Simultaneous determination of intact cisplatin and its metabolite monohydrated cisplatin in human plasma

Cisplatin is a cytotoxic platinum compound, used in the treatment of several solid tumors. Cisplatin and to a greater extent its hydrolysis product monohydrated cisplatin are responsible for side-effects like nephrotoxicity. A sensitive, accurate and precise method was developed to simultaneously determine cisplatin and monohydrated cisplatin in plasma. The compounds were separated by high-performance liquid chromatography and quantified by off-line furnace atomic absorption spectrophotometry. The linear ranges for cisplatin and monohydrated cisplatin in deproteinized plasma were 60-600 and 87.5-700 nM, respectively. From plasma, the mean recovery of cisplatin was 83.2% and that of monohydrated cisplatin 79.1%. The lower limits of quantification of cisplatin and monohydrated cisplatin in deproteinized plasma were 60 and 87.5 nM, respectively. Over the whole calibration range, the within- and between-day accuracy of intact cisplatin ranged from 100.7 to 111.4 and 94.8-102.0%, respectively. The within- and between-day accuracy of monohydrated cisplatin ranged from 107.1 to 113.3 and 101.4-104.9%, respectively. The within-day and between-day precision of cisplatin ranged from 3.4 to 11.5 and 7.3-10.3%, respectively. For monohydrated cisplatin, the within-day and between-day precision ranged from 3.7 to 6.2 and 5.6-7.9%, respectively. Currently, the developed assay has been implemented in pharmacokinetic studies of patients treated with cisplatin alone or in combination with other drugs.     

Immunohistochemical distribution of galectin-1, galectin-3, and olfactory marker protein in human olfactory epithelium

The expression pattern of galectin-1 and galectin-3 in the human olfactory epithelium was investigated in relation to olfactory marker protein (OMP) using confocal laser immunofluorescence in human specimens and postmortem biopsies. OMP expression was found in olfactory receptor neurons (ORNs) in the olfactory mucosa and in fibers of the olfactory nerve crossing the submucous connective tissue. Galectin-1 was expressed in both the connective tissue of the nasal cavity and in the basal layer of the olfactory epithelium. In contrast, galectin-3 expression was limited to cells of the upper one-third of the olfactory epithelium. Expression of galectin-3 occurred in a subset of OMP-positive cells. However, between areas of galectin-1 and galectin-3 expression in the lower and upper portion of the epithelium, OMP-positive ORNs did not stain for both galectins. Considering the potential role of galectin-1 and galectin-3 in cell differentiation and maturation, the differential localization of galectins in the olfactory epithelium appears to be consistent with a significant role of these molecules in the physiological turnover of ORNs. Accepted: 20 December 1999     

The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit

We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, interactions with the 30S subunit suggest a mechanism for its effects on ribosome function.     

Empirical calculation of the relative free energies of peptide binding to the molecular chaperone DnaK

We describe a methodology to calculate the relative free energies of protein-peptide complex formation. The interaction energy was decomposed into nonpolar, electrostatic and entropic contributions. A free energy-surface area relationship served to calculate the nonpolar free energy term. The electrostatic free energy was calculated with the finite difference Poisson-Boltzmann method and the entropic contribution was estimated from the loss in the conformational entropy of the peptide side chains. We applied this methodology to a series of DnaK*peptide complexes. On the basis of the single known crystal structure of the peptide-binding domain of DnaK with a bound heptapeptide, we modeled ten other DnaK*heptapeptide complexes with experimentally measured K(d) values from 0.06 microM to 11 microM, using molecular dynamics to refine the structures of the complexes. Molecular dynamic trajectories, after equilibration, were used for calculating the energies with greater accuracy. The calculated relative binding free energies were compared with the experimentally determined free energies. Linear scaling of the calculated terms was applied to fit them to the experimental values. The calculated binding free energies were between -7.1 kcal/mol and - 9.4 kcal/mol with a correlation coefficient of 0.86. The calculated nonpolar contributions are mainly due to the central hydrophobic binding pocket of DnaK for three amino acid residues. Negative electrostatic fields generated by the protein increase the binding affinity for basic residues flanking the hydrophobic core of the peptide ligand. Analysis of the individual energy contributions indicated that the nonpolar contributions are predominant compared to the other energy terms even for peptides with low affinity and that inclusion of the change in conformational entropy of the peptide side chains does not improve the discriminative power of the calculation. The method seems to be useful for predicting relative binding energies of peptide ligands of DnaK and might be applicable to other protein-peptide systems, particularly if only the structure of one protein-ligand complex is available.     

Sequential analysis of cell differentials and IFN-gamma production of splenocytes from mice infected with Toxoplasma gondii

To assess the relationship between the changes of cellular components and the production of Th1 cytokine in the immune tissue, inbred C57BL/6 mice were orally infected with 40 cysts of 76K strain of Toxoplasma gondii. The sequential change of cell differentials and IFN-gamma production of splenocytes were analyzed by Diff-Quik stain and RT-PCR. There were no significant proportional changes of cellular components of splenocytes until day 4 postinfection (PI) as compared to those of day 0, and the relative percentage of macrophages and neutrophils/eosinophils increased significantly (p < 0.01) thereafter. The expression of IFN-gamma mRNA of CD3- cells was observed from day 1 PI at a low level. However, IFN-gamma production of CD3+ cells increased significantly from day 4 PI (p < 0.01) which progressively increased thereafter. These findings provide the relative percentages of granulocytes and macrophages were increased in conjunction with increase of total number of splenocytes after oral infection with T. gondii in the susceptible murine hosts, and lymphocytes were the major cellular components and the important source of IFN-gamma.     

Immunohistochemical distribution of connexin 43 in the cartilage of rats and mice

Using fluorescence immunohistochemistry, the distribution of connexin 43 was examined in hyaline cartilage and in the perichondrium of mouse and rat knee joints. In addition, rat chondrocytes were shown to be coupled in dye transfer studies with Lucifer Yellow. Connexin 43 was detected between chondrocytes in the outer layer of knee joint cartilage, between chondrocytes of the growth plate and between fibrocartilage-like cells at tendon and ligament insertions and in the tendons and ligaments proper. However, in the hyaline cartilage of the hind limbs of mature rats, the degree of connexin 43 immunoreactivity was diminished. These data suggest a possible involvement of connexins in cartilage development. © 1998 Chapman & Hall     

Bovine Milk Proteome in the First 9 Days: Protein Interactions in Maturation of the Immune and Digestive System of the Newborn

In order to better understand the milk proteome and its changes from colostrum to mature milk, samples taken at seven time points in the first 9 days from 4 individual cows were analyzed using proteomic techniques. Both the similarity in changes from day 0 to day 9 in the quantitative milk proteome, and the differences in specific protein abundance, were observed among four cows. One third of the quantified proteins showed a significant decrease in concentration over the first 9 days after calving, especially in the immune proteins (as much as 40 fold). Three relative high abundant enzymes (XDH, LPL, and RNASE1) and cell division and proliferation protein (CREG1) may be involved in the maturation of the gastro-intestinal tract. In addition, high correlations between proteins involved in complement and blood coagulation cascades illustrates the complex nature of biological interrelationships between milk proteins. The linear decrease of protease inhibitors and proteins involved in innate and adaptive immune system implies a protective role for protease inhibitor against degradation. In conclusion, the results found in this study not only improve our understanding of the role of colostrum in both host defense and development of the newborn calf but also provides guidance for the improvement of infant formula through better understanding of the complex interactions between milk proteins.     

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.