Thomas Marshall

Chinese Hamster Mouse Hybrid Cells Segregating Mouse Chromosomes and Isozymes

Hybrid cells are readily formed by fusing clonal Chinese hamster cells to fresh, noncultured, adult mouse spleen cells followed by isolation in selective medium. The vast majority of such hybrids retain Chinese hamster chromosomes and isozymes while segregating mouse chromosomes and isozymes. The growth, plating efficiency, ease of karyology, and rapid segregation of mouse markers allows linkage tests in primary clones. Analysis of 13 isozymes showed 12 to be asyntenic and one pair (PGD-PGM2) to be syntenic. This system will allow extensive somatic cell hybrid gene mapping in the mouse and permit a comparison of human and mouse linkage relationships.

Coomassie blue protein dye-binding assays measure formation of an insoluble protein-dye complex

The solubility of the protein-Coomassie brilliant blue (CBB) complex formed upon Bradford (Anal. Biochem. 72, 248-254, 1976) or Sedmak and Grossberg (Anal. Biochem. 79, 544-552, 1977) protein assay has been investigated by centrifugation or filtration of the assay mix within 10 min of adding dye reagent. The results show complete loss of color yield in the respective supernates and filtrates. This indicates that the protein-CBB complexes are insoluble at the time of absorbance measurement. Protein solubility in the dye reagent may dictate the relative response of the assay to an individual protein and the requirement for macromolecular structure.

Liquid chromatographic assay for cerebrospinal fluid serotonin

A novel liquid chromatographic method was developed for accurate and precise routine quantification of serotonin in human cerebrospinal fluid. Serotonin is derivatized with an amine-specific reagent leaving its hydroxy group free for electrochemical detection and making it lipophilic enough for efficient extraction into organic solvents. The method is reproducible, linear and free of interferences by endogenous amines other than serotonin. The limit of sensitivity is 100 fmol/ml CSF or 20 parts per trillion. Serotonin concentrations in lumbar cerebrospinal fluid from drug free, depressed patients were in the low nanomolar range.

Electrophoresis of serum isoenzymes and proteins following acute myocardial infarction

The clinical significance of the serum enzymes creatine kinase (CK, EC 2.7.3.2), lactate dehydrogenase (LD, EC 1.1.1.27) and aspartate aminotransferase (EC 2.6.1.1), and the isoenzymes CK 1-3 and LD 1-5, in acute myocardial infarction (AMI) is reviewed. Particular attention is given to electrophoretic analysis of the isoenzymes (and the CK isoforms/subforms) following AMI and thrombolytic therapy. Other protein markers for the monitoring of AMI, including myoglobin and muscle contractile proteins, are also discussed and the potential for the detection of new marker proteins using high-resolution two-dimensional electrophoretic methods is demonstrated. Whilst emphasis is placed upon electrophoretic methods the value of complementary immunoassays is acknowledged in order to maintain a balanced perspective.

Electrochemical detection of biogenic amines following acylation by N-hydroxysuccinimide esters

A general methodology for the selective derivatization of amines, to enable quantitation by high pressure liquid chromatography with electrochemical detection, is presented. N‐Hydroxysuccinimide active esters present in large excess are suitably mild acylating agents to derivatize selectively trace quantities of amines for electrochemical detection. The 2 separate problems of extraction yield and detectability can be solved by this derivatization method. Due to its lipophilicity the resulting N‐acylated amine, as demonstrated with serotonin, is extracted efficiently into organic solvents during sample preparation for chromatography. Moreover, the acyl group introduced can be designed to be electroactive, thus extending the procedure to amines not readily oxidized, e.g., histamine and phenylethylamine.

Total Protein Determination in Urine: Aminoglycoside Interference

The aminoglycosides gentamicin and tobramycin interfere in the Dade Behring pyrogallol red-molybdate (PRM) assay used for determination of urinary protein (1). This is an important finding because aminoglycosides are nephrotoxic and accurate determination of urinary protein is necessary to detect renal damage in patients receiving aminoglycosides. We have investigated interference in the PRM assay (2)(3) from other aminoglycosides (dihydrostreptomycin, geneticin, kanamycin, neomycin, paromomycin, and streptomycin) and extended the study to the Coomassie Brilliant Blue (CBB) and the benzethonium chloride (BEC) protein assays, which are also used routinely for urinary protein determination (4)(5)(6). Dihydrostreptomycin (cat. no. D7253), geneticin (cat. no. G5013), gentamicin (cat. no. G3632), kanamycin (cat. no. K4000), neomycin (cat. no. N6386), paromomycin (cat. no. P9297), streptomycin (cat. no. S6501), and tobramycin (cat. no. T1783) were purchased from Sigma-Aldrich Co. Ltd., and solubilized in 0.1 mol/L phosphate buffer (pH …

Liquid chromatographic assay for cerebrospinal fluid normetanephrine

A method for quantitation of normetanephrine in human cerebrospinal fluid is described. An amine-specific reagent, sulfosuccinimidyl propionate, is used to obtain the lipid soluble N-propionyl derivative of normetanephrine, which can be separated and quantitated in presence of other biogenic amines by liquid chromatography with electrochemical detection. The method is reproducible, linear, and precise at the relatively low concentrations of unconjugated normetanephrine occurring in human cerebrospinal fluid. Hospitalized, drug-free, alcoholic patients were found to have cerebrospinal fluid unconjugated normetanephrine concentrations in the 0.5-1.5 nanomolar range. The practical limit of sensitivity for the method is about 0.025 pmole per ml of CSF.

Regulation of expression of type C virion DNA polymerase (reverse transcriptase) in human × mouse and human × rat hybrid cells

Human cells from 13 different individuals were fused to mouse and rat cells producing abundant type C viral particles. The results demonstrate that incorporation of active DNA polymerase (reverse transcriptase) into mature type C particles is suppressed in many of the hybrid clones but not in the parental mouse cell clones. This low particle-associated DNA polymerase phenotype was a heritable trait for over 100 cell generations but reversion to a high particle-associated DNA polymerase phenotype was possible. In contrast, no evidence for suppression of viral p30 antigen was found. These results suggest that human cells contain a factor (s) capable of interfering with the normal maturation of the mouse retrovirus DNA polymerase protein; however, it was not possible to assign this function to any of 20 different human chromosomes tested. It is suggested that these somatic cell hybrids may be useful in examining individual events in retrovirus packaging and release.