstitial solute concentrations as present during antidiuresis.     

Evaluation of the in vitro and in vivo proinflammatory activities of gold (+) and gold (−) nanoparticles

Objective and design

The aim of this study was to determine potential effects of gold (+) and gold (?) nanoparticles, AuNP(+) and AuNP(?), on neutrophil biology.

Material or subjects

Freshly isolated human neutrophils were used for the in vitro aspects and CD-1 mice were used in the in vivo murine air pouch model of acute neutrophilic inflammation.

Treatment

Human neutrophils were treated with the indicated concentrations of AuNP(+) or AuNP(?) in vitro and mice received 100 or 500 µg/ml AuNP(+) or AuNP(?) into air pouches.

Methods

Cellular uptake of AuNP by neutrophils was confirmed by transmission electron microscopy and the ability of the NP to modulate apoptosis, gelatinase activity, and chemokine production and chemotaxis was determined by cytology, zymography, ELISArray, antibody array, and ELISA and by a micro-chemotaxis chamber, respectively. In vivo, exudates were harvested after 6 h to determine the leukocyte infiltration to detect the production of several cytokines by an antibody array approach and ELISA. One-way analysis of variance was used for statistical analysis.

Results

AuNP possess proinflammatory activities in vitro and induce mainly a neutrophil influx in vivo, albeit at different degrees.

Conclusions

AuNP(+) and AuNP(?) should be added as new candidates into a growing list of NP having proinflammatory activities by themselves.

     

Usefulness of Negative and Weak–Diffuse Pattern of SDHB Immunostaining in Assessment of SDH Mutations in Paragangliomas and Pheochromocytomas

This is a confirmatory study about usefulness of SDHB and SDHA immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas. Paraganglioma/pheochromocytoma syndrome (PGL/PCC syndrome) consists of different entities, associated with germline mutations in five different genes: SDHD, SDHAF2, SDHC, SDHA and SDHB. It has been suggested that negative immunostaining of SDHB can be taken as an indicator of the presence of a mutation in one of the five SDH genes. We have performed SDHB and SDHA immunohistochemical staining in a series of paragangliomas and pheochromocytomas from 64 patients. The patients had been previously checked for mutations in SDHD, SDHC and SDHB, but also for mutation in RET and VHL. All 14 patients with SDH mutations (9 with SDHB and 5 with SDHD mutations) exhibited negative or weak–diffuse SDHB staining pattern in tumour tissue, whereas cells of the 23 RET mutated and 8 VHL mutated tumours showed a positive SDHB immunostaining. Sixteen of the patients that did not exhibit a mutation in any gene showed positive SDHB immunostaining in tumour tissue, while only three of the patients without mutation exhibited negative staining. All patients exhibited positive pattern of SDHA immunostaining. The results confirm the value of SDHB immunohistochemical status in assessment of germline mutations in PGL/PCC syndrome.     

Diverse roles of TGF-β receptor II in renal fibrosis and inflammation in vivo and in vitro

TGF-β1 binds receptor II (TβRII) to exert its biological activities but its functional importance in kidney diseases remains largely unclear. In the present study, we hypothesized that TβRII may function to initiate the downstream TGF-β signalling and determine the diverse role of TGF-β1 in kidney injury. The hypothesis was examined in a model of unilateral ureteral obstructive (UUO) nephropathy and in kidney fibroblasts and tubular epithelial cells in which the TβRII was deleted conditionally. We found that disruption of TβRII inhibited severe tubulointerstitial fibrosis in the UUO kidney, which was associated with the impairment of TGF-β/Smad3 signalling, but not with the ERK/p38 MAP kinase pathway. In contrast, deletion of TβRII enhanced NF-κB signalling and renal inflammation including up-regulation of Il-1β and Tnfα in the UUO kidney. Similarly, in vitro disruption of TβRII from kidney fibroblasts or tubular epithelial cells inhibited TGF-β1-induced Smad signalling and fibrosis but impaired the anti-inflammatory effect of TGF-β1 on IL-1β-stimulated NF-κB activation and pro-inflammatory cytokine expression. In conclusion, TβRII plays an important but diverse role in regulating renal fibrosis and inflammation. Impaired TGF-β/Smad3, but not the non-canonical TGF-β signalling pathway, may be a key mechanism by which disruption of TβRII protects against renal fibrosis. In addition, deletion of TβRII also enhances NF-κB signalling along with up-regulation of renal pro-inflammatory cytokines, which may be associated with the impairment of anti-inflammatory properties of TGF-β1.     

Expression and Localization of α-amylase in the Submandibular and Sublingual Glands of Mice

In the major salivary glands of mice, acinar cells in the parotid gland (PG) are known to be the main site for the production of the digestive enzyme α-amylase, whereas α-amylase production in the submandibular gland (SMG) and sublingual gland (SLG), as well as the cell types responsible for α-amylase production, has been less firmly established. To clarify this issue, we examined the expression and localization of both the mRNA and protein of α-amylase in the major salivary glands of male and female mice by quantitative and histochemical methods. α-amylase mRNA levels were higher in the order of PG, SMG, and SLG. No sexual difference was observed in α-amylase mRNA levels in the PG and SLG, whereas α-amylase mRNA levels in the female SMG were approximately 30% those in the male SMG. Using in situ hybridization and immunohistochemistry, signals for α-amylase mRNA and protein were found to be strongly positive in acinar cells of the PG, serous demilune cells of the SLG, and granular convoluted tubule (GCT) cells of the male SMG, weakly positive in seromucous acinar cells of the male and female SMG, and negative in mucous acinar cells of the SLG. These results clarified that α-amylase is produced mainly by GCT cells and partly by acinar cells in the SMG, whereas it is produced exclusively by serous demilune cells in the SLG of mice.     

Conversion of β-alanine in vivo and in isolated liver tissue

Summary -alanine was incubated with chopped liver tissue of rats in oxygen for 160 minutes at 37°C. It was established that a certain part of added -alanine (about 12%) disappears, causing an excessive production of urea and of a small quantity of -alanine.In parenteral administration of -alanine an increased urinary excretion of this amino acid is observed in B₆-deficient rats in which the process of -alanine transformation is disturbed.     

Role of Interferon-γ in GVHD and GVL

Interferon （IFN）-γ, a potent proinflammatory cytokine produced by multiple types of cells （e.g., activated T, NK and NKT cells）, plays important and complex roles in both innate and adaptive immune responses. There may be a correlation between the IFN-γ level and GVHD severity in patients receiving allogeneic hematopoietic cell transplantation. However, such a correlation may just reflect the presence of large numbers of activated T cells, and does not necessarily imply a harmful role of IFN-γ in the pathogenesis of GVHD. There has been increasing experimental evidence that IFN-γ is not required and may even inhibit GVHD. Paradoxically, IFN-γ facilitates graft-versus-leukemic （GVL） effects. Thus, IFN-γ blockade is likely deleterious in patients after allogeneic hematopoietic cell transplantation, and not beneficial as previously suggested.     

Expression and localization of E-cadherin and β-catenin in uterine carcinosarcoma

This study was designed to analyze the subcellular localization of E-cadherin and β-catenin both of which play a critical role in cell–cell adhesion in uterine carcinosarcoma (UCS). We performed an immunohistochemical reaction analysis of the subcellular localization of E-cadherin and β-catenin proteins in 46 cases of UCSs consisting of 28 UCSs with heterologous sarcoma and 18 UCSs with homologous sarcoma and compared their clinicopathological features. In most UCSs, membranous expression of E-cadherin and β-catenin was completely lost in sarcomatous components, but it was preserved in carcinomatous components. Nuclear β-catenin expression was observed significantly more frequently in sarcomatous components (31/46, 67.4%) than in carcinomatous components (22/46, 47.8%; P = 0.0025). In sarcomatous components, nuclear β-catenin expression was found significantly more frequently in heterologous sarcoma (23/28, 82.1%) than in homologous sarcoma (8/18, 44.4%; P = 0.0279). The stage was the only independent prognostic significant factor. These results suggest that reduced membranous expression of E-cadherin and β-catenin may contribute to the biphasic morphology of UCS. Furthermore, although the precise mechanism is unclear, nuclear β-catenin expression in sarcomatous components may also be associated with biphasic morphology and heterologous sarcomatous differentiation.     

Actions of 17β-estradiol and testosterone in the mitochondria and their implications in aging

A decline in the mitochondrial functions and aging are two closely related processes. The presence of estrogen and androgen receptors and hormone-responsive elements in the mitochondria represents the starting point for the investigation of the effects of 17β-estradiol and testosterone on the mitochondrial functions and their relationships with aging. Both steroids trigger a complex molecular mechanism that involves crosstalk between the mitochondria, nucleus, and plasma membrane, and the cytoskeleton plays a key role in these interactions. The result of this signaling is mitochondrial protection. Therefore, the molecular components of the pathways activated by the sexual steroids could represent targets for anti-aging therapies. In this review, we discuss previous studies that describe the estrogen- and testosterone-dependent actions on the mitochondrial processes implicated in aging.     

Expressions of Estrogen Receptorαand β in the Development and Maturation of Rat Heart

1 IntroductionPhysiological effects of estrogen on myocardium are mediated by two intracellular estrogen receptors (ER), alpha (ERα) and beta (ERβ). Their role in cardiovascular physiology is not well understood. For this reason,we investigated the expressions of ERα and ERβ in the development and maturation of rat heart.2 Materials and Methods2.1 Experimental animals The study on changes of ERs was performed in six newborn rats with both sexes and six adult female Wistar rats respedively.2.2 Semiquantitati...     

Effects of β-carotene and aspartame on clustogenic activity of cyclophosphamide and dioxidine in mice

Antimutagenic effects of combination of aspartame (0.4 and 4 mg/kg) and -carotene (0.15-15 mg/kg) were studied by estimation of chromosome aberrations in bone marrow cells of C57Bl/6 mice. Single and 5-day treatment with this combination decreased the clastogenic effects of dioxidine and cyclophosphamide and produced a more potent and universal antimutagenic effect than its constituents.     

Immunohistochemical analysis of TGF-β1 and VEGF in gingival and periodontal tissues: a role of these biomarkers in the pathogenesis of scleroderma and periodontal disease

Periodontal disease is characterized by inflammation and bone loss. The balance between inflammatory mediators and their counter-regulatory molecules may be fundamental for determining the outcome of the immune pathology of periodontal disease. Transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) represent a family of polypeptide proteins involved in the inflammation and regulation of immune responses, especially in rheumatic disease. The relationship between these growth factors and periodontitis has resulted in a new field of osteoimmunology and provides a context for better understanding the pathogenesis of periodontal disease. Therefore, the aim of this study was to compare the protein expression profile of these inflammatory mediators in 90 patients divided in three groups: healthy control, chronic periodontitis and in rheumatic disease, scleroderma. The findings presented here highlight that biomarkers, such as TGF-β1 and VEGF, play a key role in the evolution of the immune response, which in turn influences the outcome of disease establishment.     

Effects of β-carotene and aspartame on clustogenic activity of cyclophosphamide and dioxidine in mice

Antimutagenic effects of combination of aspartame (0.4 and 4 mg/kg) and β-carotene (0.15–15 mg/kg) were studied by estimation of chromosome aberrations in bone marrow cells of C57Bl/6 mice. Single and 5-day treatment with this combination decreased the clastogenic effects of dioxidine and cyclophosphamide and produced a more potent and universal antimutagenic effect than its constituents. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 130, No. 11, pp. 570–573, November, 2000     

Involvement of IL-1β and IL-6 in antiarrhythmic properties of atorvastatin in ouabain-induced arrhythmia in rats

Purpose: Evidence show that statins possess wide beneficial cardioprotective and anti-inflammatory effects; therefore, in the present experiment, we investigated the antiarrhythmic properties of atorvastatin in ouabain-induced arrhythmia in isolated rat atria and the role of several inflammatory cytokines in this effect.

Materials and methods: Male rats were pretreated with either of atorvastatin (10?mg/kg) or vehicle, orally once daily for 6 weeks. After induction of anesthesia, we isolated the atria and after incubation with ouabain, time of onset of arrhythmia and asystole as well as atrial beating rate and contractile force were recorded. We also measured the atrial levels of IL-1β, IL-6, and TNF-α after the injection of ouabain to animals.

Results: Pretreatment with atorvastatin significantly delayed the onset of arrhythmia and asystole compared with vehicle-treated group (p?p?p?p?>?.05). Injection of ouabain elevated the atrial levels of IL-1β, IL-6, and TNF-α, while pretreatment of animals with atorvastatin could reverse the ouabain-induced increase in atrial IL-1β and IL-6 (p?p?Conclusions: It is concluded that observed antiarrhythmic effects of atorvastatin might be attributed to modulation of some inflammatory cytokines, at least IL-1β and IL-6.     

Stability and immunoreactivity of glycinin and β-conglycinin to hydrolysis in vitro

The present study was to compare the stability and immunoreactivity of glycinin and β-conglycinin to hydrolysis with pepsin, trypsin or cooperation of the two enzymes for different time intervals (0.5, 1, 15, 30, 60 and 120 min) at different ratios of enzyme/substrate (1:100, 1:10, 1:1 and 10:1) in vitro. The results showed that the immunoreactivity was positively related with stability of glycinin (r=0.776, P<0.05) and β-conglycinin (r=0.851, P<0.05). B polypeptide chain of glycinin was resistant to hydrolysis with trypsin, and β subunit of β-conlycinin was not liable to hydrolysis with pepsin. The two above proteins were little or not affected by incubation time and the enzyme/substrate ratio, while others' hydrolysed degree got higher with prolonging incubation time, and the hydrolysis accelerated with increasing enzyme/substrate ratio. Our results indicated digestive enzyme, incubation time and enzyme/substrate ratio had effects on stability and immunoreactivity of allergenic proteins. The most effective hydrolysis was cooperation of the two enzymes for glycinin and trypsin for β-conglycinin.     

Electropherotypes and serotypes of human rotavirus in Estonia in 1989–1992

Summary The electropherotypes and serotypes of human rotaviruses circulating in Tallinn (Estonia) in 1989–1992 have been studied. Rotaviruses were found in 372 (25.8%) of 1 442 faecal specimens of pediatric patients with acute diarrhea. Polyacrylamide gel electrophoresis of RNA from 318 isolates has revealed 41 electropherotypes. Frequent genomic alterations, including a shift of predominant electropherotypes, were observed during the study period. The serotype of 158 (50.3%) of 314 tested rotavirus isolates was identified using ELISA with VP7-specific monoclonal antibodies against 4 established human rotavirus serotypes. Serotype G1 was found to be largely predominant and accounted for 70.9% of the typeable specimens, serotypes G4, G2, and G3 accounted for 12%, 9.5%, 7.6%, respectively. A major shift to serotype G4 took place in 1990–1991. Serotype G1 was represented by the largest number of electropherotypes. All G1, G3 and G4 isolates were of long and all G2 isolates were of short electropherotypes. According to our results the isolates of an identical electropherotype belong to the same serotype.     

Function and regulation of IL-1α in inflammatory diseases and cancer

The interleukin (IL)-1 family of cytokines is currently comprised of 11 members that have pleiotropic functions in inflammation and cancer. IL-1α and IL-1β were the first members of the IL-1 family to be described, and both signal via the same receptor, IL-1R. Over the last decade, much progress has been made in our understanding of biogenesis of IL-1β and its functions in human diseases. Studies from our laboratory and others have highlighted the critical role of nod-like receptors (NLRs) and multi-protein complexes known as inflammasomes in the regulation of IL-1β maturation. Recent studies have increased our appreciation of the role played by IL-1α in inflammatory diseases and cancer. However, the mechanisms that regulate the production of IL-1α and its bioavailability are relatively understudied. In this review, we summarize the distinctive roles played by IL-1α in inflammatory diseases and cancer. We also discuss our current knowledge about the mechanisms that control IL-1α biogenesis and activity, and the major unanswered questions in its biology.     

Expression and significance of HIF-1α and VEGF-C in gastric carcinoma

Objective The present studies aimed at studying the expression and significance of HIF-1α and VEGF-C in gastric carcinoma. Methods SP method was used for the immunohistochemical detection of HIF-1α and VEGF-C proteins in paraffin embedded archival samples from 44 patients with gastric carcinoma and 20 normal gastric tissues. Results In 44 cases, the positive rate of HIF-1αwas 59.09%(26/44)and 23 cases 52. 27%(23/44)showed VEGF-C positive in cytoplasm and they were significantly higher than those in gastric normal tissue. The positive rate of HIF-1α in gastric cancer tissue expression with lymph node metastasis was higher than that without lymph node metastasis(72. 41% 21/29 vs 33.33 % 5/15;x2 = 6.25, P ＜ 0. 05).The positive rate of VEGF-C expression with lymph node metastasis and that without lymph node metastasis was 65. 52%(19/29)and 26. 67%(4/15)respectively(x2 = 4. 53, P ＜ 0. 05). The expression of HIF-1α and VEGF-C was closely correlated with lymph node metastasis and the depth of tumor invasion(x2 = 5. 14,P ＜ 0. 05, and x2 = 4. 38, P ＜ 0. 05), but no relationship with tumor position, sex and age. The positive expression of HIF-1α was consistent with the positive expression of VEGF-C. Conclusion The up regulated expression of HIF-1α and VEGF-C were observed in gastric cancer. The expression of HIF-1α and VEGF-C were correlated with lymph node metastasis and they were of some value for predicting prognosis of gastric carcinoma.     

Roles of autophagy in lymphocytes： reflections and directions

Recent studies have revealed that autophagy, a fundamental intracellular process, plays many different roles in lymphocyte development and function. Autophagy regulates naive T-lymphocyte homeostasis, specifically by regulating mitochondrial quality and turnover, and is necessary for the proliferation of mature T cells. Autophagy also acts as a cellular death pathway in lymphocytes, both upon prolonged cytokine withdrawal and during acute antigen-receptor stimulation if improperly regulated. Furthermore, during HIV infection, hyperinduction of autophagy leads to massive T-cell death in uninfected CD4⁺ T cells, and is rescued by inhibiting autophagic initiation. Constitutively high levels of autophagy in thymic epithelial cells are necessary for optimal processing and presentation of endogenous antigens, and required for proper positive and negative selection of developing thymocytes. Autophagy also promotes the survival of B lymphocytes, as well as the development of early B-cell progenitors. In B cells, autophagy is an alternative death pathway, as antigen-receptor stimulation in the absence of costimulation induces a potent autophagic death. Thus, autophagy plays a complex role in lymphocytes and is regulated during their lifespan to ensure a healthy immune system.