Photochemical reactions in interstellar grains photolysis of co,NH3, and H2O

A simulation of the organic layer accreted onto interstellar dust particles was prepared by slow deposition of a CO:NH₃:H₂O gas mixture on an Al block at 10K, with concomitant irradiation with vacuum UV. The residues were analyzed by GC-MS, HPLC, and near IR; a reaction pathway leading from NH₃ to complex alcohol, fatty acid, and amide products in 27 stages is postulated. The astronomical relevance and significance of the observations are discussed.     

Mechanism of complement-induced stimulation of prostacyclin production by isolated rabbit peritoneum

The interaction between the complement system and prostaglandin synthesis has not thoroughly been explored, although both mediators are known to be involved in inflammatory reactions and endotoxic shock. When rabbit peritoneum, a rich source of prostacyclin forming activity was incubated in serum in which the complement system was activated (CVF, LPS, zymosan), the tissue produced significantly more PGI2, when compared with appropriate controls, indicating that by activation of the complement, factors were generated that stimulated PGI2 biosynthesis. Further results indicated that tryptic cleavage products of complement factor C3 and C5 also led to the appearance of PGI2 releasing principles with a molecular weight of about 7000-11000. The stimulation of PGI2 biosynthesis was explained by enhanced release of AA, and not due to increased activity of cyclo-oxygenase or PGI2 synthetase. Our results suggest that complement-derived products may promote the supply of prostaglandins at the site of inflammation.     

Influence of vasoconstriction, temperature, and flow on the kinetics of serotonin uptake in rabbit lungs

In the process of estimating the kinetic parameters of the pulmonary endothelial serotonin (5-HT) uptake, it is critically important to distinguish the effects of hemodynamic changes from endothelial injury. Therefore, the effects of changes in flow rate (1.7-5.0 ml/s), hemodynamics (vasoconstriction by norepinephrine), and temperature (39 vs. 33 degrees C) were investigated in isolated rabbit lungs. Indicator-dilution data were expressed in terms of the Michaelis-Menten equation for the two 5-HT uptake pathways in the preparation. The maximum uptake velocity (Vmax1) and the 5-HT concentration at half-maximum velocity (Km1) of the first pathway as well as the first-order constant (Vmax2/Km2) of the linear part of the second pathway were determined. Neither vasoconstriction nor flow variations had any effect on Km1, whereas increasing the flow rate caused extensive recruitment, with a concomitant increase in Vmax1 and Vmax2/Km2. Furthermore, all the kinetic parameters were significantly decreased at the lower temperature. We conclude that Km1 is independent of organ hemodynamics (vasoconstriction and flow) but susceptible to changes in 5-HT uptake capacity caused by a change in temperature. Vmax1 and Vmax2/Km2 respond to alterations in 5-HT uptake capacity and perfused organ volume. These are prerequisites to apply kinetic modeling as a method for the investigation of pulmonary endothelial function and integrity.     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60°C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 μm) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-μl samples) and 1 μg/ml in saliva (using 250-μl samples) and the method is linear up to 100 μg/ml in plasma and saliva. At a concentration of 5 μg/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 μg/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.     

Nitric oxide function in atherosclerosis

Atherosclerosis is a chronic inflammatory process in the intima of conduit arteries, which disturbs the endothelium-dependent regulation of the vascular tone by the labile liposoluble radical nitric oxide (NO) formed by the constitutive endothelial nitric oxide synthase (eNOS). This defect predisposes to coronary vasospasm and cardiac ischaemia, with anginal pain as the typical clinical manifestation. It is now appreciated that endothelial dysfunction is an early event in atherogenesis and that it may also involve the microcirculation, in which atherosclerotic lesions do not develop. On the other hand, the inflammatory environment in atherosclerotic plaques may result in the expression of the inducible NO synthase (iNOS) isozyme. Whether the dysfunction in endothelial NO production is causal to, or the result of, atherosclerotic lesion formation is still highly debated. Most evidence supports the hypothesis that constitutive endothelial NO release protects against atherogenesis e.g. by preventing smooth muscle cell proliferation and leukocyte adhesion. Nitric oxide generated by the inducible isozyme may be beneficial by replacing the failing endothelial production but excessive release may damage the vascular wall cells, especially in combination with reactive oxygen intermediates.     

Immunohistochemical analysis of macroautophagy

Transmission electron microscopy (TEM) is an indispensable standard method to monitor macroautophagy in tissue samples. Because TEM is time consuming and not suitable for daily routine, many groups try to identify macroautophagy in tissue by conventional immunohistochemistry. The aim of the present study was to evaluate whether immunohistochemical assessment of macroautophagy-related marker proteins such as LC3, ATG5, CTSD/cathepsin D, BECN1/Beclin 1 or SQSTM1/p62 is feasible and autophagy-specific. For this purpose, livers from starved mice were used as a model because hepatocytes are highly sensitive to autophagy induction. ATG7-deficient mouse livers served as negative control. Our findings indicate that unambiguous immunodetection of LC3 in paraffin-embedded tissue specimens was hampered due to low in situ levels of this protein. Maximum sensitivity could only be obtained using high-quality, isoform-specific antibodies, such as antibody 5F10, in combination with Envision+ signal amplification. Moreover, LC3 stains were optimal in neutral-buffered formalin-fixed tissue, immersed in citrate buffer during antigen retrieval. However, even when using this methodology, LC3 monitoring required overexpression of the protein, e.g., in GFP-LC3 transgenic mice. This was not only the case for the liver but also for other organs including heart, skeletal muscle, kidney and gut. Immunohistochemical detection of the autophagy-related proteins ATG5, CTSD or BECN1 is not recommendable for monitoring autophagy, due to lack of differential gene expression or doubtful specificity. SQSTM1 accumulated in autophagy-deficient liver, thus it is not a useful marker for tissue with autophagic activity. We conclude that TEM remains an indispensable technique for in situ evaluation of macroautophagy, particularly in clinical samples for which genetic manipulation or other in vitro techniques are not feasible.