Catecholamine-containing neurons in the peripheral nervous system of Aplysia

Numerous green-fluorescent neurons have been revealed by means of the glyoxylic acid histochemical method in cryostat sections of several organs of two Adriatic aplysiid gastropods, Aplysia depilans and A. fasciata. Catecholamine-containing perikarya and their processes have been found to be especially abundant in the vaginal portion of the large hermaphrodite duct, in the penis and its sheath, and in the gill. In the reproductive organs, two subpopulations of catecholamine-containing neurons could be distinguished according to their size and location. Axons of larger neurons formed bundles which seemed to project at the CNS.     

Organization and nucleotide sequence of the Streptococcus mutans galactose operon

The galactose operon encoding a repressor and genes for the Leloir pathway for galactose metabolism (galactokinase, galactose-1-phosphate-uridyl transferase and UDP glucose-4-epimerase) was located adjacent to the multiple sugar metabolism (msm) operon on the chromosome of Streptococcus mutans Ingbritt (serotype c) and the complete nucleotide sequence of this 5-kilobase region was determined. The Leloir pathway was induced by the presence of galactose in the growth medium or following the release of intracellular galactose after uptake and cleavage of -galactosides by the multiple sugar metabolism system. Analysis of the mechanism of galactose transport confirmed the absence of a galactose-specific phosphotransferase system and suggested the presence of an inducible galactose permease. Evidence is presented that galactose transport is independent of the proton motive force and may be ATP-dependent.     

Insulin Mimetic Effect of Tungsten Compounds on Isolated Rat Adipocytes

Investigations of effective, orally active, and safe antidiabetic metallopharmaceuticals have been carried out during the last two decades. It has been reported that tungsten compounds mimic the action of insulin in intact cell systems. As insulin mimetics, the most investigated tungsten compound was sodium tungstate (ST), rarely investigated was tungstophosphoric acid (WPA), but never alanine complex of tungstophosphoric acid (WPA-A). In this study, the insulin mimetic activity of three different tungsten compounds, ST, WPA, and WPA-A, was evaluated by means of in vitro measurements of the glucose uptake and inhibition of free fatty acids release from epinephrine-treated isolated rat white adipocytes. We investigated the influence of concentration (lower and higher, 0.1 and 1.0 mM, respectively) and solvent: isotonic salt solution—saline (0.9% w/v of NaCl) and dimethyl sulfoxide (DMSO; 2% v/v), on the biological effect of tested compounds. Our experimental data showed that all of the three investigated tungsten compounds possess insulin mimetic activity in vitro on the isolated adipocytes. Influence of concentration and solvents on insulin mimetic effect for the certain tungsten compounds were: WPA was shown effect independently of concentration and solvents; higher concentration and DMSO were significant decreasing insulin mimetic effect of ST; lower concentration and saline led to decreasing effect of WPA-A. Generally, there were no differences in insulin mimetic effect of three tungsten compounds in lower concentration and dissolved in DMSO. When saline was used as solvent, it was needed higher concentration of investigated compounds to accomplish the same effect. In conclusion, our results suggest that low concentration (0.1 mM) of ST, WPA, and WPA-A dissolved in 2% DMSO could be the good candidates for in vivo investigation of their antidiabetic properties.     

Hippocampal Hypertrophy and Sleep Apnea: A Role for the Ischemic Preconditioning?

The full impact of multisystem disease such as obstructive sleep apnoea (OSA) on regions of the central nervous system is debated, as the subsequent neurocognitive sequelae are unclear. Several preclinical studies suggest that its purported major culprits, intermittent hypoxia and sleep fragmentation, can differentially affect adult hippocampal neurogenesis. Although the prospective biphasic nature of chronic intermittent hypoxia in animal models of OSA has been acknowledged, so far the evidence for increased ‘compensatory’ neurogenesis in humans is uncertain. In a cross-sectional study of 32 patients with mixed severity OSA and 32 non-apnoeic matched controls inferential analysis showed bilateral enlargement of hippocampi in the OSA group. Conversely, a trend for smaller thalami in the OSA group was noted. Furthermore, aberrant connectivity between the hippocampus and the cerebellum in the OSA group was also suggested by the correlation analysis. The role for the ischemia/hypoxia preconditioning in the neuropathology of OSA is herein indicated, with possible further reaching clinical implications.     

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction

Background AimsViral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct.MethodsAssay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations.ResultsThe limit of detection was 10 copies/μL, whereas negative samples showed <1.3 copies/μL. Within and between assay imprecision coefficients of variation across the reportable range (10–10 000 copies/μL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R² >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations).ConclusionsDDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.     

Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^? ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^? species initiate the formation of peroxynitrite (ONOO^? ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^? decomposition and ONOO^? -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^? is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL,MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.