Using Model Systems To Study Rat Lens Membrane Damage During Cortical Cataract Formation

Cataract formation is a complex, multifactorial and multistep process: it is associated with cellular and biochemical changes in the lens. In the present work, steps in the pathway to cataract development were followed in vitro using two different reagents which induce cataract formation by two different mechanisms: elevated glucose (55.6 mM), as a model for in vivo diabetic cataractogenesis and an actin monomer-stabilizer, cytochalasin D (10{dollar}\sp{lcub}-5{rcub}{dollar} M, CD). Findings were utilized to classify and arrange the events in a common pathway and to focus on the mechanism of cataract production.;The development of cortical subcapsular opacity in cultured lenses was elucidated as a time-dependent phenomenon by measuring different parameters: (i) distribution of the living and dead cells in the lens through a confocal microscope following their fluorescent staining, (ii) appearance of the lens observed daily by a dissection microscope, and (iii) changes in and/or leakage of intracellular components ({dollar}\gamma{dollar}-crystallin, LDH, and magnesium ion). The findings suggested possible mechanisms for the action of the cataract-causative agents, glucose and CD, which initiate membrane damage in the lens cells in both model systems and help locate these factors at common steps in their pathways to cataract.;Using this reproducible lens incubation system, various potential anti-cataract agents e.g., antioxidants, sorbitol-lowering agent, and other agent were tested in an in vitro diabetic model systems. All these agents were relatively effective in preventing cataract formation in the in vitro diabetic model system. These findings support the hypothesis that the damage processes in the presence of glucose involve glycation and oxidative stress. The effect of an antioxidant, Vitamin C (VC), was also tested to explore the factors involved in the mechanism of CD-cataract formation. Prevention of damage by VC is consistent with the hypothesis that oxidative stress is associated with the disorganization of actin and cytoskeletal network during cataract formation.;The timing and localization of calcium uptake in the lens was studied in both cataractous model systems by radioactive and fluorescent labelling techniques. The lens calcium uptake increased parallel to the incubation time in cataract causative agents. The effect of CD was earlier and more severe than the glucose effect. The results of lens glucose-treatment, as well as inhibitors showed that elevated calcium could be one of the factors leading to cataract development. Finally, the role of calpain-proteolysis of spectrin/fodrin was studied during cataract formation process in both model systems. The results suggest that the proteolysis of spectrin/fodrin does not precede cataract formation but it is a consequence of precataractous changes

Responses of Plasma Catecholamine, Serotonin, and the Platelet Serotonin Transporter to Cigarette Smoking

Cigarette smoking is one of the major causes of coronary heart disease with a thirty percent mortality rate in the United States. Cigarette smoking acting on the central nervous system (CNS) to stimulate the sympathetic nervous system (SNS) through, which facilitates the secretion of serotonin (5-HT) and catecholamines to supraphysiological levels in blood. The enhanced levels of 5-HT and catecholamines in smokers’ blood are associated with increases in G protein-coupled receptor signaling and serotonylation of small GTPases, which in turn lead to remodeling of cytoskeletal elements to enhance granule secretion and promote unique expression of sialylated N-glycan structures on smokers’ platelets. These mechanisms enhance aggregation and adhesion of smokers’ platelets relative to those of non-smokers. This review focuses on the known mechanisms by which 5-HT and SERT, in coordinated signaling with catecholamines, impacts cigarette smokers’ platelet biology

In Vitro Assay and Characterization of the Farnesylation-Dependent Prelamin a Endoprotease

The 72-kDa nuclear lamina protein lamin A is synthesized as a 74-kDa farnesylated precursor. Conversion of this precursor to mature lamin A appears to be mediated by a specific endoprotease. Prior studies of overexpressed wild-type and mutant lamin A proteins in cultured cells have indicated that the precursor possesses the typical carboxyl-terminal S- farnesylated, cysteine methyl ester and that farnesylation is required for endoproteolysis to occur. In this report, we describe the synthesis of an S- farnesyl, cysteinyl methyl ester peptide corresponding to the carboxyl- terminal 18 amino acid residues of human prelamin A. This peptide acts as a substrate for the prelamin A endoprotease in vitro, with cleavage of the synthetic peptide at the expected site between Tyr657 and Leu658. Endoproteolytic cleavage requires the S-prenylated cysteine methyl ester and, in agreement with transfection studies, is more active with the farnesylated than geranylgeranylated cysteinyl substrate. N-Acetyl farnesyl methyl cysteine is shown to be a noncompetitive inhibitor of the enzyme. Taken together, these observations suggest that there is a specific farnesyl binding site on the enzyme which is not at the active site

The Cellular Distribution of Serotonin Transporter Is Impeded on Serotonin-Altered Vimentin Network

BACKGROUND:The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments. METHODOLOGY/PRINCIPAL FINDINGS:We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin. To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Delta14 and Delta20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S(611), T(613), and T(616) arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter. CONCLUSIONS/SIGNIFICANCE:Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network

Clinical presentation of abdominal tuberculosis in HIV seronegative adults

BACKGROUND: The accurate diagnosis of abdominal tuberculosis usually takes a long time and requires a high index of suspicion in clinic practice. Eighty-eight immune-competent patients with abdominal tuberculosis were grouped according to symptoms at presentation and followed prospectively in order to investigate the effect of symptomatic presentation on clinical diagnosis and prognosis. METHODS: Based upon the clinical presentation, the patients were divided into groups such as non-specific abdominal pain & less prominent in bowel habit, ascites, alteration in bowel habit, acute abdomen and others. Demographic, clinical and laboratory features, coexistence of pulmonary tuberculosis, diagnostic procedures, definitive diagnostic tests, need for surgical therapy, and response to treatment were assessed in each group. RESULTS: According to clinical presentation, five groups were constituted as non-specific abdominal pain (n = 24), ascites (n = 24), bowel habit alteration (n = 22), acute abdomen (n = 9) and others (n = 9). Patients presenting with acute abdomen had significantly higher white blood cell counts (p = 0.002) and abnormalities in abdominal plain radiographs (p = 0.014). Patients presenting with alteration in bowel habit were younger (p = 0.048). The frequency of colonoscopic abnormalities (7.5%), and need for therapeutic surgery (12.5%) were lower in patients with ascites, (p = 0.04) and (p = 0.001), respectively. There was no difference in gender, disease duration, diagnostic modalities, response to treatment, period to initial response, and mortality between groups (p > 0.05). Gastrointestinal tract alone was the most frequently involved part (38.5%), and this was associated with acid-fast bacteria in the sputum (p = 0.003). CONCLUSION: Gastrointestinal tract involvement is frequent in patients with active pulmonary tuberculosis. Although different clinical presentations of patients with abdominal tuberculosis determine diagnostic work up and need for therapeutic surgery, evidence based diagnosis and consequences of the disease does not change

Subcellular Localization and Partial Purification of Prelamin a Endoprotease: An Enzyme Which Catalyzes the Conversion of Farnesylated Prelamin a to Mature Lamin A

The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease

Regulation of Prelamin a Endoprotease Activity by Prelamin A

The maturation of lamin A is completed by the endoproteolytic cleavage of its farnesylated precursor protein, prelamin A. In the absence of this cleavage, prelamin A can neither give rise to lamin A nor assemble into the nuclear lamina. We call the enzyme which catalyzes this endoproteolytic step the \u27prelamin A endoprotease\u27. In this study, we begin characterization of the regulation of prelamin A endoprotease. In particular, we address the question as to whether prelamin A endoprotease activity is constitutive in cells or responds to expression of prelamin A. To do this, we compared the activity of this novel endoprotease in cells which express prelamin A with those that do not. Our data shows that the enzymatic activity of prelamin A endoprotease is enhanced by the expression of prelamin A

Prevention of microbial colonization of feeding tubes in the intensive care unit

Background Various microorganisms which increase the mortality rate in the intensive care unit (ICU) cause microbial colonization of the nasogastric tube (NGT) and use the NGT as a reservoir. Aim To detect the colonization on the NGT and to determine the effect that training regarding hand hygiene, NGT management, and enteral feeding (EF) provided to ICU nurses and auxiliary service staff (ASS) has on the level of NGT colonization. Methods A quasi-experimental pre-test and post-test control design was used in this study. Microbial samples were taken from the outer and inner parts of NGT. The microorganisms were categorized as: group 1, no risk; group 2, low risk pathogenic; group 3, high-risk pathogenic group. The training was given to nurses (n = 15) and ASS (n = 7). Hand hygiene, NGT, and EF care training are provided to nurses and ASS by researchers. A total of three training sessions were scheduled to be held in 3 weeks so that all health care staff members were trained. Each session lasted 2 h in total. Patients were assigned to a group if one of the microorganisms presented on the outer surface of the patient's feeding tube and/or on the hub. The hand hygiene compliance was evaluated by direct observation according to the World Health Organization hand hygiene indications. Results The study was conducted with 46 patients. Evaluating the patients for the presence of microorganisms before education revealed that 4.3% were in group 1, 21.8% were in group 2, and 73.9% were in group 3. After the education, evaluating the samples for the presence of microorganisms revealed that 39.1% were in group 1, 13% were in group 2, and 47.8% were in group 3. A statistically significant difference was found between the number of samples included in the groups after the participants had received training (H = 8.186; p = .017). Conclusions An NGT could act as a reservoir of microbial colonization and high-risk microorganisms could be on the tube. Providing training not only to nurses but also to ASS will help reduce the risk of colonization. Relevance to Clinical Practice Eliminating such colonization with effective hand hygiene during NGT feeding is a cost-effective method. Providing training not only to nurses but also to ASS will help obtain the optimum benefit from patient care