THE GENUS THALASSIOSIRA (BACILLARIOPHYCEAE): MORPHOLOGY OF HETEROVALVATE RESTING SPORES OF T. SCOTIA

Resting spores of Thalassiosira scotia Fryx. et Hoban were abundant in phytoplankton net hauls collected from the Scotia Sea south of South Georgia in December 1981. Primary valves are distinguished from secondary valves by the presence of marginal striae with rounded, shoelike protrusions, villi surrounding marginal areolae and marginal striae, greater numbers of central strutted processes, and differences in structure of occluded processes. Densities of marginal strutted processes also tend to differ, though ranges overlap. Thalassiosira scotia is most closely related to T. antarctica, differing from that taxon by having coarser areolation, heavier silicification, and more prominent occluded processes. Rudimentary valves of T. scotia differ from those of T. antarctica.     

Catalytic properties of cysteine proteinases from Trypanosoma cruzi and Leishmania infantum: a pre-steady-state and steady-state study

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationship. Moreover, they appear as promising targets for antiparasite chemotherapy. Here, the first quantitative investigation on the steady-state and pre-steady-state kinetics of the papain-like cysteine proteinases from epimastigotes of Trypanosoma cruzi (cruzipain), the agent of Chagas' disease, and from promastigotes of Leishmania infantum, an agent of visceral and cutaneous leishmaniases, is reported. The results indicate that kinetics for the parasite proteinase catalyzed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) may be consistently fitted to the minimum three-step mechanism involving the acyl.enzyme intermediate E.P: [mechanism: see text] At neutral pH, the k(+3) step (deacylation process) is rate limiting in enzyme catalysis, whereas, at pH<6, the k(+2) step (acylation process) becomes rate limiting. This illustrates the potential danger in interpreting both kcat versus pH profile, given that the acylation or the deacylation step is rate limiting throughout the whole pH range explored, and Km as the true affinity constant for the E:S complex formation. Comparison with the steady-state and pre-steady-state kinetics of homologous plant enzymes suggests that the parasite cysteine proteinase catalytic behavior appears to be of general significance.     

The anti-parasitic effects of nitric oxide

Endogenous and exogenous nitric oxide (NO) possesses antiparasitic effects on both Protozoa and Metazoa. However, NO production requires a tight control to limit cytotoxic damage to the host's own cells. The best known parasitic macromolecular targets for NO(-donors) are cysteine proteases, which are relevant in several aspects of the parasite life cycle and parasite-host relationships, and appear as promising targets for anti-parasitic chemotherapy.     

Development of selective tolerance to interleukin-1beta by human chondrocytes in vitro

Interleukin-1 induces release of NO and PGE(2) and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1beta. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1beta (0.05-10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1beta declined over the pretreatment period. In response to IL-1beta (0.1 ng/ml), NO and PGE(2) release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1beta on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1beta-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1beta increased glucose consumption and lactate production throughout the treatment. IL-1beta stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE(2) and MMP-13 in response to IL-1beta than OA cultures. Like the OA, IL-1beta-induced NO and PGE(2) release decreased over time. In conclusion, with prolonged exposure to IL-1beta, human chondrocytes develop selective tolerance involving NO and PGE(2) release but not MMP-13 production, metabolic activity, or matrix metabolism.     

Arterial intima-media thickness: site-specific associations with HRT and habitual exercise

We determined the site-specific relations of hormone replacement therapy (HRT) and habitual exercise status with intima-media thickness (IMT) in both elastic (carotid) and muscular (femoral) arteries in 77 healthy postmenopausal women: 43 women were sedentary (20 no-HRT and 23 HRT users) and 34 women were endurance trained (14 no-HRT and 20 HRT users). Femoral IMT was not different among the sedentary HRT and endurance-trained no-HRT and HRT groups, but was lower (P < 0.005) in these three groups than in the sedentary no-HRT women. There were no significant group differences in carotid IMT. However, in older women (> or =65 yrs) carotid IMT was smaller (P < 0.05) in HRT compared with no-HRT women. We conclude that both endurance training and HRT status are independently associated with a smaller IMT and these effects are evident primarily in muscular arteries. These results suggest that HRT and habitual exercise may protect postmenopausal women against cardiovascular disease through influences on IMT. The site-specific relations may be due to a greater number of smooth muscle cells and plasticity of muscular arteries compared with elastic arteries and/or differences in heterogeneous influences such as metabolic requirements and hydrostatic pressures.     

Absorption and fluorescence spectra of ring-substituted indole-3-acetic acids

The absorption and fluorescence spectra of indole-3-acetic acid (1), a plant growth regulator (auxin) and experimental cancer therapeutic, 29 ring-substituted derivatives and the 7-aza analogue (1H-pyrrolo[2,3b]pyridine-3-acetic acid) are compared. Two to four absorbance maxima in the 260-310-nm range are interpreted as overlapping vibronic lines of the 1La<--1A and 1Lb<--1A transitions. Two further maxima in the 200-230-nm region are assigned to the 1Ba<--1A and 1Bb<--1A transitions. 4- and 7-Fluoroindole-3-acetic acid exhibit blue shifts with respect to 1, most other derivatives show red shifts. All indole-3-acetic acids studied, with the exception of chloro-, bromo- and 4- or 7-fluoro-derivatives, fluoresce at 345-370 nm when excited at 275-280 nm. 7-Azaindole-3-acetic acid emits at 411 nm. The fluorescence quantum yield of 6-fluoroindole-3-acetic acid significantly exceeds that of 1 (0.3); the other derivatives have lower quantum yields. The plant-growth promoting activity of the ring-substituted indole-3-acetic acids studied correlates with the position of the 1Bb<--1A transition band.     

Kinetics of parasite cysteine proteinase inactivation by NO-donors

NO-donors block Plasmodium, Trypanosoma, and Leishmania life cycle inactivating parasite cysteine proteinases. In this study, the inactivation of falcipain, cruzipain, and Leishmania infantum cysteine proteinase by S-nitroso-5-dimethylaminonaphthalene-1-sulphonyl (dansyl-SNO), S-nitrosoglutathione (GSNO), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitrosoacetylpenicillamine (SNAP) is reported. With NO-donors in excess over the parasite cysteine proteinase, the time course of enzyme inactivation corresponds to a pseudo-first-order reaction for more than 90% of its course. The concentration dependence of the pseudo-first-order rate constant is second-order at low NO-donor concentrations but tends to first-order at high NO-donor concentrations. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting pseudo-first-order process. Kinetic parameters of cruzipain inactivation by GSNO were affected by the acidic pK shift of one ionizing group (from pKunl = 5.7 to pKlig = 4.8) upon GSNO-induced enzyme inactivation. Falcipain, cruzipain, and L. infantum cysteine proteinase inactivation by dansyl-SNO, GSNO, NOR-3, and SNAP is prevented and reversed by dithionite and l-ascorbic acid. However, the incubation of L. infantum cysteine proteinase with dansyl-SNO does not result in the appearance of fluorescence of the enzyme. More than 90% of the S-transnitrosylation product GSH existed in the inactivation reaction, suggesting that S-transnitrosylation is the favorite process for parasite cysteine proteinase inactivation. Furthermore, the fluorogenic substrate N-alpha-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) protects L. infantum cysteine proteinase from inactivation by SNAP. These results indicate that parasite cysteine proteinase inactivation by NO-donors occurs via NO-mediated S-nitrosylation of the Cys25 catalytic residue.     

Heme impairs allosterically drug binding to human serum albumin Sudlow's site I

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand (e.g., heme and drugs) binding capacity. Here, the binding of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, and tolbutamide to HSA and ferric heme-HSA is reported. Moreover, ferric heme binding to HSA in the absence and presence of drugs has been investigated. Values of the association equilibrium constant for drug binding to Sudlow’s site I of ferric heme-HSA (ranging between 1.7 × 10³ and 1.6 × 10⁵ M⁻¹) are lower by one order of magnitude than those for drug binding to ferric heme-free HSA (ranging between 1.9 × 10⁴ and 1.8 × 10⁶ M⁻¹). According to linked functions, the value of the association equilibrium constant for heme binding to HSA decreases from 7.8 × 10⁷ M⁻¹, in the absence of drugs to 7.0 × 10⁶ M⁻¹, in the presence of drugs. These findings represent a clear-cut evidence for the allosteric inhibition of drug binding to HSA Sudlow’s site I by the heme. According to linked functions, drugs impair allosterically heme binding to HSA. These results appear to be relevant in the drug therapy and management.