Use of lectins to characterize plasma membrane preparations from boar spermatozoa: a novel technique for monitoring membrane purity and quantity

The object of this study was to develop a method to quantify the amount of outer acrosomal membrane material in isolated plasma membranes from boar sperm cells. The cells were fractionated by nitrogen cavitation, and plasma membranes were isolated by subsequent differential centrifugation steps. Marker enzyme measurement showed that the plasma membrane isolates were enriched in plasma membrane markers and did not contain nuclei, inner acrosomal membranes, or mitochondria. Since there is no marker enzyme known for the outer acrosomal membrane, lectins were used for the detection of this membrane. The membrane specificity of a number of lectin conjugates was tested with fluorescence microscopy and transmission electron microscopy. Membrane binding of these lectin conjugates was quantified with flow-cytometry and an enzyme-linked lectin binding assay. Wheat germ agglutinin was specific for the plasma membrane while peanut agglutinin was specific for the outer acrosomal membrane. The use of these lectins made it possible for the first time to discriminate between these two membranes. The isolated plasma membrane fraction was enriched more than 10-fold (17-fold after further purification by a sucrose gradient) in plasma membrane material compared to outer acrosomal membrane material. Highly purified sperm plasma membranes should prove to be useful for research on primary sperm-zona interactions.     

Mandibular reconstruction with transforming growth factor-beta1

This is a case report of a 43-year-old woman who received a transplant for end-stage liver disease due to hereditary hemorrhagic telangiectasia and fibropolycystic liver disease. This is an uncommon association of two autosomal-dominant conditions with defined genetic and molecular defects. The liver showed extensive vascular malformations of arteries and veins as well as telangiectasia and fibrosis. In addition, there were cystically dilated ducts containing inspissated bile and extensive von Meyenburg complexes. This case raises interesting questions about the possible relationship of these genes and their gene products, both of which are related to cell-matrix interactions and are strongly associated with blood vessels, one of them being expressed on endothelial cells and the other being developmentally important in blood vessels.     

Antiretroviral therapies

We describe an algorithm which enables us to search the conformational space of the side chains of a protein to identify the global minimum energy combination of side chain conformations as well as all other conformations within a specified energy cutoff of the global energy minimum. The program is used to explore the side chain conformational energy surface of a number of proteins, to investigate how this surface varies with the energy model used to describe the interactions within the system and the rotamer library. Enumeration of the rotamer combinations enables us to directly evaluate the partition function, and thus calculate the side chain contribution to the conformational entropy of the folded protein. An investigation of these conformations and the relationships between them shows that most of the conformations near to the global energy minimum arise from changes in side chain conformations that are essentially independent; very few result from a concerted change in conformation of two or more residues. Some of the limitations of the approach are discussed.     

Structural analysis of substrate binding by the molecular chaperone DnaK

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.     

Microscopic and electrochemical characterization of lead film electrode applied in adsorptive stripping analysis

Lead film electrodes (PbFEs) deposited in situ on glassy carbon or carbon paste supports have recently found application in adsorptive stripping voltammetric determination of inorganic ions and organic substances. In this work, the PbFE, prepared in ammonia buffer solutions, was investigated using scanning electron microscopy, atomic force microscopy and various voltammetric techniques. The microscopic images of the lead films deposited on the glassy carbon substrate showed a considerable variability in microstructure and compactness of the deposited layer depending on the selected experimental conditions, such as the concentration of Pb(II) species, the nucleation and deposition potential, and the time applied. The catalytic adsorptive systems of cobalt and nickel in a solution containing 0.1 ammonia buffer, 2.5 × 10⁻⁵ M nioxime and 0.25 M NaNO₂ were employed to investigate the electrochemical characteristics and utility of the in situ prepared lead films.The optimal parameters, i.e. the lead concentration in the solution, the procedure of film removal, and the time and potential of lead nucleation and film deposition for the adsorptive determination of metal traces, were selected, resulting in the very good reproducibility (RSD = 4.2% for 35 scans) of recorded signals. The voltammetric utility of the lead film electrode was compared to that of glassy carbon, mercury film and bismuth film electrodes, and was subsequently evaluated as superior.     

Long-term inhalable particles and other air pollutants related to mortality in nonsmokers

Long-term ambient concentrations of inhalable particles less than 10 microm in diameter (PM10) (1973- 1992) and other air pollutants-total suspended sulfates, sulfur dioxide, ozone (O3), and nitrogen dioxide-were related to 1977-1992 mortality in a cohort of 6,338 nonsmoking California Seventh-day Adventists. In both sexes, PM10 showed a strong association with mortality for any mention of nonmalignant respiratory disease on the death certificate, adjusting for a wide range of potentially confounding factors, including occupational and indoor sources of air pollutants. The adjusted relative risk (RR) for this cause of death as associated with an interquartile range (IQR) difference of 43 d/yr when PM10 exceeded 100 microg/m3 was 1.18 (95% confidence interval [CI]: 1.02, 1.36). In males, PM10 showed a strong association with lung cancer deaths-RR for an IQR was 2.38 (95% CI: 1.42, 3.97). Ozone showed an even stronger association with lung cancer mortality for males with an RR of 4.19 (95% CI: 1.81, 9.69) for the IQR difference of 551 h/yr when O3 exceeded 100 parts per billion. Sulfur dioxide showed strong associations with lung cancer mortality for both sexes. Other pollutants showed weak or no association with mortality.