Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[alpha-32P]ATP

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[alpha-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 microM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 microM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A-Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-beta-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.     

The relationship between excitotoxicity and oxidative stress in the central nervous system

Excitotoxicity and oxidative stress are two phenomena that have been repeatedly described as being implicated in a wide range of disorders of the nervous system. Such disorders include several common idiopathic neurological diseases, traumatic brain injury, and the consequences of exposure to certain neurotoxic agents. While there is evidence that metabolic derangements can laed to these adverse processes, and that these processes may synergize in their damaging effects, the degree of interdependence, and the causal relation between them is not clear. The intent of this review is to delineate potential mechanisms which may unit hyperexcitation to the excessive generation of reactive oxygen species. The degree of linkage between these events appears rather strong. It is likely that excitoxicity frequency leads to a pro-oxidant condition but that high rates of generation of reactive oxygen species are not invariably accompanied by a hyperexcited neuronal state Both excitoxic and ‘oxidotoxic’ states result from the failure of normal compensatory anti excitatory and antioxidant mechanisms to maintain cellular homeostatis.     

Biologic Response to Peripheral and Central Administration of Recombinant Human Leptin in Dogs

LEBEL, CARL, AMY BOURDEAU, DAVID LAU, AND PAMELA HUNT. Biologic response to peripheral and central administration of recombinant human leptin in dogs. Obes Res. Objective: Because leptin is believed to act within the central nervous system, the objective of this study was to test that presumption by comparing the biologic responses to recombinant human leptin (rHuLeptin) when delivered either subcutaneously or intrathecally in a large animal species, the beagle dog. Methods and Procedures: Adult beagle dogs were used for all studies (n = 3 to 14). Treatment with rHuLeptin was either as daily subcutaneous or intermittent intrathecal injections. Results: Subcutaneously administered rHuLeptin was absorbed with peak concentrations appearing at 2 to 4 hours. After intrathecal administration, cerebral spinal fluid concentrations declined in a bi-phasic manner with a terminal half-life of ?6 to 8 hours. When lean beagles were given leptin subcutaneously, at 0. 05 to 5 gkglday for up to 6 months, reductions in body weight (up to 30%) and food intake (up to 75%) were observed. Body fat loss was observed in both lean and obese dogs, and confirmed by dual energy X-ray absorptiometry and histology of adipose tissue. When rHuleptin was delivered intrathecally at 4 to 1000 μg/dose for up to 3 months, the primary effects observed were reductions in body weight and food intake. In general all findings reported in the intrathecal studies were consistent with those noted in the subcutaneous studies; however, the required intrathecal dose was substantially lower than that for subcutaneous delivery. Discussion: These studies demonstrate that both subcutaneous and intrathecal treatment of rHuLeptin was associated with effects on body weight, food intake, and body fat in dogs. These results support the concept that the central nervous system is the probable primary site of action for leptin and suggest that rHuLeptin has similar physiologic activities that influence body weight, body fat, and metabolism in large animals to those reported previously in rodents.     

Suicin 90-1330 from a Nonvirulent Strain of Streptococcus suis: a Nisin-Related Lantibiotic Active on Gram-Positive Swine Pathogens

Streptococcus suis serotype 2 is known to cause severe infections (meningitis, endocarditis, and septicemia) in pigs and is considered an emerging zoonotic agent. Antibiotics have long been used in the swine industry for disease treatment/prevention and growth promoters. This pattern of utilization resulted in the spread of antibiotic resistance in S. suis worldwide. Interestingly, pigs may harbor S. suis in their tonsils without developing diseases, while North American strains belonging to the sequence type 28 (ST28) are nonvirulent in animal models. Consequently, the aim of this study was to purify and characterize a bacteriocin produced by a nonvirulent strain of S. suis serotype 2, with a view to a potential therapeutic and preventive application. S. suis 90-1330 belonging to ST28 and previously shown to be nonvirulent in an animal model exhibited antibacterial activity toward all S. suis pathogenic isolates tested. The bacteriocin produced by this strain was purified to homogeneity by cationic exchange and reversed-phase fast protein liquid chromatography. Given its properties (molecular mass of <4 kDa, heat, pH and protease stability, and the presence of modified amino acids), the bacteriocin, named suicin 90-1330, belongs to the lantibiotic class. Using a DNA-binding fluorophore, the bacteriocin was found to possess a membrane permeabilization activity. When tested on other swine pathogens, the suicin showed activity against Staphylococcus hyicus and Staphylococcus aureus, whereas it was inactive against all Gram-negative bacteria tested. Amino acid sequencing of the purified bacteriocin showed homology (90.9% identity) with nisin U produced by Streptococcus uberis. The putative gene cluster involved in suicin production was amplified by PCR and sequence analysis revealed the presence of 11 open reading frames, including the structural gene and those required for the modification of amino acids, export, regulation, and immunity. Further studies will evaluate the ability of suicin 90-1330 or the producing strain to prevent experimental S. suis infections in pigs.     

The major protein of pancreatic zymogen granule membranes (GP-2) is anchored via covalent bonds to phosphatidylinositol

GP-2, the major integral protein characteristic of the pancreatic zymogen granule membrane can be released from the membrane by the action of a phosphatidylinositol specific phospholipase C (PI-PLC). In a hydrophobic/hydrophilic phase separation system using the non-ionic detergent Triton X-114, the membrane-bound form of the protein went from the detergent phase into the hydrophilic phase upon action of the phospholipase. PI-PLC solubilization of GP-2 unmasked an antigenic determinant similar to the cross-reacting determinant of the trypanosome variant surface glycoproteins. This determinant being a distinctive feature of the glycan moiety of phosphatidyl-inositol anchored membrane proteins, it established the glycosyl-phosphatidyl-inositol nature of the GP-2 membrane anchor. Since soluble GP-2 is also found in the contents of the granule and is secreted intact into the pancreatic juice, it is likely that one of the mechanisms responsible for its release could be a specific phospholipase. GP-2 is the first glycosyl-phosphatidyl-inositol-anchored protein that is integral to the membrane of an organelle and not located at the surface of the cell.     

Identification of the proteins exposed on the cytoplasmic surface of the pancreatic zymogen granule

Lactoperoxidase-catalyzed ¹²⁵I-iodination was used to label pancreatic zymogen granules. Membrane proteins facing the cytoplasmic surface were specifically labeled. Two low molecular weight proteins of 17000 and 15000 were intensely labeled at 0°C. Another small 13 kDa protein was strongly iodinated at 25°C along with some others, including the 29 kDa subunit of the ATP diphosphohydrolase. The major glycoprotein of the granule membrane was not iodinated but the presence of an iodinated 80 kDa protein suggests that proteolytic fragments of the 92 kDa glycoprotein were accessible to iodination on the intact granule. These proteins localized on the cytoplasmic surface of the granule are believed to play a major role in the exocytotic phenomenon of the exocrine pancreas.     

Perturbations in cerebral oxygen radical formation and membrane order following vitamin E deficiency

The effects of dietary vitamin E deficiency on mouse cerebral membrane order and oxygen reactive species were studied. Quantitation of vitamin E levels in several brain regions showed greatest deficiencies in striatum and cerebellum, followed by substantia nigra, and cortex. Vitamin E deficiency increased central-core membrane order in cerebral P2 fraction, but was without effect in the superficial hydrophilic membrane domain. Oxygen radical formation was studied using the probe 2',7'-dichlorofluorescein diacetate. Basal generation rates of oxygen reactive species were 2.5-fold higher when compared to control animals. While hepatic levels of vitamin E are much more reduced than brain levels, in deficient mice, the rate of oxygen radical formation in the liver was unaltered. This implies an special susceptibility of the brain to deficiency of this lipophilic antioxidant vitamin. Data demonstrate that endogenous levels of free radical scavengers, such as vitamin E, may play an important role in maintaining basal oxygen radical levels and membrane integrity. The dietary vitamin E depletion paradigm suggests that a relation exists between elevated levels of oxygen radicals and more rigid hydrophobic central-cores in cerebral membranes, effects that may play a role in mechanisms underlying the neuropathologic lesions observed following vitamin E deficiency.     

Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas

An ATP diphosphohydrolase (EC 3.6.1.5) from the pancreas of the pig has been characterized and purified. The enzyme which has an optimum pH between 8 and 9 is specific for diphospho- and triphosphonucleosides. The Km values for ADP and ATP are 7.4 and 7.3 x 10(-4) M, respectively, and the purified enzyme has specific activities of 13 and 15.2 mumol of Pi/min/m of protein, respectively. It requires calcium or magnesium ions and it is insensitive to ATPase inhibitors, namely oligomycin, ouabain, and ruthenium red, and to levamisole, an inhibitor of alkaline phosphatase. Denaturation experiments, by heat and trypsin treatments, indicated that only one enzyme is involved. This is confirmed by the solubilization and purification process and by polyacrylamide gel electrophoresis. A 270-fold purification was obtained by centrifugation and successive column chromatography on Sepharose 4B and Affi-Gel blue. It is a glycoprotein with a molecular weight of 65,000 as estimated by polyacrylamide gel electrophoresis.     

Translocation of CaMKII to dendritic microtubules supports the plasticity of local synapses

The processing of excitatory synaptic inputs involves compartmentalized dendritic Ca²⁺ oscillations. The downstream signaling evoked by these local Ca²⁺ transients and their impact on local synaptic development and remodeling are unknown. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is an important decoder of Ca²⁺ signals and mediator of synaptic plasticity. In addition to its known accumulation at spines, we observed with live imaging the dynamic recruitment of CaMKII to dendritic subdomains adjacent to activated synapses in cultured hippocampal neurons. This localized and transient enrichment of CaMKII to dendritic sites coincided spatially and temporally with dendritic Ca²⁺ transients. We show that it involved an interaction with microtubular elements, required activation of the kinase, and led to localized dendritic CaMKII autophosphorylation. This process was accompanied by the adjacent remodeling of spines and synaptic AMPA receptor insertion. Replacement of endogenous CaMKII with a mutant that cannot translocate within dendrites lessened this activity-dependent synaptic plasticity. Thus, CaMKII could decode compartmental dendritic Ca²⁺ transients to support remodeling of local synapses.     

A convenient method for the ATPase assay.

A new method for the determination of inorganic phosphorus released in ATPase assay has been evaluated. The method is based on the reduction of a phosphomolybdate complex by Elon in a copper acetate buffer. In contrast to current methods, there is no interference by ATP with color development. There is also less or no interference by other compounds usually present in ATPase assay media. The method is simple, sensitive, and reproducible.