Influence of food intake on the absorption of diftalone in man

An average 2.2-fold increase in the peak plasma concentrations of the non-steroidal anti-inflammatory agent diftalone in the presence of food was observed in three studies carried out with healthy volunteers who received an oral dose of 0.75 g (6 subjects, study 1), 0.25 g (10 subjects, study II) and 0.5 g (6 subjects, study V) of the compound at 9:00 a.m. both in fasting conditions and after a meal. The effect does not depend on the unusual time (8:00 a.m., selected for experimental needs) at which the subjects were given the meal. In fact, a 2.5-fold increase in plasma concentrations was observed when an oral dose of 0.75 g of diftalone was administered to 2 subjects (study II) both at 8:00 a.m. in fasting conditions and at 1:00 p.m. after a meal. A similar enhancement in the absorption of diftalone was observed when 5 healthy volunteers (study VI) received an oral dose of 0.5 g of the compound both as plain capsules and as capsules containing dry ox bile. However, the absorption of diftalone was not modified when the compound was administered orally as an aqueous suspension or in tensioactive vehicles, or after 20 mg of metoclopramide (study II). Also, the results of a study (IV) on 2 subjects partly deprived of bile after surgery, showed that diftalone does not undergo enterohepatic circulation. The hypothesis that the increase in diftalone absorption is mainly due to bile flow following food intake is supported by all the above experimental results.     

Ocular squamous cell carcinoma in Simmental cattle in Zimbabwe

In Zimbabwe, ocular squamous cell carcinoma (OSCC) was frequently observed in 5 breeding herds of Simmental cattle, a Bos taurus breed originating from Switzerland. In these herds, initial signs of OSCC were already noticeable in cattle about 3 years old. Gradually, OSCC prevalence increased, and 36 to 53% of cattle over 7 years old had 1 or more tumors. More tumors developed in Simmental cattle with periorbital white skin than in cattle with periorbital pigmented skin. Other breeds of cattle (eg, Friesian) also are partly white-faced and live in Zimbabwe in a comparable environment; yet, OSCC prevalence was lower in those breeds.     

Semi-automated detection of the factor V mutation by allele specific amplification and capillary electrophoresis

Recently a point mutation (G1691A) in the coagulation factor V gene was shown to cause resistance for cleavage by activated protein C. The mutation is associated with an increased thrombotic risk and thus-far the most common genetic cause of thrombophilia. Current techniques to investigate the single base pair mutation at the DNA level use an assay based upon the polymerase chain reaction followed by restriction enzyme digestion or Southern blotting and allele specific probing. The method we describe here consists of a single PCR in which two specially designed allele specific primers and two consensus primers were used in one reaction to distinguish between homozygous normal, heterozygous and homozygous mutant individuals. Amplification products were analysed using Capillary Electrophoresis and on line UV monitoring. The Allele Specific Amplification Protocol and subsequent CE analysis (ASAP-CE) is a convenient, fast, automated and highly reproducible method that can be used in a routine laboratory setting.     

Involvement of integrins with adhesion-promoting, heparin-binding peptides of type IV collagen in cultured human corneal epithelial cells

PURPOSE: The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential role of integrins in cell adhesion to the above proteins and peptides. RESULTS: FACS analysis identified the beta 1, beta 4, alpha 2, alpha 3, alpha 5, alpha 6, and alpha v integrin subunits on human corneal epithelial cells grown as primary cultures. The anti-beta 1 antibody inhibited cell adhesion to heparin-binding peptides of type IV collagen, type IV collagen, fibronectin, and GRGDSP: Antibodies to the alpha 2 integrin subunit inhibited cell adhesion to the heparin-binding peptides of type IV collagen and slightly inhibited cell adhesion to intact type IV. Antibodies to the alpha 3 integrin subunit exhibited a somewhat lesser effect compared to the anti-alpha 2 integrin antibody. CONCLUSIONS: These data show that the alpha 2 beta 1 integrin of human corneal epithelial cells recognize heparin-binding peptide sequences derived from human type IV collagen. It seems likely that these sequences play an important role in integrin-mediated corneal epithelial cell adhesion. In addition, the alpha 3 beta 1 integrin may mediate similar events.     

Imaging biomolecule arrays by atomic force microscopy

We describe here a method for constructing ordered molecular arrays and for detecting binding of biomolecules to these arrays using atomic force microscopy (AFM). These arrays simplify the discrimination of surface-bound biomolecules through the spatial control of ligand presentation. First, photolithography is used to spatially direct the synthesis of a matrix of biological ligands. A high-affinity binding partner is then applied to the matrix, which binds at locations defined by the ligand array. AFM is then used to detect the presence and organization of the high-affinity binding partner. Streptavidin-biotin arrays of 100 x 100 microns and 8 x 8 microns elements were fabricated by this method. Contact and noncontact AFM images reveal a dense lawn of streptavidin specific to the regions of biotin derivatization. These protein regions are characterized by a height profile of approximately 40 A over the base substrate with a 350-nm edge corresponding to the diffraction zone of the photolithography. High resolution scans reveal a granular topography dominated by 300 A diameter features. The ligand-bound protein can then be etched from the substrate using the AFM tip, leaving an 8 A shelf that probably corresponds to the underlying biotin layer.     

Nonpeptide cyclic cyanoguanidines as HIV-1 protease inhibitors: synthesis, structure-activity relationships, and X-ray crystal structure studies

Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.     

Erythropoietin therapy in the perioperative setting

Recombinant human erythropoietin has been approved for use in patients undergoing autologous donation in Japan, Europe, and Canada since 1993, 1994, and 1996, respectively, and for perisurgical adjuvant therapy without autologous donation in Canada and the United States since 1996. Early clinical trials of erythropoietin therapy in the setting of autologous donation have provided important information regarding clinical safety, erythropoietin dose, and erythropoietic response. Later trials of perisurgical erythropoietin therapy without autologous donation provided data on efficacy (reduced allogeneic blood exposure) that led to approval of erythropoietin in patients undergoing surgery. However, the erythropoietin doses (300 U/kg subcutaneous x14 days) used in these trials, and their subsequent inclusion in labeling for the use of this product, are costly and tedious to administer. A recent study reported that a weekly regimen of erythropoietin (600 U/kg) for 4 weeks is less costly but just as effective at reducing allogeneic blood exposure in elective orthopaedic surgery. The most cost effective regimen that has been shown to minimize allogeneic exposure is preoperative erythropoietin therapy (600 U/kg subcutaneous weekly x2 and 300 U/kg subcutaneous on day of surgery) coupled with acute normovolemic hemodilution in patients undergoing radical retropubic prostatectomy. A similar regimen of erythropoietin therapy in patients undergoing coronary artery bypass grafting (2500 U/kg subcutaneous in divided doses for 2 weeks preoperatively) coupled with hemodilution also was effective. Low dose erythropoietin therapy coupled with acute normovolemic hemodilution ultimately may be shown to be cost equivalent to the predonation of three autologous blood units before elective surgery.     

A cis-acting element that directs the activity of the murine methylation modifier locus Ssm1

Silencing of chromosomal domains has been described in diverse systems such as position effect variegation in insects, silencing near yeast telomeres, and mammalian X chromosome inactivation. In mammals, silencing is associated with methylation at CpG dinucleotides, but little is known about how methylation patterns are established or altered during development. We previously described a strain-specific modifier locus, Ssm1, that controls the methylation of a complex transgene. In this study we address the questions of the nature of Ssm1's targets and whether its effect extends into adjacent sequences. By examining the inheritance of methylation patterns in a series of mice harboring deletion derivatives of the original transgene, we have identified a discrete segment, derived from the gpt gene of Escherichia coli, that is a major determinant for Ssm1-mediated methylation. Methylation analysis of sequences adjacent to a transgenic target indicates that the influence of this modifier extends into the surrounding chromosome in a strain-dependent fashion. Implications for the mechanism of Ssm1 action are discussed.     

High-pressure liquid chromatographic determination of acetaminophen in biological fluids

A method for the rapid estimation of free acetaminophen in biological fluids is described. The assay involves either extraction and high-pressure liquid chromatographic analysis on a 10-mum particle-size silica gel column, using a mobile phase of 10% chloroform in tetrahydrofuran. The procedure was used to determine acetaminophen levels in urine from two healthy volunteers who ingested 650 mg of 14C-acetaminophen (20 muCi), and the accuracy of the method was compared with the carbon-14 determination. The limit of detectability for acetaminophen is 1 mug/ml.