Proteinase-sensitive release of enzymes from pancreatic microsomal fraction.

Suspensions of rat pancreatic microsomal fraction release alpha-amylase and ribonuclease on incubation at 37 degrees C, but not at 2 degrees C. The release is abolished by proteolytic enzymes. Ribonuclease associated with the microsomal fraction is protected from subtilisin BPN' attack, but is sensitive after release.     

PURIFICATION AND PROPERTIES OF A PROLYL ENDOPEPTIDASE FROM RABBIT BRAIN

Abstract— An enzyme with the specificity of a prolyl endopeptidase was purified about 880-fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl-peptide and peptidylprolyl-amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N -benzyloxycarbonylglycyl-L-prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N -benzyloxycarbonylglycyl-L-proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.     

Production of IL-10 by CD4+ T lymphocytes correlates with down-regulation of Th1 cytokine synthesis in helminth infection.

After the onset of parasite egg deposition, mice infected with the helminth Schistosoma mansoni mount strong Th2 cytokine responses in the absence of significant Th1 cytokine synthesis. To examine the basis of this immunoregulatory state, spleen or lymph node cells from schistosome-infected mice were stimulated with parasite-specific Ag and the supernatants tested for their capacity to suppress IFN-gamma synthesis by a Th1 cell line. Strong inhibition was observed that was neutralized by a mAb against IL-10, a cytokine previously shown to down-regulate Th1 cytokine synthesis. By means of ELISA measurements the production of IL-10 in schistosome infection was confirmed and shown to depend on CD4+ T cells. IL-10 synthesis stimulated by either mitogen or Ag was observed only at those stages of infection when Th2 response induction and Th1 cytokine down-regulation also occurred and was not detected in mice vaccinated with attenuated parasites. Moreover, the addition of the neutralizing anti-IL-10 mAb to Ag-stimulated spleen cell cultures from infected mice caused a dramatic augmentation in IFN-gamma synthesis. These findings suggest that IL-10 is responsible for the down-regulation of Th1 responses observed in schistosome infections, a phenomenon that may enable the parasite to escape potentially harmful cell-mediated responses.     

Population Genetic Models of Genomic Imprinting

The phenomenon of genomic imprinting has recently excited much interest among experimental biologists. The population genetic consequences of imprinting, however, have remained largely unexplored. Several population genetic models are presented and the following conclusions drawn: (i) systems with genomic imprinting need not behave similarly to otherwise identical systems without imprinting; (ii) nevertheless, many of the models investigated can be shown to be formally equivalent to models without imprinting; (iii) consequently, imprinting often cannot be discovered by following allele frequency changes or examining equilibrium values; (iv) the formal equivalences fail to preserve some well known properties. For example, for populations incorporating genomic imprinting, parameter values exist that cause these populations to behave like populations without imprinting, but with heterozygote advantage, even though no such advantage is present in these imprinting populations. We call this last phenomenon "pseudoheterosis." The imprinting systems that fail to be formally equivalent to nonimprinting systems are those in which males and females are not equivalent, i.e., two-sex viability systems and sex-chromosome inactivation.     

An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase

X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural changes occurring in interrenal tissue of the duck (Anas platyrhynchos) following adenohypophysectomy and treatment in vivo and in vitro with corticotropin

Adrenal glands from ACTH-treated intact ducks and chronically adenohypophysectomized ducks showed clear zonation into a subcapsular zone (SCZ) and an inner zone (IZ). Adenohypophysectomy caused ultrastructural changes in the IZ but not in the SCZ cells. These included increases in lipid droplets, changes in mitochondrial cristae from tubular to shelf-like, and changes in the shape of the nuclei from spherical to crenated. These changes were reversed by treatment with ACTH. Also, cells of the IZ, but not the SCZ, of adrenals from intact birds given ACTH showed more SER, more dense bodies, fewer lipid droplets and more prominent Golgi complexes. IZ cells incubated in buffer containing no ACTH developed mitochondria with shelf-like cristae and numerous opaque granules in the matrix. Exposure to buffer containing ACTH caused the mitochondrial cristae to become tubular and the matrix granules either decreased in number or disappeared. The granules could be extracted by incubating sections with chelating agents. The mitochondria in SCZ cells did not respond structurally to the presence of ACTH in the incubation medium but the matrix granules, like those in IZ cells, responded to the presence of chelating agents.     

Nitrogen deposition enhances moss growth,but leads to an overall decline in habitat condition of mountain moss‐sedge heath

Ecosystems are subject to multiple, natural and anthropogenic environmental influences, including nitrogen (N) deposition, land use and climate. Assessment of the relative importance of these influences on biodiversity and ecosystem functioning is crucial for guiding policy and management decisions to mitigate global change; yet, few studies consider multiple drivers. In the UK, ongoing loss of the internationally important arctic/alpine moss‐sedge community, Racomitrium heath, has been linked to elevated N deposition, high grazing pressures and their combination; however, the relative importance of these drivers remains unclear. We used environmental gradients across the habitat's European distribution (UK, Faroes, Norway and Iceland) to investigate the relative impact of N deposition and grazing pressure, as well as climate, on the condition of the dominant moss species, Racomitrium lanuginosum. Key variables including tissue chemistry, growth and cover were measured at 36 sites, and multiple linear regressions were used to examine the relative importance of the drivers across sites. Our results clearly show that regional variation in the condition of R. lanuginosum across Europe is primarily associated with the impacts of N deposition, with climate (air temperature) and grazing pressure playing secondary roles. In contrast to previous experimental studies, we found moss growth to be stimulated by elevated N deposition; this apparent discrepancy may result from the use of artificially high N concentrations in many experiments. Despite increased growth rates, we found that moss mat depth and cover declined in response to N deposition. Our results suggest that this is due to increased decomposition of material in the moss mat, which ultimately leads to loss of moss cover and habitat degradation. This study clearly demonstrates both the key role of N deposition in degradation of Racomitrium heath and the importance of observational studies along natural gradients for testing predictions from experimental studies in the real world.     

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae. International Journal for Parasitology16: 491–510. Naive guinea pigs and guinea pigs vaccinated 4 weeks previously with highly irradiated cercariae were challenged percutaneously with normal cercariae. Skin samples from the challenge site were then harvested at varying times to provide histological, quantitative and ultrastructural data on the respective cellular responses to cercarial invasion. The primary cutaneous reaction was characterised by neutrophils; these cells reached peak numbers (16% of total cells) by 18 h. Eosinophils and basophils made only a small contribution to the infiltrate (2.9 and 5.7% respectively). Some basophils showed evidence of anaphylactic degranulation, others seemed to be damaged, but most appeared normal. Mononuclear cells of varied morphology were present at all times, but activated fibroblasts were prominent, and collagen deposition increased with time. Degranulating mast cells were recognised at 24 and 48 h. Dead schistosomula were never seen in naive-challenged skin, although one or two of the observed larvae showed minor tegumental abnormalities. In vaccinated guinea pigs, the cutaneous cellular response to challenge was significantly enhanced, with basophils dominating the reaction (33% of total cells at 24 h). Many basophils were already degranulating by the anaphylactic mechanism at 3 h post challenge, and free basophil granules were seen frequently. Both intact cells and free granules congregated in close proximity to invading larvae. Eosinophils were also present in greater numbers at secondary reaction sites, but they never exceeded 6% of the total infiltrate. Mononuclear cells believed to be immature eosinophils were prominent from 3 h. For the first time, the mechanism of eosinophil attachment and degranulation onto a multicellular target, described previously only from in vitro investigations, was recognized in vivo. Neutrophil numbers matched those recorded in naive-challenged skin at 3 and 6 h, but declined thereafter, while mast cells were seen degranulating at these early times. Mononuclear cells again presented a variety of morphological appearances; of particular note were large cells that had phagocytosed debris and were presumed to be macrophages, and small rounded cells with scant cytoplasm and few organelles, that may have been lymphocytes. By 12 h, large areas of the dermis had become severely disorganised and numerous, free basophil granules were distributed amongst the other cellular constituents. Dead schistosomula, denuded of tegument, were clearly recognised at 6 h, and such individuals invariably had neutrophils attached to their exposed muscle layers. Since dead schistosomula were not identified in naive-challenged guinea pig skin, it is concluded that a percentage of the challenge larvae, however small, is preferentially killed in the skin of the vaccinated animals.