Biomedical subjects
Publications and source records attributed to T Wehr.
Anthranilic acid derivatives bearing basic amines were prepared and evaluated in vitro and in vivo as inhibitors of MMP-1, MMP-9, MMP-13, and TACE. Piperazine 4u has been identified as a potent, selective, orally active inhibitor of MMP-9 and MMP-13.
A novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, MMP-13, and TACE was prepared and evaluated. Selective inhibitors of MMP-9, MMP-13, and TACE were identified, including the potent, orally active MMP-13 inhibitor 4p.
To investigate the influence of taurine and caffeine containing drinks and physical stress on the cortical movement-preparation, the readiness potentials or "Bereitschaftspotentiale" (BPs), preceding voluntary self-placed pedalling movements, were examined after different states of exhaustion on an ergometer. 15 (13 right-handed) healthy men, aged between 22-30, participated in a randomised, cross over, double-blind, placebo controlled study. BPs were averaged out of artefact free EEG-segments from more than 90 triggered events, measured at 17 electrodes of the 10:20 system. With increasing effort the BPs were enlarged differently depending on the drink consumed. In placebo trials after exhaustive exercise premovement negative potential curves could be seen even in frontal areas. With caffeine the BPs increased after lower workload, achieving a level, which was reached in the placebo trials only after submaximal physical activation. Furthermore a significant shortening of premovement-brain-potentials in frontal and parietal regions could be seen in the caffeine trials at rest. Taurine admixture seems to inhibit this effects.
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Capillary isoelectric focusing (CIEF) provides excellent resolution of proteins with the advantage that separations are carried out in a capillary format with on-tube detection and automated analysis. Recent advances in the technique provide improved resolution, reproducibility and reliability. This review summarizes improvements in sample preparation, capillary selection, and focusing and zone mobilization procedures which have helped CIEF become a more robust analytical method. New applications are reviewed, in particular the use of CIEF in analysis of biopharmaceutical products.
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Capillary isoelectric focusing is a useful analytical technique for characterization of protein mixtures and determination of protein isoelectric points. It is particularly useful in separation of protein glycoforms (Fig. 5), characterizing protein microheterogeneity (Fig. 6), and resolution of charge variants (Fig. 7). The capillary focusing process is analogous to conventional isoelectric focusing in gels, while the requirement for zone mobilization is unique to the capillary format with on-tube detection. A variety of mobilization methods have been described, and the selection of the mobilization method for a particular application depends on the capillary type, the instrument configuration, and the type of proteins to be analyzed. Capillary IEF is generally successful for proteins with a molecular weight up to about 150,000 that exhibit good solubility in aqueous buffers, but may be unsatisfactory for large or hydrophobic proteins. Because of precipitation and variation in mobilization efficiencies, use of internal standards is recommended in most applications. Capillary IEF can be compared to conventional gel IEF in terms of sample throughput and sensitivity. Conventional gels require approximately 4-6 hr to cast, run, and stain the gel, depending on whether silver or Coomassie staining is used. A typical gel contains 10 sample lanes, yielding a throughput of 25-35 min/sample. Capillary IEF separations (including focusing and mobilization) are typically 15-20 min. The mass sensitivity of conventional gel IEF is 36-47 ng for Coomassie staining and 0.5-1.2 ng for silver staining. In capillary IEF, sensitivity will depend on the volume of sample injected; assuming a capillary with a volume of 100 nl is completely filled with sample prior to focusing, the limit of detection will be approximately 1 microgram/ml or 0.1 ng injected. Thus capillary IEF compares favorably with conventional gel IEF in terms of detectivity and analysis time, and has the additional benefit of complete automation of the process including separation and data reduction.
Capillary electrophoresis was evaluated for separation of hemoglobin species associated with alpha-thalassemias, and for identification of hemoglobin variants commonly found in the same human populations. Separation of hemoglobins was achieved using capillary isoelectric focusing with chemical mobilization; visible-wavelength absorbance detection was used to identify hemoglobins against a background of nonheme-containing proteins. This technique could easily differentiate hemoglobins Bart's and H (associated with alpha-thalassemias) from hemoglobin variants. Analysis of globin chains derived from intact hemoglobins was performed by free zone capillary electrophoresis under denaturing conditions. This technique was useful for distinguishing Hb Bart's and Hb H, and for confirming the identity of hemoglobin variants.
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Capillary isoelectric focusing (cIEF) and free zone capillary electrophoresis were evaluated for separation of native hemoglobins and globin chains. High-resolution separations of adult human hemoglobin A, fetal human hemoglobin F, and hemoglobin variants S and C were obtained using cIEF with cathodic mobilization. Absorbance detection in the UV and visible regions were compared, and on-line fast UV or visible-wavelength scanning detection was used to obtain spectral information on separated components. Globin chain analysis was performed on the same hemoglobin species by free zone capillary electrophoresis following precipitation of the protein with acidic acetone. Free zone separations were carried out at low pH in the presence of 7 M urea.
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Successful separations of proteins by capillary electrophoresis in uncoated fused-silica capillaries is limited by adsorption and variable rates of electroendosmosis, which can compromise quantitative accuracy and precision. Operation at extremes of pH to minimize these problems is useful in special cases but is not a general strategy for protein separations. Three alternative strategies are described: use of capillaries coated with a linear hydrophilic polymer, the use of acidic solutions to wash the capillary between runs, and the incorporation of additives into the electrophoresis buffer to minimize adsorption during analysis. Applications of these techniques to protein samples is demonstrated.
High performance isoelectric focusing in capillaries provides rapid, high resolution separation of proteins based on their isoelectric points. Results can be obtained in a matter of minutes with little or no sample preparation. The technique requires the use of coated capillaries to reduce electroendosmosis so that stable, focused zones can be attained. Once focused, protein zones may be mobilized by the addition of salt to the catholyte or anolyte buffer. On-tube UV monitoring enables direct detection of sample components during mobilization with mass sensitivity equal to that of silver staining. The linear relationship between mobilization time and isoelectric point allows the technique to be used for estimation of protein pI. Demonstrated applications include separation of proteins in biological fluids with possible clinical applications, and characterization of biopharmaceutical proteins and monoclonal antibodies.
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We have examined the accumulation of polyphosphorylated nucleotides in Bacillus subtilis in relation to the function of the rel gene. Our results are as follows. (i) During inhibition of isoleucine activation by O-methylthreonine, wildtype B. subtilis cells accumulate unusual nucleotides with the chromatographic and chemical properties of pppApp, ppApp, pppGpp, ppGpp, pGpp, and ppGp. (ii) During the carbon source downshift elicited by inhibiting glucose uptake, we observed accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iii) At the end of long phase in sporulation medium, we observed a small transient accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iv) We were unable to detect a nucleotide with chromatographic behavior expected for pppAppp under any conditions. (v) The rel mutant of Swanton and Edlin (Biochem. Biophys. Res. Commun. 46-583-588, 1972) did not accumulate any of these polyphosphorylated nucleotides under any of the conditions examined. (vi) the rel mutant is unimpaired in sporulation. We conclude that one or more of the nucleotides we have detected may be involved in controlling the specificity of transcription during the stringent response, but none of them are required for sporogenesis.
Normal subjects were admitted to a psychiatric unit devoted to the study and treatment of affective illness. Initially the authors were concerned about the difficulties volunteers might have living with psychiatric patients. However, the volunteers adjusted with relative ease, while the patients' depressive symptoms were exacerbated. Their confrontation with the volunteers "normality" triggered an acute awareness of their underlying sense of failure. This response is analogous to depressed patients' reactions before discharge, when they struggle not only with their special vulnerability to separation and loss but with inevitable challenges to their fragile self-esteem during reintegration into the outside world. The presence of normal volunteers highlighted these issues and led to increased therapeutic work and considerable resolution.
The authors studied the effect of a catecholamine-controlled diet on the urinary level of 3-methoxy-4-hydroxyphenylglycol (MHPG) of 6 depressed patients and 6 normal volunteers. The normal subjects showed no change in MHPG levels on or off the diet; the depressed patients showed a significant increase in MHPG while off the diet.
The authors studied pituitary thyrotropin, i.e., thyroid-stimulating hormone (TSH), response to thyrotropin-releasing hormone (TRH) in patients with primary affective disorder. There were no overall differences between either depressed or manic patients and normal controls; however, the TSH response was significantly lower in the unipolar depressed patients than in either bipolar depressed patients or normal subjects. Bipolar patients in the manic phase tended to have a lower response than bipolar depressed patients. In the unipolar group, the TSH response showed a significant negative correlation with the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the CSF. These neuroendocrine responses may constitute markers of specific monoamine dysfunction in subgroups of patients with affective illness.