Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

Isoleucyl transfer ribonucleic acid synthetase. Competitive inhibition with respect to transfer ribonucleic acid by blue dextran.

The inhibitory effects of blue dextran and a small dye molecule derived from it (F3GA-OH) on the steady-state reaction catalyzed by Escherichia coli isoleucy-tRNA synthetase have been studied. Blue dextran gave uncompetitive inhibition with respect to Mg.ATP, mixed inhibition with respect to L-isoleucine, and competitive inhibition with respect to tRNA. The small dye molecule (F3GA-OH) was also competitive with respect to tRNA. These inhibition patterns were not consistent with the bi-uni-uni-bi Ping Pong mechanism generally accepted for aminoacyl-tRNA synthetases. They were consistent with a mechanism in which a second L-isoleucine is bound after isoleucyl-AMP synthesis and before transfer of the isoleucyl moiety to tRNA. Enzyme-bound L-isoleucine lowered the affinity of the enzyme for blue dextran approximately fivefold, a value comparable to the ninefold lowering of the enzyme's affinity for tRNA upon binding L-isoleucine. The affinity of the synthetase for F3GA-OH (K1 = 1.0 X 10(-7) M) is approximately fivefold higher than its affinity for blue dextran (K1 = 5.3 X 10(-7) M). These results indicate that blue dextran and its derivatives may be useful for kinetic and physical studies of polynucleotide binding sites on proteins as well as NAD and ATP sites.     

Hazards in the use of Cibacron Blue F3GA in studies of proteins.

Cibacron Blue F3GA from several commercial sources is shown to be heterogeneous. This crude dye inactivates both phosphoglycerate kinase and isoleucyl-tRNA synthetase. Purification of Cibacron Blue F3GA to homogeneity results in a dramatic decrease in inactivation of these enzymes. The inactivation is shown to be due to covalent modification of phosphoglycerate kinase and probably isoleucyl-tRNA synthetase by a minor component present in crude Cibacron Blue F3GA.     

Over‐Expression of RNA Processing,Heat Shock,and DNA Repair Proteins in Breast Tumor Compared to Normal Tissue

This study identifies the main changes in protein expression in human breast tumors compared to normal breast tissue. Malignant tumors (32) and normal breast tissue samples (23), from formaldehyde‐fixed, paraffin‐embedded specimens are subjected to discovery proteomics using liquid chromatography/tandem mass spectrometry, with spectral counts for quantitation. The dataset contains 1406 proteins. Differential expression is measured using a method that takes advantage of estimates of the percentage of tumor on a slide. This analysis shows that the major classes of proteins over‐expressed by tumors are RNA‐binding, heat shock and DNA repair proteins. RNA‐binding proteins, including heterogeneous nuclear ribonucleoproteins (HNRNPs), SR splice factors (SRSF) and elongation factors form the largest group. Comparison with results from another study demonstrates that the RNA‐binding proteins are associated specifically with malignant transformation, rather than with cell proliferation. HNRNP and SRSF proteins help define splice sites in normal cells. Their over‐expression may dysregulate splicing, which in turn has the potential to promote malignant transformation.     

Effect of Yokukansan on sleep disturbance and neuropathic pain in chronic constriction injury using a rat model

Sleep and Biological Rhythms - Patients with chronic pain develop peripheral neuropathy and experience sleep disturbance. Yokukansan is used to treat insomnia and control neuropathic pain. We...     

Assessment of human adenovirus removal by qPCR in an advanced water reclamation plant in Georgia,USA

Aims

To assess human adenoviruses (HAdVs) removal in an advanced wastewater treatment facility and compare two parallel tertiary treatment methods for the removal of HAdVs.

Methods and Results

Tangential flow ultrafiltration was used to concentrate the water samples, and HAdVs were precipitated by polyethylene glycol. HAdVs were detected only by TaqMan real‐time PCR, and HAdV genotype was determined by DNA sequence. HAdVs were detected in 100% of primary clarification influent, secondary clarification effluent and granular media (GM) filtration effluent samples but only in 31·2% of membrane filtration (MF) effluent and 41·7% of final effluent (FE) samples, respectively. The average HAdVs loads were significantly reduced along the treatments but HAdVs were still present in FE. Comparison of two parallel treatments (GM vs MF) showed that MF was technically superior to GM for the removal of HAdVs.

Conclusions

These findings indicate that adenoviruses are not completely removed by treatment processes. MF is a better treatment for removal of adenoviruses than GM filtration. Because only qPCR was used, the results only indicate the removal of adenovirus DNA and not the infectivity of viruses.

Significance and Impact of the Study

Presence of HAdVs in FE by qPCR suggests a potential public health risk from exposure to the treated wastewater and using the FE for recreational or water reuse purposes should be cautious.     

A Systematic Review of Risk Factors Associated with Surgical Site Infections among Surgical Patients

Importance

Surgical site infection (SSI) complicates 2-5% of surgeries in the United States. Severity of SSI ranges from superficial skin infection to life-threatening conditions such as severe sepsis, and SSIs are responsible for increased morbidity, mortality, and economic burden associated with surgery. Staphylococcus aureus (S. aureus) is a commonly-isolated organism for SSI, and methicillin-resistant S. aureus SSI incidence is increasing globally.

Objective

The objective of this systematic review was to characterize risk factors for SSI within observational studies describing incidence of SSI in a real-world setting.

Evidence Review

An initial search identified 328 titles published in 2002-2012; 57 were identified as relevant for data extraction. Extracted information included study design and methodology, reported cumulative incidence and post-surgical time until onset of SSI, and odds ratios and associated variability for all factors considered in univariate and/or multivariable analyses.

Findings

Median SSI incidence was 3.7%, ranging from 0.1% to 50.4%. Incidence of overall SSI and S. aureus SSI were both highest in tumor-related and transplant surgeries. Median time until SSI onset was 17.0 days, with longer time-to-onset for orthopedic and transplant surgeries. Risk factors consistently identified as associated with SSI included co-morbidities, advanced age, risk indices, patient frailty, and surgery complexity. Thirteen studies considered diabetes as a risk factor in multivariable analysis; 85% found a significant association with SSI, with odds ratios ranging from 1.5-24.3. Longer surgeries were associated with increased SSI risk, with a median odds ratio of 2.3 across 11 studies reporting significant results.

Conclusions and Relevance

In a broad review of published literature, risk factors for SSI were characterized as describing reduced fitness, patient frailty, surgery duration, and complexity. Recognition of risk factors frequently associated with SSI allows for identification of such patients with the greatest need for optimal preventive measures to be identified and pre-treatment prior to surgery.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

SHORT Syndrome with Partial Lipodystrophy Due to Impaired Phosphatidylinositol 3 Kinase Signaling

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.