Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

Measurement error affects risk estimates for recruitment to the Hudson River stock of striped bass

We examined the consequences of ignoring the distinction between measurement error and natural variability in an assessment of risk to the Hudson River stock of striped bass posed by entrainment at the Bowline Point, Indian Point, and Roseton power plants. Risk was defined as the probability that recruitment of age-1+ striped bass would decline by 80% or more, relative to the equilibrium value, at least once during the time periods examined (1, 5, 10, and 15 years). Measurement error, estimated using two abundance indices from independent beach seine surveys conducted on the Hudson River, accounted for 50% of the variability in one index and 56% of the variability in the other. If a measurement error of 50% was ignored and all of the variability in abundance was attributed to natural causes, the risk that recruitment of age-1+ striped bass would decline by 80% or more after 15 years was 0.308 at the current level of entrainment mortality (11%). However, the risk decreased almost tenfold (0.032) if a measurement error of 50% was considered. The change in risk attributable to decreasing the entrainment mortality rate from 11 to 0% was very small (0.009) and similar in magnitude to the change in risk associated with an action proposed in Amendment #5 to the Interstate Fishery Management Plan for Atlantic striped bass (0.006)--an increase in the instantaneous fishing mortality rate from 0.33 to 0.4. The proposed increase in fishing mortality was not considered an adverse environmental impact, which suggests that potentially costly efforts to reduce entrainment mortality on the Hudson River stock of striped bass are not warranted.     

Excitation-contraction coupling in isolated locomotor muscle fibres from the pelagic tunicate Doliolum which lack both sarcoplasmic reticulum and transverse tubular system

In the locomotor muscle of the pelagic tunicate Doliolum, both the sarcoplasmic reticulum (SR) and the transverse-tubular (T-tubular) system are absent. The mechanism of excitation-contraction (E-C) coupling was studied in single muscle fibres enzymatically dissociated from Doliolum denticulatum. Whole cell voltage clamp experiments demonstrated an inward ionic current associated with membrane depolarisation. This current was blocked by 5 mmol.l(-1)Co(2+), a calcium current blocker, and suppressed by nifedipine, a specific L-type calcium channel blocker. An increase in the external K(+) concentration to 200 mmol.l(-1) (K(+)-depolarisation) induced a rise in the intracellular Ca(2+) level detected with fluo-3, a Ca(2+)-sensitive dye. However, when 5-10 mmol.l(-1) Co(2+) or 10-15 micro mol.l(-1) nifedipine was present in the external solution, K(+)-depolarisation did not induce a rise in the intracellular Ca(2+) level. Externally applied 5-10 mmol.l(-1) caffeine or 20 micro mol.l(-1) ryanodine had no effect on the intracellular Ca(2+) level. K(+)-depolarisation induced a rise in the intracellular Ca(2+) level in the presence of caffeine or ryanodine. Replacement of external Na(+) with Li(+) increased intracellular Ca(2+) levels. Our results show that contraction of the locomotor muscle in Doliolum is solely due to the influx of Ca(2+) through L-type calcium channels, and that relaxation is due to extrusion of Ca(2+) by Na(+)/Ca(2+) exchange across the sarcolemma.     

Hydroxyethyl starch does not cross the blood-brain or the placental barrier but the perineurium of peripheral nerves in infused animals

Therapy with hydroxyethyl starch (HES) is associated with a high incidence of persistent pruritus due to HES storage in cutaneous nerves. Up to now it has been unknown if HES also accumulates in the extracutaneous peripheral or central nervous system. To study this, five rats including one pregnant one were infused with a single dose (34-150 mg) of HES (70/200/450 kDa molecular weight) conjugated with fluorescein isothiocyanate (FITC). In addition, four sheep were infused with a cumulative dosage of 30 g, 120 g, and 420 g HES (200 kDa), respectively. After 7-13 days, biopsies from the adult rats, four fetal rats and sheep were taken from various organs. The specimens were analyzed by light, electron, and confocal laser scanning microscopy. Typical HES storage vacuoles were found in macrophages of the skin, liver, spleen, lung, and kidney. HES storage in healthy animals was not associated with signs of either inflammation or apoptosis contrary to a previously described animal hemorrhagic shock model. Beyond that, fetus biopsies did not show any storage phenomenon, confirming that HES does not cross the placental barrier. Deposits of HES could be detected in Schwann cells of cutaneous nerve fibers as well as in perineural and endoneural cells of sciatic nerve in one rat (HES 450 kDa) and three of four sheep. No HES storage was found in the central nervous system. Our findings clearly demonstrate that storage of HES is detectable only in small peripheral nerves, suggesting a cutaneous origin of the HES-induced pruritus.     

Arteriovenous carboxyhemoglobin difference in critical illness: fiction or fact?

It is still unclear whether the paradoxical arteriovenous carboxyhemoglobin (COHb) difference found in critical illness is due to increased COHb production by the lung, or whether this gradient is caused by technical artifacts using spectrophotometry. In healthy and matched endotoxemic sheep, blood gases were analyzed with a standard ABL 625 and the updated version, an ABL 725. The latter one was accurately calibrated for COHb wavelengths (SAT 100) to eliminate the FCOHb dependency on oxygen tension. All endotoxemic sheep exhibited a hypotensive-hyperdynamic circulation and a pulmonary hypertension. Interestingly, arteriovenous COHb difference occurred in both healthy and endotoxemic sheep (P<0.001 each). Arterial and central venous COHb concentrations determined with the ABL 625 were significantly lower than those measured with the ABL 725 (P<0.001 each). We conclude that (a) arteriovenous COHb difference per se does not reflect critical illness and (b) measurements with an ABL 625 underestimate COHb concentrations.     

Disease in scleractinian corals: a new problem in the reef at Cayo Sombrero, Morrocoy National Park, Venezuela?

At the beginning of 1996 coral reefs in Morrocoy National Park, Venezuela, suffered an unprecedented mass mortality event. As a consequence, live coral cover dropped to 2-10%. One of the few reefs that kept live coral cover over 35% was Cayo Sombrero; nonetheless, the presence of some coral diseases has been detected within the past 2 years, representing a new source of coral mortality. Due to this situation, this study started a monitoring program on the incidence of coral diseases and syndromes in the reef of Cayo Sombrero. The CARICOMP protocol was used in order to evaluate reef health. Ten parallel band-transects (20 x 2m) where established at two depth intervals: Five between 3-8 m and five between 8-12 m, and the frequency of both, healthy and unhealthy colonies of each coral species was recorded along each band transect. In addition to other sources of coral damage (predation, siltation, etc), significant differences in disease incidence between the two depths intervals were tested with a Kruskall-Wallis test. The main problems observed were coral diseases such as yellow band (4.2%), dark spots (1.61%) and white plague-II (1.4%), mainly affecting Montastraea faveolata, M. annularis and Siderastrea siderea. Siltation, affecting massive colonies, such as Colpophyllia natans and Diploria strigosa; algae overgrowth, predation, anchor damage, and bleaching. Significant differences were found in the incidence of unhealthy (Kruskall-Wallis, p < 0.05) bleached (Kruskall-Wallis, p < 0.05) and colonies affected by siltation (Kruskall-Wallis, p < 0.05). More than 60% of the 585 coral colonies surveyed at both depths were found to be healthy, indicating that the Cayo Sombrero reef is still in good conditions compared to other localities in the Park. This study stresses the need to conduct early monitoring programs that survey coral disease incidence as a source of mortality for this coral reef.     

Increased carbon monoxide in exhaled air of critically ill patients

Heme oxygenase produces carbon monoxide (CO) during breakdown of heme molecules primarily in liver and spleen. Recent data suggest that CO is also produced in the lungs. CO is excreted by exhalation via the lungs. A number of inflammatory agents induce the expression of heme oxygenase, possibly leading to increased CO production. To investigate whether critical illness results in increased CO production we measured the CO concentration in exhaled air in 30 critically ill patients and in healthy controls (n = 6). Critically ill patients showed a significantly higher CO concentration in exhaled air (median 2.4 ppm, 95% CI 1.0-7.0 ppm vs median 1.55 ppm, 95% CI 1.2-1.7 ppm, P = 0.01) as well as total CO production (median 20 ml/min, 95% CI 8 to 90 ml/min vs median 13.5 ml/min, 95% CI 11 to 19 ml/min, P = 0.026) compared to healthy controls. No correlation was found between CO concentration in exhaled air and carboxyhemoglobin concentration in arterial and central venous blood (P > 0.05). The increase of CO concentration in exhaled air in critical illness suggests an induction of inducible heme oxygenase (HO-1) and might reflect the severity of illness.     

Interaction of α-Phenyl-N-tert-Butyl Nitrone and Alternative Electron Acceptors with Complex I Indicates a Substrate Reduction Site Upstream from the Rotenone Binding Site

Abstract: Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O₂ to hydrogen peroxide (H₂O₂) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H₂O₂ formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O₂ consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H₂O₂ generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H₂O₂ flux was accelerated by rotenone. α-Phenyl- N-tert -butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I-stimulated H₂O₂ flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H₂O₂ synthesis, possessing an "electron leak" site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H₂O₂ production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.