pH dependence of the enzymatic processing of collagen I by MMP-1 (fibroblast collagenase), MMP-2 (gelatinase A), and MMP-14 ectodomain

The proteolytic processing of collagen I by three matrix metalloproteinases (MMPs), a collagenase (MMP-1), a gelatinase (MMP-2), and the ectodomain of a membrane-type metalloproteinase (MMP-14), has been investigated at 37 °C between pH 6.0 and 9.2, a pH range reflecting conditions found in different body compartments under various physiopathological processes. In the proteolytic degradation the native collagen triple helix must be partially unwound to allow the binding of α chains to the protease’s active-site cleft. We have found that MMP-1 interacts with the two types of collagen I α chains in a similar fashion, whereas both MMP-2 and MMP-14 bind the two α chains in a different way. The overall enzymatic activity is higher on the α-2 chain for both MMP-1 and MMP-2, whereas the MMP-14 ectodomain preferentially cleaves the α-1 chain. In MMP-2 a marked difference for substrate affinity (higher for the α-1 chain) is overwhelmed by an even more marked propensity to cleave the α-2 chain. As a whole, the three classes of MMPs investigated appear to process collagen I in a significantly different fashion, so various MMPs play different roles in the collagen homeostasis in various compartments (such as bloodstream, synovial fluid, normal and tumoral tissues), where different pH values are observed.     

Development and Evaluation of Emulsions from <Emphasis Type="Italic">Carapa guianensis</Emphasis> (Andiroba) Oil

Carapa guianensis, a popular medicinal plant known as “Andiroba” in Brazil, has been used in traditional medicine as an insect repellent and anti-inflammatory product. Additionally, this seed oil has been reported in the literature as a repellent against Aedes aegypti. The aim of this work is to report on the emulsification of vegetable oils such as “Andiroba” oil by using a blend of nonionic surfactants (Span 80® and Tween 20®), using the critical hydrophilic–lipophilic balance (HLB) and pseudo-ternary diagram as tools to evaluate the system’s stability. The emulsions were prepared by the inverse phase method. Several formulations were made according to a HLB spreadsheet design (from 4.3 to 16.7), and the products were stored at 25°C and 4°C. The emulsion stabilities were tested both long- and short-term, and the more stable one was used for the pseudo-ternary diagram study. The emulsions were successfully obtained by a couple of surfactants, and the HLB analysis showed that the required HLB of the oil was 16.7. To conclude, the pseudo-ternary diagram identified several characteristic regions such as emulsion, micro-emulsion, and separation of phases.     

Shifts in phenology of Bythotrephes longimanus and its modern success in Lake Maggiore as a result of changes in climate and trophy

In Lake Maggiore, the density of the invertebrate predator Bythotrepheslongimanus increased following lake re-oligotrophication inthe late 1980s. This "invasion" was followed by dramatic changesin the pelagic food web, consistent with those that followedthe establishment of B. longimanus in North American lakes whereit is not native. In this contribution, we explore the modernsuccess of B. longimanus in Lake Maggiore by investigating itsphenology and population density, and their correlations withabiotic and biotic factors during the period from 1981 to 2003.A 10-fold increase in the abundance of B. longimanus followedan earlier start and longer duration of annual population growth.Increased prey resources and decreased predation pressure werenot observed during the B. longimanus density increase. Instead,a rise in lake temperature may have altered the reproductivecycle of this species. Furthermore, the depth and duration ofa refuge from visually orienting fish predators increased duringthese 20 years, as a result of changes in the thermal stratificationregime of the lake. This case study provides a timely exampleof how climatic changes may interact with biotic drivers (e.g.fish predation) to influence the density and phenology of aninvertebrate predator.     

Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela

A longitudinal epidemiological and entomological study was carried out in Ocamo, Upper Orinoco River, between January 1994 and February 1995 to understand the dynamics of malaria transmission in this area. Malaria transmission occurs throughout the year with a peak in June at the beginning of the rainy season. The Annual Parasite Index was 1,279 per 1,000 populations at risk. Plasmodium falciparum infections accounted for 64% of all infections, P. vivax for 28%, and P. malariae for 4%. Mixed P. falciparum/P. vivax infections were diagnosed in 15 people representing 4% of total cases. Children under 10 years accounted for 58% of the cases; the risk for malaria in this age group was 77% higher than for those in the greater than 50 years age group. Anopheles darlingi was the predominant anopheline species landing on humans indoors with a biting peak between midnight and dawn. A significant positive correlation was found between malaria monthly incidence and mean number of An. darlingi caught. There was not a significant relationship between mean number of An. darlingi and rainfall or between incidence and rainfall. A total of 7295 anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite (CS) protein. Only An. darlingi (55) was positive for CS proteins of P. falciparum (0.42%), P. malariae (0.25%), and P. vivax-247 (0.1%). The overall estimated entomological inoculation rate was 129 positive bites/person/year. The present study was the first longitudinal entomological and epidemiological study conducted in this area and set up the basic ground for subsequent intervention with insecticide-treated nets.     

Analysis of the 5q31 33 locus shows an association between single nucleotide polymorphism variants in the IL-5 gene and symptomatic infection with the human blood fluke, Schistosoma japonicum

Genetic studies of human susceptibility to Schistosoma (blood fluke) infections have previously identified a genetic locus determining infection intensity with the African species, Schistosoma mansoni, in the 5q31-33 region of the human genome that is known to contain the Th2 immune response cluster, including the genes encoding the IL-4, IL-5, and IL-13 cytokines. These cytokines are key players in inflammatory immune responses and have previously been implicated in human susceptibility to infection with the Asian species, S. japonicum. In a nested case control study, we genotyped 30 HapMap tagging single nucleotide polymorphisms (SNPs) across these three genes in 159 individuals identified as putatively susceptible to reinfection with S. japonicum and in 133 putatively resistant individuals. A third group comprising 113 individuals demonstrating symptomatic infection was also included. The results provided no significant association at a global level between reinfection predisposition and any of the individual SNPs or haplotype blocks. However, two tagging SNPs in IL-5 demonstrated globally significant association with susceptibility to symptomatic infection. They were in strong linkage disequilibrium with each other and were found to belong to the same haplotype block that also provided a significant association after permutation testing. This haplotype was located in the 3'-untranslated region of IL-5, suggesting that variants in this region of IL-5 may modulate the immune response in these individuals with symptomatic infection.     

Acute exposure to long-chain fatty acids impairs {alpha}2-adrenergic receptor-mediated antilipolysis in human adipose tissue

The acute in vitro and in vivo effects of long-chain fatty acids (LCFAs) on the regulation of adrenergic lipolysis were investigated in human adipose tissue. The effect of a 2 h incubation, without or with LCFA (200 mumol/l), on basal and hormonally induced lipolysis was tested in vitro on isolated fat cells. The lipolytic response to epinephrine was enhanced by suppression of the antilipolytic alpha(2)-adrenergic effect. Then, healthy lean and obese male subjects performed a 45 min exercise bout at 50% of their heart rate reserve either after an overnight fast or 3 h after a high-fat meal (HFM: 95% fat, 5% carbohydrates). Subcutaneous adipose tissue lipolysis was measured by microdialysis in the presence or absence of an alpha-antagonist (phentolamine). In vivo, a HFM increased plasma levels of nonesterified fatty acids in lean and obese subjects. In both groups, the HFM did not alter hormonal responses to exercise. Under fasting conditions, the alpha(2)-adrenergic antilipolytic effect was more pronounced in obese than in lean subjects. The HFM totally suppressed the alpha(2)-adrenergic antilipolytic effect in lean and obese subjects during exercise. LCFAs per se, in vitro as well as in vivo, suppress alpha(2)-adrenergic-mediated antilipolysis in adipose tissue. LCFA-mediated suppression of antilipolytic pathways represents another mechanism whereby a high fat content in the diet might increase adipose tissue lipolysis.     

Programmed cell death and stem cell differentiation are responsible for midgut replacement in <Emphasis Type="Italic">Heliothis virescens</Emphasis> during prepupal instar

We have analyzed midgut development during the fifth larval instar in the tobacco budworm Heliothis virescens. In prepupae, the midgut formed during larval instars undergoes a complete renewal process. This drastic remodeling of the alimentary canal involves the destruction of the old cells by programmed cell-death mechanisms (autophagy and apoptosis). Massive proliferation and differentiation of regenerative stem cells take place at the end of the fifth instar and give rise to a new fully functioning epithelium that is capable of digesting and absorbing nutrients and that is maintained throughout the subsequent pupal stage. Midgut replacement in H. virescens is achieved by a balance between this active proliferation process and cell-death mechanisms and is different from similar processes characterized in other insects. This work was supported by FAR 2006 (University of Insubria) to G.T., by a MIUR-FIRB-COFIN grant (no. RBNE01YXA8/2004077251), and by the Centro Grandi Attrezzature (University of Insubria).     

SAR studies of 1,5-diarylpyrazole-based CCK1 receptor antagonists

A high throughput screening campaign revealed compound 1 as a potent antagonist of the human CCK(1) receptor. Here, we report the syntheses and SAR studies of 1,5-diarylpyrazole analogs with various structural modifications of the alkane side chain of the molecule. The difference in affinity between the two enantiomers for the CCK(1) receptor and the flexible nature of the linker led to the design of constrained analogs with increased potency.     

Metal binding of metallothioneins in human astrocytomas (U87 MG,IPDDC-2A)

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl₂ (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.     

The intervention of antioxidant therapy on platelet adhesion and immunomodulation in experimental physical stress

During effort overstress the reactive oxygen species act chiefly on unsaturated lipids, inducing the formation of certain peroxidation products. We have investigated malondialdehide (MDA), platelet adhesion index, and immunological activation parameters during effort overstress and administration of vitamins E and C. Biochemical measurements were performed on erythrocytes and heart homogenate. In the vitamin E supplemented group, the platelet adhesion index was constantly correlated with the MDA level (p < 0.001). There is a protecting effect concerning the oxidative stress in animals pretreated with vitamin E and C, which is expressed through the diminution of the MDA quantity both in the erythrocyte and in the heart. The physical effort required by swimming led to a decrease in the NBT test values and in the activity of the serum complement. The steady administration of vitamin E in the effort overstress, due to its antioxidant properties, causes the progressive decrease in peroxidation and platelet adhesion.