Dexmedetomidine protects the uterus against ischemia-reperfusion injury in rats.

OBJECTIVE: This study aimed to analyze the histopathological and biochemical effects of dexmedetomidine on the rat uteri exposed to experimental ischemia-reperfusion injury. MATERIALS AND METHODS: Twenty-four female rats were randomly divided into three groups. Group 1 was defined as the control group. An experimental uterine ischemia-reperfusion model was created in Group 2. Group 3 was assigned as the treatment group. Similar uterine ischemia-reperfusion models were created for the rats in Group 3, and then, unlike the other groups, 100 µg/kg of dexmedetomidine was administered intraperitoneally immediately after the onset of reperfusion. In blood biochemical analysis, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA), interleukin 1beta (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels were measured. In the histopathological analyses, endometrial epithelial glandular changes (leukocytosis, cell degeneration) and endometrial stromal changes (congestion, edema) were analyzed using the tissue damage scoring system. RESULTS: It was observed that IL-1ß, IL-6, and TNF-α levels were significantly suppressed in Group 3 compared to Group 2 (p=0.001, p<0.001 and p=0.001, respectively). MDA level was noted as the highest in Group 2. The MDA value in Group 3 was measured at 5.37±0.82, which was significantly decreased compared to Group 2 (p<0.001). An increase in antioxidant enzyme activities (SOD and GSH-PX) was observed in Group 3 compared to Group 2 (p=0.001 and p=0.006, respectively). In our histopathological analysis, a significant improvement in endometrial epithelial glandular and endometrial stromal changes was revealed in Group 3 compared to Group 2 (p<0.001). CONCLUSIONS: In our study, it has been documented that dexmedetomidine protects the uterine tissue against ischemia-reperfusion injury.

Evaluation of glutathione peroxidase and superoxide dismutase enzyme polymorphisms in celiac disease patients.

Celiac disease (CD) is a multifactorial, inflammatory small bowel disorder characterized by nutrient malabsorption resulting from mucosal damage, the latter induced by cereal products like barley, oat, and wheat. Oxidative stress has previously been reported to play an important role in the pathogenesis of CD. In the present study, we aimed to evaluate the frequency of polymorphisms that affects the structure of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), with levels being dependent on the amount of oxidative stress and whether or not there is an association with the mutations DQA1*0501, DQB1*0201, and DRB1*04 that are frequently reported for CD. SOD and GSH-Px polymorphisms were investigated by real-time quantitative polymerase chain reaction in 265 cases. Of the 117 cases that had at least one of DQA1*0501, DQB1*0201, or DRB1*04, 98 (83.75%) also had SOD enzyme polymorphisms and 68 (58.12%) also had GSH-Px polymorphisms. In conclusion, although the etiology of CD is not yet entirely clear, many mechanisms have been suggested. This study supports the notion that SOD and GSH-Px polymorphisms are involved in CD development, even though our findings were not statistically significant, and, furthermore, are influenced at various levels. SOD polymorphisms and activities were more frequently identified than those of GSH-Px.

The protective effects of endothelin-A receptor antagonist BQ-123 in pentylenetetrazole-induced seizure in rats.

Endothelin-1 has been shown to increase neuronal activity and glutaminergic synaptic transmission by endothelin-A receptors (ETAR) in the nucleus tractus solitarius neurons that play an important role in epileptic seizures. Therefore, BQ-123 as an ETAR antagonist might attenuate neuronal excitability and glutaminergic synaptic transmission. The main purpose of the present study is to investigate the protective effect of acute BQ-123 treatment against pentylenetetrazole (PTZ)-induced tonic-clonic seizures. Wistar albino rats were divided into three groups: control, PTZ, and PTZ + BQ-123 groups. BQ-123 (3 mg/kg, intravenously) was administered for 15 min before injecting with PTZ (50 mg/kg, intraperitoneally). We determined a delay resulting from BQ-123 in "duration of the seizure onset." "Number of rats with major seizure" also decreased according to scoring with video camera in PTZ + BQ-123 group. In BQ-123-treated group, there were eight rats without a major seizure, but only one rat had a delayed major seizure. The brain tissue glutathione peroxidase activity was significantly decreased in the PTZ and PTZ + BQ-123 groups. According to the results of the control group, there was a significant increase in the protein carbonyl levels of the PTZ group and a significant increase in the nitric oxide levels of the PTZ + BQ-123 group. Histological examination showed an increase in the number of neuronal hyperchromatic nucleus especially in hippocampal gyrus dentatus region of BQ-123-treated group. We concluded that BQ-123 impeded the formation and spread of seizure to a great degree. The beneficial effects of BQ-123 were comparatively supported with biochemical parameters and histological examinations.

Attenuation of iNOS mRNA exacerbates hypoperfusion and upregulates endothelin-1 expression in hippocampus and cortex after brain trauma.

Nitric oxide (NO, a vasodilator) and endothelin-1 (ET-1, a powerful vasoconstrictor) participate in the regulation of brain's microcirculation influencing each other's expression and synthesis. Following injury to the brain, NO is derived largely from the inducible form of nitric oxide synthase (iNOS). We used Marmarou's model of traumatic brain injury (TBI) to study the cerebral blood flow and expression (mRNA) of ET-1 in rats that were pretreated with antisense iNOS oligodeoxynucleotides (ODNs). Intracerebroventricular application of iNOS ODNs resulted in reduced synthesis of iNOS as detected by Western blot analysis. The cerebral blood flow (measured by laser Doppler flowmetry), generally decreased after TBI, was further markedly reduced in the treated animals and remained at low levels up to 48 h post-TBI. The expression of ET-1 (detected by in situ hybridization in cortex and hippocampus) was increased 2-3-fold following TBI alone and this increase reached 5-6-fold in animals pretreated with antisense iNOS ODNs. The results indicate that most likely, NO, generated primarily by iNOS, suppresses ET-1 production and that a decrease of NO results in upregulation of ET-1 via transcriptional and translational mechanisms. Increased availability of ET-1 at the vascular bed and the neuropil may contribute to the altered microvascular reactivity and reduced perfusion of the brain following TBI.

ERM proteins of the lens.

Ezrin and radixin and protein 4.1 were detected in the lens of the eye. These proteins were mainly present in the young elongating cortical fiber cells and localized to the plasma membranes. Moesin was not detected. Ezrin, radixin, and protein 4.1 provide another means whereby actin is linked to the plasma membrane in addition to the known adherens junctions in the lens.

Paralemnin of the lens.

Paralemmin was identified in the chicken lens as a protein with mol. wt 65 kDa and a splice variant of 60 kDa, both soluble in Triton X-100. Paralemmin is localized to the plasma membrane of fiber cells, and was not detected in the annular pad cells. Thus in the chick lens it is another feature of fiber cell differentiation. Its localization to the short side of the fiber cell and the sites of fiber cell interlocking suggests that paralemmin may play a role in the development of such interdigitating processes.

Associated proteins of lens adherens junction.

Cytoplasmic proteins associated with adherens junctions were identified in the chicken ocular lens. The catenins, alpha, beta, and gamma, were present in epithelial and fiber cells, although their pattern of distribution changed with fiber cell differentiation. The sharp decline in alpha-catenin with fiber cell formation and the increasing Triton-insolubility of N-cadherin suggests that another subtype of alpha-catenin exists in the lens.

Heat shock proteins of adult and embryonic human ocular lenses.

We investigated the presence and distribution of heat shock proteins, HSP-70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449-10453], HSP-40, HSc-70, HSP-27, and alphabeta-crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS-PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP-27 and alphabeta-crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP-27. The presence of HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age-related cataract.

Expression of membrane-type matrix metalloproteinases 4, 5, and 6 in mouse corneas infected with P. aeruginosa.

PURPOSE: To investigate the expression and regulation of membrane-type matrix metalloproteinases (MT-MMPs) 4, 5, and 6 in the mouse corneas infected with Pseudomonas aeruginosa. METHODS: C57BL/6J mice were intracorneally infected with P. aeruginosa. The expression of MT4-, MT5-, and MT6-MMP was detected at both the mRNA and protein levels by RT-PCR and immunoblot analysis. Immunohistochemical staining was performed to localize the expression of MT4- and MT5-MMP in the mouse corneas. RESULTS: Expression of MT4- and MT5-MMP was detected in the normal (uninfected) cornea by RT-PCR and immunoblot analysis. When infected with P. aeruginosa, the corneas showed significant induction of each MT-MMP. Localization of MT4- and MT5-MMP revealed that the expression of MT5-MMP was restricted to the epithelial tissue in the normal cornea, whereas the induced expression of MT4- and MT5-MMP was predominantly in the substantia propria, which contained most of the infiltrating cells. MT6-MMP expression was not detected in the uninfected cornea but was upregulated in the infected corneas. CONCLUSIONS: Expression of MT4-, MT5-, and MT6-MMP was induced in corneas infected with P. aeruginosa. Immunohistochemistry showed predominant immunoreactivity of MT4- and MT5-MMP in the substantia propria. Previous histologic studies have revealed different patterns of inflammatory cell infiltration with an increased number of polymorphonuclear neutrophils (PMNs) during the early stage of inflammation and increased macrophages during the late stage. These results indicate a good correlation between the overexpression of the MT-MMPs in the infected corneas and the inflammatory response-that is, leukocyte infiltration-indicating that inflammatory cells such as macrophages and PMNs may play a role in the upregulation of MT-MMPs during corneal infection, which in turn can cause the destruction of corneal tissue.

Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosa.

C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection.

Heat shock proteins of chicken lens.

The presence of heat shock proteins HSP-40, HSP-70, and HSc-70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP-40, HSP-70, and HSc-70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP-40, HSP-70, and HSc-70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunofluorescence technique. Data obtained from these experiments revealed that HSP-40, HSP-70, and HSc-70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP-40, HSP-70, and HSc-70 can interact with protein of these areas and protect them from stress induced denaturation.

Plasminogen Activators and Inhibitors in the Corneas of Mice Infected with Pseudomonas aeruginosa.

PURPOSE: To characterize the presence of plasminogen activators and their inhibitors in the corneas during the inflammatory response in naïve and immunized mice intracorneally infected with Pseudomonas aeruginosa. METHODS: RT-PCR was used to detect gene expression for plasminogen activators and their inhibitors in naïve and immunized mice. Immunoblot analysis, zymography, and ELISA were used to demonstrate the syntheses of these proteins. RESULTS: Naïve mice intracorneally infected with P. aeruginosa showed a temporally enhanced expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), over a several-day holding period. Immunized mice demonstrated a lower and shorter expression of these factors over the same period. Expression of these factors at the mRNA and protein levels may have been due to enzymes and inhibitors present in inflammatory cells and in resident corneal cells. CONCLUSIONS: These results show a correlation between the overexpression of the PA system in infected naïve mice as part of the inflammatory response, with eventual ocular destruction. Immunized mice exhibit a more balanced and shorter expression of the PA system, which may contribute to the restoration of corneal clarity.

A heat shock transcription factor like protein in the nuclear matrix compartment of the tissue cultured mammalian lens epithelial cell.

This investigation characterizes a prominent nuclear matrix protein isolated from tissue cultured mouse lens epithelial cells. The nuclear matrix protein was isolated using a modified Penman technique. Total nuclear matrix proteins were further separated by SDS-polyacrylamide gel electrophoresis. The SDS-PAGE profile of the nuclear matrix proteins displayed a prominent doublet band at 60 kDa region. Nonequilibrium 2D gel electrophoresis revealed that this protein is a basic nuclear protein. This 60 kDa protein was further characterized by comparing its internal peptide amino acid sequence with known protein sequence using the BLAST technique, and this study demonstrated that 60 kDa nuclear matrix protein displays significant sequence similarity with Xenopus Laevis heat shock transcription factor. We also raised antibodies against 60 kDa nuclear matrix protein. Immunofluorescence, studies showed that this 60 kDa nuclear matrix protein preferably decorates nucleus, and puncted pattern of fluorescence suggest presence of this protein in the discrete areas of the nucleus. Heat shock transcription factors upregulate synthesis of heat shock proteins and many of these protein act as molecular chaperones. Thus, presence of a nuclear matrix protein with significant sequence similarity with heat shock transcription factor suggests sustained heat shock protein synthesis in the mouse lens cells.

Membrane-type matrix metalloproteinases in mice intracorneally infected with Pseudomonas aeruginosa.

PURPOSE: To establish the presence of membrane-type matrix metalloproteinases (MT-MMPs) in the cornea and their expression in naive and immunized mice intracorneally infected with Pseudomonas aeruginosa. METHODS: Naive (unimmunized) and immunized C57BL/6J mice were infected with P. aeruginosa, and gene expression of MT-MMPs were detected by RT-PCR. Immunoblot analysis and immunostaining were also used to characterize the MT-MMP response in both sets of animals. RESULTS: Expression of MT1-MMP, MT2-MMP, and MT3-MMP (MMP 14, 15, and 16) was detected by RT-PCR and immunoblot analysis. Of the three MT-MMPs detected, MT1-MMP exhibited the greatest expression at protein levels. In general, a bell-shaped curve was obtained for each of the MT-MMPs in naive mice, but all of them showed much less expression in the immunized mice. MT1-MMP was localized in the epithelial tissue of the cornea, whereas MT2-MMP and MT3-MMP were mainly found in the interface between the epithelium and substantia propria. CONCLUSIONS: MT1-MMP was detected and expressed to a greater extent in naive mice than MT2-MMP and MT3-MMP. Peak expression of all three MT-MMPs showed a good correlation with the overall inflammatory response.

Nonchromatin nuclear proteins of mammalian lens epithelial cells.

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determined possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [35S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development.

Sequence-specific DNA binding activities of nuclear matrix proteins of mammalian lens epithelial cells.

This study examines matrix and nonmatrix nuclear proteins of the rabbit lens epithelial cells. The nuclear matrix proteins were isolated by modified Penman technique, which requires presence of detergents and nucleases, whereas nonmatrix nuclear proteins were obtained by high salt extraction. The data from these experiments revealed presence of DNA binding activities for SP-1 and OCT-1 proteins in both matrix and non-matrix compartments of rabbit lens epithelial cells. Comparison of the relative abundance of SP-1 and OCT-1 binding activities in nuclear matrix and nonmatrix fractions suggest the distribution between these two compartments is cell type specific and possibly related to the control of cell growth.

NCAM of the mammalian lens.

NCAM is present in the plasma membranes of human and rat lens epithelial cells and superficial fiber cells. The predominant isoform in epithelial cells is NCAM 140, while NCAM 120 appears only in the superficial fiber cells. The immunofluorescence patterns are consistent with a decreasing concentration of NCAM associated with fiber cell differentiation.

Reversal of the limited proteolysis of MP26 during the reversal and prevention of the galactose cataract in rat lenses.

The reversal and prevention of the galactose-induced cataract in rats were employed to study their effects on the acceleration of the limited proteolysis of MP26 into MP23-24 previously observed in cataractous lenses of galactose-fed animals. Lenses of rats on a cataract reversal-diet demonstrated the reversal of MP23-24 and MP26 levels to control levels in the clearing cortical areas but not in remaining cataractous nuclear areas. Acceleration of the limited proteolysis of MP26 was observed in the nucleus but not the cortex in the clear lenses of animals on a cataract prevention-diet. The results demonstrated that the limited proteolysis of MP26 may form part of a gradual aging process that although not directly (causally) related to cataractogenesis may at least be accelerated by cataractogenic agents or conditions.

Enolase in the avian and turtle lens.

Enolase is a dimeric enzyme of molecular weight of 100,000 daltons, which plays an important role in the glycolytic cycle. The aim of this study was to characterize the enzyme of the chicken lens epithelium and to compare its distribution in different regions of the chicken, duck and turtle lens. Enolase of the chicken lens epithelium was found to be an enzymatically active dimeric protein of molecular weight 100,000 daltons and representing alpha-enolase. It is a major component of the epithelium comprising 4%, 12% and 46% of the water-soluble protein of chicken, duck and turtle epithelium respectively. Enolase is found in trace amount in the fiber cells of the chicken and duck, but is retained in much greater concentration in the turtle fiber mass as a predominantly inactive enzyme.

Ankyrin of the ocular lens.

Ankyrin was identified in the human, bovine and chicken lens as a protein of molecular weight 216 kilodaltons. It is specifically extracted from association with the fiber cell plasma membranes by high-ionic-strength salt solution and is predominantly found in young fiber cells. A protein which is unrelated to plakoglobin, protein 4.1 or ankyrin is also specifically extracted by high-salt solution.