Neuron-specific aminopeptidase and puromycin-sensitive aminopeptidase in rat brain development

Neuron-specific aminopeptidase (NAP) and the ubiquitous puromycin-sensitive aminopeptidase (PSA) were compared in the rat hippocampus during early development. Hippocampus contains the highest amount of NAP determined by a fast-protein liquid chromatography-aminopeptidase analyzer using Leu -naphthylamide as substrate. Both enzymes were found in the hippocampus in all ages. NAP was lower in immature rat; the 19th embryonic-day fetus contained the least. It increased steeply during the prenatal through the early postnatal period, 9-fold by the first month. The rate of increase diminished subsequently, increasing 20% in the second month and 13% in the third. The age-dependent increase in NAP activity was parallel to its protein expression as determined by Western blot. The specific molecular activity (hydrolytic activity/NAP antigenicity) in newborn, 15-day-old, and 30-day-old rats were 1.00, 0.88, and 1.00, respectively. The PSA developmental profile without linear increase in activity was distinct from NAP. PSA activity was higher than NAP in decreasing order, 100–4 times, during the same development span. Similarly, different growth profiles for NAP and PSA were also found in the primary culture of developing cerebellar granule cells. Puromycin (1–5 M) blocked neurite outgrowth and caused apoptosis by nonantibiotic effects. Our data suggest that the synaptosome-enriched NAP plays a role in neuron growth, differentiation, and information programming.     

Atomic force microscopy of pea starch granules: granule architecture of wild-type parent, r and rb single mutants, and the rrb double mutant

AFM studies have been made of the internal structure of pea starch granules. The data obtained provides support for the blocklet model of starch granule structure (Carbohydr. Polym. 32 (1997) 177-191). The granules consist of hard blocklets dispersed in a softer matrix material. High-resolution images have yielded new insights into the detailed structure of growth rings within the granules. The blocklet structure is continuous throughout the granule and the growth rings originate from localised defects in blocklet production distributed around the surface of spheroidal shells within the granules. A mutation at the rb locus did not lead to significant changes in granule architecture. However, a mutation at the r locus led to loss of growth rings and changed blocklet structure. For this mutant the blocklets were distributed within a harder matrix material. This novel composite arrangement was used to explain why the granules had internal fissures and also changes in gelatinisation behaviour. It is suggested that the matrix material is the amylose component of the granule and that both amylose and amylopectin are present within the r mutant starch granules in a partially-crystalline form. Intermediate changes in granule architecture have been observed for the double mutant rrb.     

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs

A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca(2+) levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2(f) during formation of the new CGFD. Moreover, clamping intracellular Ca(2+) did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD.     

Performance of upflow anaerobic sludge blanket (UASB) reactor treating wastewaters containing carbon tetrachloride

The anaerobic biodegradation of carbon tetrachloride (CT) was investigated during the granulation process by reducing the hydraulic retention time, increasing the chemical oxygen demand (COD) and CT loadings in a 2l laboratory-scale upflow anaerobic sludge blanket (UASB) reactor. Anaerobic unacclimated sludge and glucose were used as seed and primary substrate, respectively. Granules were developed 4 weeks after start-up, which grew at an accelerated rate for 8 months, and then became fully grown. The effect of operational parameters such as influent CT concentrations, COD, CT loading, food to biomass ratio and specific methanogenic activity (SMA) were also considered during granulation. The granular sludge cultivated had a maximum diameter of 2.1 mm and SMA of 1.6 g COD/g total suspended solid (TSS) day. COD and CT removal efficiencies of 92 and 88% were achieved when the reactor was firstly operating at CT and COD loading rates of 17.5 mg/l day and 12.5 g/l day, respectively. This corresponds to hydraulic retention time of 0.28 day and food to biomass ratio of 0.5 g COD/g TSS day. Kinetic coefficients of maximum specific substrate utilization rate, half velocity coefficient, growth yield coefficient and decay coefficient were determined to be 2.4 × 10^–3 mg CT/TSS day^–1, 1.37 mg CT/l, 0.69 mg TSS/mg CT and 0.046 day^–1, respectively for CT biotransformation during granulation.     

AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro . The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet α-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes.     

The Evolution of the Ribonucleotide Reductases: Much Ado About Oxygen

Ribonucleotide reduction is the only known biological means for de novo production of deoxyribonucleotides, the building blocks of DNA. These are produced from ribonucleotides, the building blocks of RNA, and the direction of this reaction has been taken to support the idea that, in evolution, RNA preceded DNA as genetic material. However, an understanding of the evolutionary relationships among the three modern-day classes of ribonucleotide reductase and how the first reductase arose early in evolution is still far off. We propose that the diversification of this class of enzymes is inherently tied to microbial colonization of aerobic and anaerobic niches. The work is of broader interest, as it also sheds light on the process of adaptation to oxygenic environments consequent to the evolution of atmospheric oxygen.     

Dantrolene protects neurons against kainic acid induced apoptosis in vitro and in vivo

Apoptotic cell death induced by kainic acid (KA) in cultures of rat cerebellar granule cells (CGC) and in different brain regions of Wistar rat pups on postnatal day 21 (P21) was studied. In vitro , KA (100–500 μM) induced a concentration-dependent loss of cell viability in MTT assay and cell death had apoptotic morphology as studied by chromatin staining with propidium iodide (PI). In vivo , twenty-four hours after induction of status epilepticus (SE) by an intraperitoneal KA injection (5 mg/kg) we quantified apoptotic cells in hippocampus (CA1 and CA3), parietal cortex and cerebellum using PI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. We report that dantrolene, a specific ryanodine receptor antagonist, was able to significantly reduce the apoptotic cell death in CGC cultures and in hyppocampal CA1 and parietal cortex regions. Our finding can be valuable for neuroprotective therapy strategies in patients with repeated generalized seizures or status epilepticus.     

Cardiorespiratory performance in salmonids during exercise at high temperature: insights into cardiovascular design limitations in fishes

Studies in the laboratory with salmonids and now in the field with wild salmon clearly show that critical swimming performance has an optimum temperature. This temperature optimum is coincident with maximum aerobic scope and maximum cardiac scope. At a temperature that is higher than this optimum, however, whole animal performance declines abruptly. Evidence is presented here to suggest that this is directly associated with a decline in cardiac scope which limits oxygen supply to tissues. It is further suggested that the decline in maximum cardiac performance could reflect problems with the heart's own oxygen supply. The reasoning behind this suggestion is that, at temperatures at or below the optimum and probably because of a limitation on oxygen diffusion in skeletal muscle during exercise, venous oxygen does not fall below a threshold level during exercise, and so the heart receives just enough oxygen for its own muscular activity via the cardiac circulation (i.e. the venous return to the heart). However, because high temperature favours increased oxygen extraction by skeletal muscle, which consequently lowers venous oxygen, cardiac oxygen supply may become insufficient to meet cardiac oxygen demand. The hypoxic myocardium then cannot maintain cardiac scope and internal oxygen delivery to tissue declines.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.