Cytoskeletal regulation of Caco-2 intestinal monolayer paracellular permeability

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and processes that regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments, and microtubules. Cytochalasin-b (5 m?g/ml) and colchicine (2 × 10^?5M) at the doses used caused marked depolymerization and disruption of actin microfilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracellular permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway “re-tightening” following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercellular junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin microfilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes. This re-closure process did not require protein synthesis or microtubule-mediated shuttling process. © 1995 Wiley-Liss, Inc.     

Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake

Summary Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.Abbreviations bar/PAT bialaphos resistance gene/phosphinotricin acetyltransferase - 2,4-D 2,4-di-chlorophenoxyacetic acid - dhfr/DHPR dihydrofolate reductase gene/enzyme - gus/GUS -glucuronidase gene/enzyme - hpt/HPT hygromycin phosphotransferase gene/enzyme - Kn kinetin - PEG polyethylene glycol - RH relative humidity     

Fine needle aspiration cytology in malignant Sertoli cell tumor of the testis. A case report.

A rare case of malignant Sertoli cell tumor of the testis occurred in which the diagnosis was initially suggested on fine needle aspiration cytology. Smears of the testicular, inguinal and cervical lymph node aspirates showed cells resembling normal Sertoli cells arranged in nests and tubules. Histopathology of the orchidectomy specimen confirmed the diagnosis. The literature is briefly reviewed.     

Marine Bacterial Compounds Evaluated by In Silico Studies as Antipsychotic Drugs Against Schizophrenia

Schizophrenia (SCZ) is one of the brain disorders which affects the thinking and behavioral skills of patients. This disorder comes along with an overproduction of kynurenic acid in the cerebrospinal fluid and the prefrontal cortex of SCZ patients. In this study, marine bacterial compounds were screened for their suitability as antagonists against human kynurenine aminotransferase (hKAT-1) which causes the synthesis of kynurenic acid downstream which ultimately causes the SCZ disorder according to the kynurenic hypothesis of SCZ. The marine actinobacterial compound bonactin shows more promising results than other tested marine compounds such as the histamine H2 blocker famotidine and indole-3-acetic acid (IAC) from docking and in silico toxicological studies carried out here. The obtained results of the Grid-based Ligand Docking with Energetics (Glide) scores of extra-precision (XP) Glide against the target protein hKAT-1 on IAC, famotidine, and bonactin were ??6.581, ??6.500 and ??7.730 kcal/mol where Glide energies were ??29.84, ??28.391, and ??47.565 kcal/mol, respectively. Bonactin is known as an antibacterial and antifungal compound being extracted from a marine Streptomyces sp. Comparing tested compounds against the drug target hKAT-1, bonactin alone showed the best Glide score and Glide energy on the target protein hKAT-1.     

Staged Reimplantation of a Total Hip Prosthesis After Co-infection with <Emphasis Type="Italic">Candida tropicalis</Emphasis> and <Emphasis Type="Italic">Staphylococcus haemolyticus</Emphasis>: A Case Report

Fungal prosthetic joint infection is a rare complication in total joint arthroplasty. There are no established guidelines for management of these infections. We present a case of a 53-year-old male with a hip joint prosthesis co-infected with Candida tropicalis and Staphylococcus haemolyticus. A two-stage exchange arthroplasty was performed. The patient underwent implant removal, debridement, irrigation with saline solution and application of cement spacer impregnated with vancomycin followed by aggressive antimicrobial treatment in first stage. Complete eradication of infection was demonstrated by negative culture of sonicated cement spacer fluid and negative 16S rRNA and 18S rRNA gene PCR of sonicate fluid, synovial fluid and periprosthetic tissue samples. He underwent second-stage revision hip arthroplasty after 9 months of the first stage. At the latest follow-up, there was no evidence of recurrence of infection. This case illustrates the utility of sonication of biomaterials and molecular techniques for microbiological confirmation of absence of infection in staged surgeries which is required for a successful outcome.     

Serum metabolomics reveals many novel metabolic markers of heart failure, including pseudouridine and 2-oxoglutarate

There is intense interest in the identification of novel biomarkers which improve the diagnosis of heart failure. Serum samples from 52 patients with systolic heart failure (EF < 40% plus signs and symptoms of failure) and 57 controls were analyzed by gas chromatography – time of flight – mass spectrometry and the raw data reduced to 272 statistically robust metabolite peaks. 38 peaks showed a significant difference between case and control (p < 5 × 10⁻⁵). Two such metabolites were pseudouridine, a modified nucleotide present in t- and rRNA and a marker of cell turnover, as well as the tricarboxylic acid cycle intermediate 2-oxoglutarate. Furthermore, 3 further new compounds were also excellent discriminators between patients and controls: 2-hydroxy, 2-methylpropanoic acid, erythritol and 2,4,6-trihydroxypyrimidine. Although renal disease may be associated with heart failure, and metabolites associated with renal disease and other markers were also elevated (e.g. urea, creatinine and uric acid), there was no correlation within the patient group between these metabolites and our heart failure biomarkers, indicating that these were indeed biomarkers of heart failure and not renal disease per se. These findings demonstrate the power of data-driven metabolomics approaches to identify such markers of disease. Warwick B. Dunn, David I. Broadhurst and Sasalu M. Deepak contributed equally to this work.     

Phenotypic divergence and molecular characterization of two sympatric species of genus Barilius (Hamilton) from the River Chenab of Western Himalaya

Barilius bendelisis and Barilius vagra inhabit mostly in the spring‐fed streams of Indian Himalaya and constitute an important portion of protein diet of the rural human population residing near the stream banks. In spite of large aquaculture importance of this genus very less morphometric and molecular studies have been conducted. This study addresses morphometric and molecular characterization of B. bendelisis and B. vagra by using truss analysis and mitochondrial COI gene. A total of 293 samples of Barilius bendelisis and 127 samples of Barilius vagra were collected from three different sites of Chenab river basin from March 2015 to April 2017. 14 landmarks were used to measure 90 truss measurements from digital images of specimens using network of three softwares, tpsUtil, tpsDig2 and PAST. A total of 89 distance variables exhibited significant differences among the populations. The principal component analysis generated ten components explaining 89.164% of total variance among six populations of B. bendelisis and B. vagra. 89.0% of individuals of B. bendelisis and B. vagra were classified into their original groups. The Phylogenetic analysis with other published COI sequences revealed distinct nature of these two species. The study may aid in taxonomic identification and phenotypic divergence which will be helpful in proper conservation and management of these species of hill trout in India.     

Altered lipid metabolism in post-traumatic epileptic rat model: one proposed pathway

Molecular Biology Reports - Post-traumatic epilepsy (PTE) is a common long-term risk associated with traumatic brain injury (TBI). PTE rat model, proposed by Willmore et al., is a well known model...