Enterochromaffin (EC-) cells of the mammalian gastro-entero-pancreatic (GEP) endocrine system: cellular source of pro-dynorphin-derived peptides

Summary It has long been disputed whether mammalian enterochromaffin (EC-) cells contain a peptide in addition to serotonin. Previous immunohistochemical studies have provided evidence for the presence of enkephalins in EC-cells. These findings, however, are equivocal. Therefore, the problem of opioid peptides in EC-cells has been re-examined in the gastro-intestinal mucosa of dog, guinea-pig and man. A battery of antisera against derivatives of pro-opiomelanocortin, pro-enkephalin and pro-dynorphin have been applied to semithin serial sections of the tissues, in combination with fluorescence histochemistry and serotonin immunocytochemistry. Our findings indicate that EC-cells of the investigated species contain pro-dynorphin-related peptides, i.e. dynorphin A and -neo-endorphin, but no derivatives from pro-opiomelanocortin or pro-enkephalin. Since remarkable interspecies variations occur with respect to the number and staining characteristics of opioid immunoreactive EC-cells, it is concluded that pro-dynorphin shows specific routes of post-translational processing depending upon the species and the gastro-intestinal segment investigated. Future studies should focus on the mutual relationships between serotonin and dynorphins and on the physiological significance of these peptides in the gastrointestinal tract.Part of the results were presented at the Bayliss and Starling Society National Scientific Meeting 1985, London (Cetin et al. 1985)     

Prevalence of Human Herpesvirus 6 Variants A and B in Patients with Chronic Fatigue Syndrome

Peripheral blood mononuclear cells collected from 13 patients with chronic fatigue syndrome and 13 healthy controls were analyzed for the presence of human herpesvirus 6 (HHV-6) DNA by variant-specific polymerase chain reaction and dot blot hybridization. HHV-6 DNA was detected in 7 of 13 (53%) patients, and of those 7 patients, 4 were positive for HHV-6 variant A DNA and 3 were for variant B. No HHV-6 DNA was detected in the controls. Serum antibody titers to the late antigen and antibody prevalence to the early antigen of HHV-6 were significantly higher in the patient group. These results suggest active replication of HHV-6 in patients with chronic fatigue syndrome.     

Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding site on the alpha-chain of human haemoglobin.

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.     

Localization and verification by synthesis of five antigenic sites of bovine serum albumin.

Recently we have shown that the major antigenic sites of bovine serum albumin exhibit functional equivalence progessively increasing with the time at which antibodies are obtained after the first immunization. Analysis of our recent immunochemical findings and the known covalent structure of bovine serum albumin have enabled us to predict the locations of five antigenic sites of bovine serum albumin. The predicted locations were synthesized, and immunochemical studies with late-course antisera showed them to constitute antigenic sites of native bovine serum albumin.     

The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.     

Functional characterization of human myosin-binding protein C3 variants associated with hypertrophic cardiomyopathy reveals exon-specific cardiac phenotypes in zebrafish model

Myosin-binding protein C 3 (MYBPC3) variants are the most common cause of hypertrophic cardiomyopathy (HCM). HCM is a complex cardiac disorder due to its significant genetic and clinical heterogeneity. MYBPC3 variants genotype–phenotype associations remain poorly understood. We investigated the impact of two novel human MYBPC3 splice-site variants: V1: c.654+2_654+4dupTGG targeting exon 5 using morpholino MOe5i5; and V2: c.772+1G>A targeting exon 6 using MOe6i6; located within C1 domain of cMyBP-C protein, known to be critical in regulating sarcomere structure and contractility. Zebrafish MOe5i5 and MOe6i6 morphants recapitulated typical characteristics of human HCM with cardiac phenotypes of varying severity, including reduced cardiomyocyte count, thickened ventricular myocardial wall, a drastic reduction in heart rate, stroke volume, and cardiac output. Analysis of all cardiac morphological and functional parameters demonstrated that V2 cardiac phenotype was more severe than V1. Coinjection with synthetic human MYBPC3 messenger RNA (mRNA) partially rescued disparate cardiac phenotypes in each zebrafish morphant. While human MYBPC3 mRNA partially restored the decreased heart rate in V1 morphants and displayed increased percentages of ejection fraction, fractional shortening, and area change, it failed to revert the V1 ventricular myocardial thickness. These results suggest a possible V1 impact on cardiac contractility. In contrast, attempts to rescue V2 morphants only restored the ventricular myocardial wall hypertrophy phenotype but had no significant effect on impaired heart rate, suggesting a potential V2 impact on the cardiac structure. Our study provides evidence of an association between MYBPC3 exon-specific cardiac phenotypes in the zebrafish model providing important insights into how these genetic variants contribute to HCM disease.     

Molecular Characterization of Chitinase Genes from a Local Isolate of Serratia marcescens and Their Contribution to the Insecticidal Activity of Bacillus thuringiensis Strains

The chitinase B (chiB) and C (chiC) genes and flanking regions from a local isolate of Serratia marcescens were cloned individually and sequenced. Results showed that these chiB and chiC genes have a 96 % maximum similarity with chiB and chiC from different S. marcescens species (GenBank numbers Z36295.1 and AJ630582.1, respectively). The amplified chiB fragment, including some upstream and downstream regions, is 1,689-bp long with an open reading frame of 1,500 bp. The amplified fragment of chiC is 1,844 bp with an open reading frame of 1,443 bp. These sequences were submitted to the GenBank with accession numbers JX847796 (chiB) and JX847797 (chiC). Putative promoter regions and Shine–Dalgarno sequences were identified in both genes. The genes were cloned into a shuttle vector and the constructs were designated as pHYSB and pHYSC, respectively. Both plasmids were introduced separately into kurstaki and israelensis strains of Bacillus thuringiensis and the insecticidal activities of the engineered B. thuringiensis strains were assayed in larvae of Galleria mellonella and adult of Drosophila melanogaster. Engineered B. thuringiensis strains showed higher insecticidal activity than parental strain and the parental S. marcescens. In addition, pHYSB and pHYSC were stable over 16 daily passages under non-selective conditions in transformed B. t. israelensis 5724 strain.     

Allyl isothiocyanate attenuates oxidative stress and inflammation by modulating Nrf2/HO-1 and NF-κB pathways in traumatic brain injury in mice

Molecular Biology Reports - Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in young adults and children in the industrialized countries; however, there are presently...     

Graded alterations of RBC aggregation influence in vivo blood flow resistance

Although the effects of red blood cell (RBC) aggregation on low-shear rate blood viscosity are well known, the effects on in vivo flow resistance are still not fully resolved. The present study was designed to explore the in vivo effects of RBC aggregation on flow resistance using a novel technique to enhance aggregation: cells are covalently coated with a block copolymer (Pluronic F-98) and then suspended in unaltered plasma. RBC aggregation was increased in graded steps by varying the Pluronic concentration during cell coating and was verified by microscopy and erythrocyte sedimentation rate (ESR), which increased by 200% at the highest Pluronic level. RBC suspensions were perfused through an isolated in situ guinea pig hindlimb preparation while the arterial perfusion pressure was held constant at 100 mmHg via a pressure servo-controlled pump. No significant effects of enhanced RBC aggregation were observed when studies were conducted in preparations with intact vascular control mechanisms. However, after inhibition of smooth muscle tone (using 10(-4) M papaverin), a significant change in flow resistance was observed in a RBC suspension with a 97% increase of ESR. Additional enhancements of RBC aggregation (i.e., 136 and 162% increases of ESR) decreased flow resistance almost to control values. This was followed by another significant increase in flow resistance during perfusion with RBC suspensions with a 200% increase of ESR. This triphasic effect of graded increases of RBC aggregation is most likely explained by an interplay of several hemodynamic mechanisms that are triggered by enhanced RBC aggregation.