Real-time imaging of Rab3a and Rab5a reveals differential roles in presynaptic function

We investigated the roles of two Rab-family proteins, Rab3a and Rab5a, in hippocampal synaptic transmission using real-time fluorescence imaging. During synaptic activity, Rab3a dissociated from synaptic vesicles and dispersed into neighbouring axonal regions. Dispersion required calcium-dependent exocytosis and was complete before the entire vesicle pool turned over. In contrast, even prolonged synaptic activity produced limited dispersion of Rab5a. A GTPase-deficient mutant, Rab3a (Q81L), dispersed more slowly than wild-type Rab3a, and decreased the rate of exocytosis and the size of the recycling pool of vesicles. While overexpression of Rab3a did not affect vesicle recycling, overexpression of Rab5a reduced the recycling pool size by 50%. We propose that while Rab3a preferentially associates with recycling synaptic vesicles and modulates their trafficking, Rab5a is largely excluded from recycling vesicles.     

Delayed development of humoral immunity in chickens following in ovo treatment with mibolerone

In order to determine if mibolerone causes rapid development of the humoral immune system as previously hypothesized, 12-day-old chicken embryos were injected with 0.1, 1, or 10 micrograms mibolerone. Mibolerone caused bursal atrophy in a dose-dependent manner with severe atrophy at the 10 micrograms dose, substantial atrophy at the 1 microgram dose, and very little or no atrophy at the 0.1 microgram dose. Mibolerone-treated chicks were subsequently challenged with sheep red blood cells (SRBC) and Brucella abortus at 1, 7, 14, or 21 days of age. When serum samples were analysed for agglutinins to SRBC and B. abortus, the results indicated that treated chickens had lower agglutinin titers to both antigens at younger ages. These responses improved with age in groups treated with 0.1 microgram and 1 microgram and were comparable to controls at 5 weeks of age except for low primary and secondary (IgG specific) titers to B. abortus in birds treated with 1 microgram. Chickens treated with 10 micrograms mibolerone still had considerably lower primary responses to SRBC and lower primary as well as secondary responses to B. abortus at 4 and 5 weeks of age. These results indicate that mibolerone causes delayed rather than rapid development of the humoral immune system. In control as well as in treated chickens, agglutinin responses to SRBC appeared earlier than responses to B. abortus.     

Imaging biomarkers of inflammation <Emphasis Type="Italic">in situ</Emphasis> with fun ctionalized quantum dots in the dextran sodium sulfate (DSS) model of mouse colitis

Objective and design: Myeloperoxidase (MPO) and proinflammatory cytokines play an important role in the development of inflammation. These markers are generally measured using tedious ELISA procedures. In this study, a novel technique utilizing antibody conjugated quantum dot nanoparticles was developed to detect Myeloperoxidase, Interleukin-1α (IL-1α) and Tumor Necrosis Factor-α (TNF-α) in vivo in the dextran sodium sulfate (DSS) model of experimental colitis. Materials and methods: Colitis was induced in animals (n = 8 animals/group) by feeding 4% DSS solution ad libitum for seven to eight days. Quantum Dots (QDs) exhibiting fluorescence at various wavelengths were conjugated to MPO, IL-1α and TNF-α polyclonal antibodies and tested in vivo at various stages of colitis. Tissue sections obtained were imaged with confocal microscope. The image intensity obtained from the tissue specimen was correlated with clinical activity measured as Disease Activity Index (DAI). Results: Myeloperoxidase, IL-1α and TNF-α were visualized with quantum dots on various days of disease. The intensity of quantum dots increased with the increase in inflammation. The increase in intensity showed an excellent correlation with the DAI based on the clinical parameters. Conclusion: The study demonstrated that multiple biomarkers can be detected simultaneously and their quantitative expression correlated well with clinical disease severity. This novel technology should facilitate design of a novel optical platform for imaging various biomarkers of inflammation, early detection of acute and chronic disease markers and inflammation-mediated cancer markers. This detection may also facilitate determination of therapeutic success. Received 14 March 2007; returned for revision 8 May 2007; accepted by M. Parnham 27 June 2007     

Alterations in hepatocytes after manipulation of the diet: Copper,zinc and cadmium interactions

Morphometric analyses of liver parenchymal cells were performed on male albino rats fed a semipurified diet supplemented with two levels of copper and zinc. These metals were administered in demineralized drinking water to four groups of animals at two levels. (1) 0.25 μgm Cu/ml, 2.5 μgm Zn/ml, and (2) 1.0 μgm Cu/ml, 10 μgm Zn/ml. Cadmium, also administered in the drinking water daily to rats at 17.2 μgm cadmium/ml, was given to one group of animals on each of the two semipurified diets. Two groups of rats were given laboratory chow plus 17.2 μgm Cd/ml for 71 days and for 280 days. Volume densities of all organelles diminished significantly when levels of copper and zinc were lowest. Surface densities of rough and smooth endoplasmic reticulum were significantly decreased. Higher levels of copper and zinc produced morphologic and morphometric parameters which fell just below those obtained for rats fed chow. Glycogen accumulation was highest when the levels of metal were low. Lipid inclusions were more frequent with semipurified diets than with chow. Hepatocytes from rats fed the semipurified diet with low copper and zinc plus cadmium demonstrated a significant increase in smooth endoplasmic reticulum compared to animals fed the semipurified diet alone. The smooth endoplasmic reticulum changed little when cadmium was given to rats receiving adequate copper and zinc. Smooth endoplasmic reticulum increased when diets included chow and cadmium whereas rough endoplasmic reticulum decreased. Cup-shaped and elongated mitochondria with longitudinal cristae appeared in hepatocytes from animals fed chow with cadmium. These were not seen with semipurified diets and cadmium.     

Hydrogen peroxide-induced alterations in prostaglandin secretion in the rat colon in vitro

Although the specific cause(s) of inflammatory bowel diseases (IBD) has not been identified, one theory suggests ischemia as the early event that occurs in IBD and reperfusion causes sustained release of oxyradicals, leading to inflammation and ulceration. In this study, we have confirmed that H₂O₂ in the concentration seen during ischemia/reperfusion is primarily responsible for cellular membrane damage in the rat colonic fragments in vitro. Hydrogen peroxide caused a time and dose-dependent increase in 6-keto-PGF₁ and TXB₂ release. Hydrogen peroxide-stimulated 6-keto-PGF₁ release was blocked (50%) by phospholipase A₂ (PLA₂) inhibitors quinacrine and dimethyleicosodienoic acid at 5 min. Hydrogen peroxide-stimulated 6-keto-PGF₁ release was completely blocked by idomethacin, significantly blocked (69%) by nordihydroguiaretic acid, and completely blocked by catalase. superoxide dismutase and uric acid failed to inhibit H₂O₂-stimulated 6-keto-PGF₁ release. Endogenous catalase inhibitors 3-aminotriazole and sodium azide further enhanced the release of 6-keto-PGF₁ stimulated by H₂O₂ by 29% and 73%, respectively. Xanthine-xanthine oxidase also increased 6-keto-PGF₁ release from the fragments by 110%. This release was not inhibited by superoxide dismutase and uric acid, but was completely inhibited by catalase. These studies suggest a direct effect of H₂O₂ on colonic fragments leading to submicroscopic cellular membrane damage and excess prostanoid production utilizing a PLA₂/cyclooxygenase and catalase-sensitive pathway without the formation of toxic hydroxyl ions. The quick release of 6-keto-PGF₁ also suggests an early manifestation of H₂O₂-induced damage in rat colonic fragments.     

The role of fibronectin in tumor implantation at surgical sites

Fibronectins are a family of glycoproteins with modular functional domains. They mediate cell-cell and cell-matrix interactions which are important in embryogenesis, wound healing, metastasis and other processes. We present data on the influence of fibronectin on wound implantation of a murine mammary carcinoma line, TA3Ha. Fibronectin used in these studies was derived from bovine plasma, human serum, human foreskin fibroblasts, and mouse embryo cultures. TA3Ha cells rarely form tumors in the liver of syngeneic mice when injected intravenously but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Wound implantation is markedly reduced when the cells are pre-exposed to 200 µg/ml bovine plasma fibronectin (13%, P = 0.007), human serum fibronectin (0%, P = 0.02), human cellular fibronectin (0%, P = 0.02), or mouse cellular fibronectin (0%, P = 0.04). Lung colonization is also reduced by these fibronectins. These effects are not due to a cytotoxic action of fibronectin, since intraperitoneally injected fibronectin-treated cells form ascites tumor as effectively as do control untreated cells. Local application of a solution containing 0.25 mg/ml mouse cellular fibronectin to the hepatic wound reduces the frequency of tumor implantation from 45% to 5% (1/21, P = 0.001). No tumor implantation inhibition is seen when only suspending medium or albumin in suspending medium is used. The mechanism by which topical application of fibronectin reduces hepatic wound implantation of tumor cells is unclear, but this finding raises an exciting possibility of preventing local recurrence of cancer.     

Down syndrome and trisomy 18 in the bedouins

Direct chromosome preparations of neonatal cord blood provides the unique opportunity for rapid chromosome analysis (turnaround time; 6 hr), without the necessity of bone marrow aspiration. Based on 42 samples we confirm the finding of Garnham and Sutherland [1987] for suitability of cord blood for direct chromosome preparation. Procedural modifications are provided for higher yield of cells for chromosome analysis. The procedure may well be of major significance for rapid diagnosis of neonates who suffer from aneusomy.