Organic matter characterisation and turnover in the sediment and seawater of a tourist harbour.

A survey of a Ligurian tourist harbour was carried out during winter 2006 and summer 2007 in order to study the organic matter (OM) turnover through extracellular enzymatic activity. Seawater and sediments were sampled at six stations, three inside the port boundaries, one outside the port and two in an area influenced by the outflow of a minor river (Boate). The seawater showed OM turnover times similar to other oligo-mesotrophic coastal areas, and low concentrations of chlorophyll-a and inorganic nutrients. The sediments, instead, revealed high OM loads and a predominance of proteolysis. A significant reduction of the OM loads was observed in the outside station, indicating that the OM accumulation was due to the structures and activities of the harbour and to the Boate influence. The OM biotic recycling via enzymatic activity was enhanced especially during summer. Although the carbohydrates were probably highly refractory, their turnover was notably faster, due to glycolytic enzymatic activity that was enhanced more than the proteolytic in both the sediment and in the seawater. This suggested that the removal and recycling of OM were potentially efficient, and prevented the shift to eutrophication of the Rapallo harbour area.

Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis.

Aminopeptidases responsible for blood digestion have yet to be identified in haematophagous ticks. We report here the cloning and molecular characterisation of a cDNA encoding leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from the hard tick Haemaphysalis longicornis (HlLAP). Endogenous HlLAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HlLAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HlLAP was induced by a blood-feeding process. A functional recombinant HlLAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the Km value 0.19 +/- 0.011 mM and Vmax value 157.2 +/- 3.17 nmol/min/mgprotein) and a methionyl substrate (with the Km value 0.12+/-0.0052 mM and Vmax value 171.9 +/- 2.31 nmol/min/mgprotein). Enzyme activity was found to be optimum at pH 8 and 35 degrees C. The recombinant HlLAP enzyme activity was strongly dependent on metal divalent cations, Mn2+, and was inhibited by bestatin. These results indicate that HlLAP play an important role for host's blood digestion process.

Microbial responses to long-term N deposition in a semiarid grassland.

Nitrogen (N) enrichment of the biosphere is an expanding problem to which arid ecosystems may be particularly sensitive. In semiarid grasslands, scarce precipitation uncouples plant and microbial activities, and creates within the soil a spatial mosaic of rhizosphere and cyanobacterial crust communities. We investigated the impact of elevated N deposition on these soil microbial communities at a grama-dominated study site located incentral New Mexico (USA). The study plots were established in 1995 and receive 10 kg ha(-1) year(-1) of supplemental N in the form of NH(4)NO(3). Soil samples were collected in July 2004, following 2 years of severe drought, and again in March 2005 following a winter of record high precipitation. Soils were assayed for potential activities of 20 extracellular enzymes and N(2)O production. The rhizosphere and crust-associated soils had peptidase and peroxidase potentials that were extreme in relation to those of temperate soils. N addition enhanced glycosidase and phosphatase activities and depressed peptidase. In contrast to temperate forest soils, oxidative enzyme activity did not respond to N treatment. Across sampling dates, extracellular enzyme activity responses correlated with inorganic N concentrations. N(2)O generation did not vary significantly with soil cover or N treatment. Microbial responses to N deposition in this semiarid grassland were distinct from those of forest ecosystems and appear to be modulated by inorganic N accumulation, which is linked to precipitation patterns.

Trophic status of the small intestine in young and aged rats: modulation by a yogurt-supplemented diet.

Among the multifactorial causes of undernutrition in old age, gastrointestinal mucosa altered function and resulting specific malabsorption are the most relevant. Despite numerous studies that have dealt with the effects of aging on the digestive tract of mammals, results showed discrepancies in terms of proliferation and biochemical aging small intestine events. However, the slowing-down of the maturation process and the poor adaptation of metabolism and intestinal function are obvious and there is evidence that protective mechanisms are impaired with age and contribute to affecting the trophic activity and related systemic homeostasis. Good prospects to improve gastrointestinal function in the elderly are essential and research on nutritional intervention to limit and counteract age-related impairments must be extensive. Probiotics are good candidates and fermented milks might be of great interest. In the present study we first show the main structural and functional variations between 3- and 23-month-old rat small intestines. The trophic consequences of aging and nutritional adaptation under basal conditions are also analyzed and discussed after 20 days of a yogurt-supplemented specific diet in both young and aged rats. The main variations that occur with aging and yogurt diet are located in the proximal small intestine. The present findings indicate a slight improvement of morphological trophic parameters in both young and aged rats by yogurt, whereas enzymatic changes are more discrete. Despite the obvious age-related decrease in trophicity, we suggest that assessment of probiotic potentials on trophicity requires a more altered model than normal, healthy aging animals.

Oral Candida albicans and Candida dubliniensis differentiation by multilocus enzyme electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis.

Five oral strains of Candida albicans and five C. dubliniensis, as well as their respective type-strains, were analyzed by multilocus enzyme electrophoresis (MLEE) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoreses and numerical analyses, we obtained two distinct species-specific taxa, which may justify the use of MLEE and SDS-PAGE as reliable methods for differentiation and complementary identification of C. dubliniensis.

Evidence for the presence of proteolytic activity in peroxisomes.

The presence of endo- and exoproteolytic activity in peroxisomes was detected in cell organelles purified from pea leaves. By PAGE using different exopeptidase substrates (L-aa-beta NA), one leucine aminopeptidase (AP) was found in peroxisomes. The peroxisomal AP was characterized as a serine protease and had a maximal activity at pH 7.5, a molecular mass of 56.8 kDa and a pI of 5.3. This enzyme was mainly present in the soluble fraction of peroxisomes. The occurrence of proteases in peroxisomes suggests that they might be involved in the protein turnover and processing of imported precursor polypeptides in peroxisomes.

The effect of dietary n-3 and n-6 fatty acids on tumor necrosis factor-alpha production and leucine aminopeptidase levels in rat peritoneal macrophages.

The purpose of this study was to determine the effect of dietary n-3 and n-6 fatty acids on tumor necrosis factor-alpha (TNF-alpha) production and macrophage (MO) activation state. Rats were fed diets containing 12.5% linseed oil (LO) or corn oil (CO) that are high in n-3 and n-6 fatty acids respectively. The LO diet resulted in a significant increase in basal and endotoxin (LPS)-induced levels of TNF-alpha from resident MO cultured in vitro. There was no difference between the diets in LPS-induced TNF-alpha production by complete Freund's adjuvant (CFA) elicited macrophages. Variable responses were also observed between LO and CO MO in response to prostaglandin E2, indomethacin (INDO), and the prostaglandin E receptor antagonist SC-19220. This may indicate differences in signal transducing secondary messengers due to different activation states, receptor expression or ligand binding. Fluorescence due to leucine aminopeptidase (LAP) staining was determined by flow cytometry. Resident LO MO had a 15% increase in LAP fluorescence compared to CO MO. In CFA-elicited MO, the CO MO had a 43% increase in fluorescence compared to LO MO. Resident LO MO increased in LAP fluorescence by 35% to the activated state whereas resident CO MO increased in LAP fluorescence by 93%. The smaller window of activation for the LO MO may explain some of the antiinflammatory properties of dietary n-3 fatty acids.

[Personal experience with the detection of sperm using leucylaminopeptidase]. / Nase zkusenosti s prukazem spermatu pomocí leucylaminopeptidázy (LAP).

The detection of leucylaminopeptidase (LAP) has been carried out in human male and female sexual organ tissues and in vaginal smears through the use of histochemical methods. On the ground of the performed experiments it is possible to prefer the statement that the finding of a positive result of LAP activity testing might be used as a complementary method for obtaining indirect proof of the presence of sperm in vaginal smears. LAP activity may be demonstrated until the term of twelve hours following sexual intercourse.

Immunologic characterization of human seminal leucine aminopeptidase (LAP) and its medicolegal use.

A new method for identification of seminal stains is described, based on the immunologic demonstration of leucine aminopeptidase (LAP), which is extremely abundant in human semen and specific for the prostate as well as semen. An antiserum against human seminal plasma was obtained by repeated immunization of rabbits with seminal plasma and Freund's adjuvant. Ouchterlony's double immunodiffusion test and Culliford's precipitin electrophoresis were performed to demonstrate specific proteins of seminal plasma. LAP activity was visualized with L-leucyl-beta-naphthylamide as substrate and with Fast Garnet GBC as coupler. The immunologic analysis of LAP produced two precipitin lines with enzyme activity. One was observed in kidney, jejunum, pancreas, prostate, as well as in semen, and was completely absorbed with kidney homogenates. The other was found only in semen and the prostate and was not absorbed with kidney homogenates. When the anti-seminal plasma serum absorbed with the kidney was used, the semen-specific LAP could be demonstrated by precipitin electrophoresis only in seminal stains stored for up to 2 months, whereas it was not demonstrated in stains from other human body fluids. By means of precipitin electrophoresis the detection of the semen-specific LAP was possible at semen dilutions of up to 1:32. The method described here greatly enhances the value of semen identification and is quite recommendable for the examination of stains in medico-legal practice.

Characterization of some of the enzymes in ragweed pollen.

Ragweed pollen contains 11 esterase, 5 acid phosphatase, 2 alkaline phosphatose, 2 hexokinase, 2 glucose-6-phosphate dehydrogenase isozymes and one leucine amino peptidase band which can be separated by starch gel electrophoresis. The isozymes were distinguished from one another by their electrophoretic mobility, heat inactivation temperatures and antigenic differences.