Rate of disappearance of glycerol from plasma of fetal and newborn sheep

The disappearance of glycerol from plasma was studied after a single intravenous injection to estimate its volume of distribution (Vdist), plasma clearance rate, and rate constant for irreversible loss (kd). Studies were repeated before and after birth of the lamb to test whether loss of the placenta could account for rapidly increasing plasma concentrations in the newborn. The disappearance of glycerol was closely described by a double-exponential model in each instance. In fetal sheep Vdist averaged 0.41 +/- 0.15 (SD) 1/kg fetal wt (n = 15). This volume decreased to 0.33 +/- 0.11 l/kg (n = 8) soon after functionally removing the placenta (by snaring the umbilical cord and maintaining the fetus with intrauterine ventilation), but the change was not significant. In newborn lambs 1-3 days of age, Vdist averaged 0.45 +/- 0.11 l/kg (n = 5, NS). Plasma clearance rate also did not change significantly, averaging 7.9 +/- 2.9, 7.9 +/- 3.8, and 9.0 +/- 5.9 ml.min-1.kg-1 in the fetus, after simulated birth, and in the newborn lamb, respectively, kd also was not altered measurably and averaged 0.020 +/- 0.006, 0.024 +/- 0.007, and 0.019 +/- 0.007 min-1 during the same time periods. Similar results were obtained by using three widely different amounts of infused glycerol. The results indicate that removal of glycerol does not depend on placental function to an appreciable extent. It is concluded that plasma glycerol concentration reflects principally glycerol turnover and, hence, lipolysis before and after birth.     

Synthesis of functionalized 2-aryl-5-nitro-1H-indoles and their activity as bacterial NorA efflux pump inhibitors

In order to develop structure-activity relationships and to provide access to antibacterial agents for dual action studies, a variety of aryl group-substituted 2-aryl-5-nitro-1H-indoles were synthesized and the activity of the compounds assessed as inhibitors of the NorA multidrug resistance pump in the bacterium Staphylococcus aureus. The NorA protein from the major facilitator superfamily of efflux pumps confers resistance to a variety of structurally dissimilar antimicrobials such as norfloxacin, ethidium bromide, berberine and acriflavin. The compound [4-benzyloxy-2-(5-nitro-1H-2-yl)-phenyl]-methanol was the most potent pump inhibitor.     

Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation

The objective was to evaluate apoptotic markers in ejaculated equine spermatozoa after separation by density-gradient centrifugation and after cryopreservation. Subpopulations of percoll-separated equine spermatozoa differed (P < 0.05) in the percentage of live, caspase-activated spermatozoa (2.9 ± 0.7% vs 14.2 ± 6.4%; mean ± S.E.M.), low mitochondrial membrane potential (MMP; 6.8 ± 1.1 vs 23.8 ± 3.7), altered plasma membrane permeability (1.3 ± 0.2 vs 3.0 ± 0.5), DNA fragmentation (2.0 ± 1.3 vs 14.3 ± 3.6), total motility (81.8 ± 3.3 vs 35.1 ± 5.4), and progressive motility (66.3 ± 4.3 vs 24.1 ± 4.5) for high-density versus low-density subpopulations, respectively. Phosphatidylserine externalization did not differ (P = 0.67) between the high- and low-density subpopulations (2.6 ± 0.7 vs 3.1 ± 0.9). After cryopreservation, equine spermatozoa differed (P < 0.01) in the percentage of active caspases (19.1 ± 1.6 vs 52.1 ± 2.8), low MMP (18.2 ± 2.5 vs 48.7 ± 2.6), altered plasma membrane permeability (6.8 ± 1.7 vs 17.6 ± 2.0), total motility (75.5 ± 2.4 vs 45.2 ± 5.6), and progressive motility (53.9 ± 3.1 vs 28.3 ± 4.5) for pre-freeze versus cryopreserved spermatozoa. There was no difference (P = 0.21) in percentage of DNA fragmented cells before (5.5 ± 1.2) versus after cryopreservation (6.6 ± 1.1). We concluded that apoptotic-like changes were detectable in ejaculated equine spermatozoa and were more prevalent after cryopreservation.     

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE)

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.     

Molecular cloning and characterization of ARO7-OSM2, a single yeast gene necessary for chorismate mutase activity and growth in hypertonic medium

Summary The chorismate mutase structural gene, ARO7, which is necessary for both phenylalanine and tyrosine biosynthesis was cloned by complementation in yeast. Genetic analysis showed that ARO7 was identical to a gene necessary for growth in hypertonic medium, OSM2, which mapped nearby. After restriction mapping and subcloning of the plasmid, the cloned gene was used to detect mRNA levels in several growth conditions. Enzyme activities were measured in various genotypes. At our level of detection ARO7-OSM2 is a low level constitutively expressed gene.     

Molecular modeling of cardiac glycoside binding by the human sequence monoclonal antibody 1B3

The amino acid sequences of the heavy- and light-chain variable regions of the high-affinity human sequence antidigoxin monoclonal antibody 1B3 (mAb 1B3) were determined, and a structural model for the mAb's variable region was developed by homology modeling techniques. The structural model provided the basis for computationally docking digoxin and eight related cardiac glycosides into the putative binding site of mAb 1B3. Analysis of the consensus binding mode obtained for digoxin showed that the cardenolide moiety of digoxin is deeply embedded in a predominantly hydrophobic, narrow cavity, whereas the terminal, gamma-carbohydrate group is solvent-exposed. The docking results indicated that the primary driving forces for digoxin binding by mAb 1B3 are hydrophobic interactions with the digoxin steroid ring system and hydrogen bonds with the digitoxose groups. The binding model accounts for the experimentally observed variations in mAb 1B3 binding affinity for various structural analogs of digoxin used previously to develop a 3D structure-activity relationship model of drug binding (Farr CD, Tabet MR, Ball WJ Jr, Fishwild DM, Wang X, Nair AC, Welsh WJ. Three-dimensional quantitative structure-activity relationship analysis of ligand binding to human sequence antidigoxin monoclonal antibodies using comparative molecular field analysis. J Med Chem 2002;45:3257-3270). In particular, the hydrogen bond pattern is consistent with the unique sensitivity of mAb 1B3's binding affinity to the number of sugar residues present in a cardiac glycoside. The hydrophobic environment about the steroid moiety of digoxin is compatible with the mAb's reduced affinity for ligands that possess hydrophilic hydroxyl and acetyl group modifications in this region. The model also indicated that most of the amino acid residues in contact with the ligand reside in or about the three complementarity determining regions (CDRs) of the heavy chain and the third CDR of the light chain. A comparison of the 1B3 binding model with the crystal structures of two murine antidigoxin mAbs revealed similar binding patterns used by the three mAbs, such as a high frequency of occurrence of aromatic, hydrophobic residues in the CDRs and a dominant role of the heavy chain CDR3 in antigen binding.     

Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

The sterol carrier protein-2 amino terminus: a membrane interaction domain.

Sterol carrier protein-2 (SCP2) is a small, 123 amino acid, protein postulated to play a role in intracellular transport and metabolism of lipids such as cholesterol, phospholipids, and branched chain fatty acids. While it is thought that interaction of SCP2 with membranes is necessary for lipid transfer, evidence for this possibility and identification of a membrane interaction domain within SCP2 has remained elusive. As shown herein with circular dichroism and a direct binding assay, SCP2 bound to small unilamellar vesicle (SUV) membranes to undergo significant alteration in secondary structure. The SCP2 amphipathic N-terminal 32 amino acids, comprised of two alpha-helical segments, were postulated to represent a putative phospholipid interaction site. This hypothesis was tested with a series of SCP2 N-terminal peptides, circular dichroism, and direct binding studies. The SCP2 N-terminal peptide (1-32)SCP2, primarily random coil in aqueous buffer, adopted alpha-helical structure upon interaction with membranes. The induction of alpha-helical structure in the peptide was maximal when the membranes contained a high mole percent of negatively charged phospholipid and of cholesterol. While deletion of the second alpha-helical segment within this peptide had no effect on formation of the first alpha-helix, it significantly weakened the peptide interaction with membranes. Substitution of Leu(20) with Glu(20) in the N-terminal peptide disrupted the alpha-helix structure and greatly weakened the peptide interaction with membranes. Finally, deletion of the first nine nonhelical amino acids had no effect either on formation of alpha-helix or on peptide binding to membranes. N-Terminal peptide (1-32)SCP2 competed with SCP2 for binding to SUV. These data were consistent with the N-terminus of SCP2 providing a membrane interaction domain that preferentially bound to membranes rich in anionic phospholipid and cholesterol.     

Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70 degrees C, respectively. The activity was strongly enhanced in the presence of Cu(2+), Mn(2+), and Mg(2+) and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.