Andrew O. Martinez

A rapid and simple MTT-based spectrophotometric assay for determining drug sensitivity in monolayer cultures

A rapid and sensitive spectrophotometric assay for determining viability in monolayer cultured cell lines, with specific applications in normal and drug resistant cell line determinations, is described. The assay involves conversion of the tetrazolium salt MTT by viable proliferating cells to an insoluble product, purple formazan. The chief advantage of this assay is that it requires fewer cells than standard cytotoxicity assays. In addition, it allows for multiple sample concentrations on a single 96-well plate which is then rapidly quantitated using an automated spectrophotometric microplate reader.

A comparison of the suppression of human transferrin synthesis by lead and lipopolysaccharide

Transferrin, as the major iron-transport protein in serum and other body fluids, has a central role in managing iron the body receives. Liver is a major site of transferrin synthesis, and in this study we present evidence that liver synthesis of human transferrin is suppressed by both the toxic metal lead and bacterial lipopolysaccharide, an inducer of the hepatic acute phase response. The responses of intact endogenous transferrin in the human hepatoma cell line HepG2 and chimeric human transferrin-chloramphenicol acetyltransferase genes in transgenic mice were examined. In HepG2 cells, 35S-transferrin protein synthesis and mRNA levels were suppressed by 100 microM and 10 microM lead acetate as early as 24 h after the initial treatment. Yet, synthesis of two proteins known to respond in the hepatic acute phase reaction, complement C3 and albumin, was not altered by the lead treatment. In transgenic mouse liver, lead suppressed expression of chimeric human transferrin genes at both the protein and mRNA levels, but LPS only suppressed at the protein level. The study indicates that lead suppresses human transferrin synthesis by a mechanism that differs from the hepatic acute phase response and that lead may also affect iron metabolism in humans by interfering with transferrin levels.

Extraordinarily stable disulfide-linked homodimer of human growth hormone

Although a 22‐kDa human growth hormone (hGH) is the predicted protein product of the hGH‐N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS‐PAGE under reducing conditions we isolated an unusually stable mercaptoethanol‐resistant (MER) 45‐kDa hGH. A 5‐h incubation at 100°C in the presence of 2‐mercaptoethanol was required to convert approximately 90% of MER‐45‐kDa hGH into a 22‐kDa hGH. Other reductants were not as effective in splitting MER‐45‐kDa hGH. After fracturing MER‐45‐kDa hGH, the 22‐kDa hGH fragments would spontaneously reassociate if the reductant was removed; however, alkylation of cysteine residues prevented their reassociation. Identical amino acid sequences for the first six N‐terminal residues were obtained for MER‐45‐kDa hGH and its 22‐kDa hGH cleavage product. Structural identity of MER‐45‐kDa hGH and 22‐kDa hGH was demonstrated by MALDI‐TOF mass spectrometry of tryptic digests. MER‐45‐kDa hGH did not break up upon incubation with EDTA and EGTA. The significance of this work to our understanding of the structure of hGH isoforms is that it demonstrates that MER‐45‐kDa hGH is not a single chain polypeptide but is instead a homodimer of 22‐kDa hGH monomers. The MER‐45‐kDa hGH dimer is held together by interchain disulfide bonds and not by divalent metal cation bridges. Additionally, MER‐45‐kDa hGHs interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.

Flow-cytometric analysis of mitochondria-associated fluorescence in young and old human fibroblasts

Rhodamine 123 fluorescence in populations of young and old human fibroblasts was analysed and quantified with a fluorescence-activated cell sorter (FACS). Old fibroblasts exhibited a higher mean relative fluorescence than young fibroblasts. Moreover, two distinct subpopulations were evident in the fluorescence distributions of old cells--but not in those of young cells.

Human APOE protein localized in brains of transgenic mice.

Transgenic mice carrying the three common human apolipoprotein E (APOE) alleles have been developed. In this study, brains of the transgenic mice have been analyzed by in situ histohybridization, immunohistochemistry, and immunoblots to determine sites of gene expression, to identify specific brain cells associated with human apoE protein, and to determine the relative concentrations of the human apoE. Results indicate that (1) human APOE mRNA and apoE protein occur in the gray and white matter of transgenic mouse brains; (2) in the hippocampus of transgenic brains, human apoE protein reacts immunologically within the same cells as the glial fibrillary acidic protein (GFAP), a specific marker for astrocytes; and (3) concentrations of the apoE isoforms determined in three heterozygous transgenic brains range from 22 to 250 pmol/g wet weight of brain.

Divalent metal cation chelators enhance chromatographic separation of structurally similar macromolecules: separation of human growth hormone isoforms

Human GH isoforms were separated by anion-exchange chromatography using a linear NaCl gradient in the presence and absence of EDTA and EGTA. SDS-PAGE showed that glycosylated 24-kDa hGH did not appreciably separate from other hGH variants in the absence of metal chelators. However, in the presence of metal chelators, glycosylated 24-kDa hGH separated from the bulk of the hGH isoforms. Human GH isoforms were also separated by size-exclusion chromatography in the presence and absence of metal chelators. Glycosylated 24-kDa hGH eluted with the bulk of the hGH isoforms in both separations. The inclusion of metal chelators in chromatographic buffers to alter the charge and/or size of proteins by stripping their metals may be a generally useful strategy in their fractionation.

O-Glycosylated 24 kDa human growth hormone has a mucin-like biantennary disialylated tetrasaccharide attached at Thr-60

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc2, N‐acetyl galactosamine1, Gal1). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.

Haptoglobin gene expression in human glioblastoma cell lines

Increases in cerebrospinal fluid (CSF) levels of the acute phase protein haptoglobin (Hp) occur in central nervous system (CNS) disorders such as Alzheimers disease. To establish if Hp CSF level increases can be associated with Hp expression in brain, reverse transcription-polymerase chain reaction (RT-PCR) experiments were conducted to determine if the Hp mRNA transcript is expressed in human glioblastoma cells. Furthermore, Western blots and immunoprecipitations were performed to elucidate if Hp protein is synthesized and secreted by human glioblastoma cells. The Hp mRNA (alpha2beta) transcript (1155 bp) was detected both in U-87MG and U-138MG cells, and was positively verified by nested PCR in which a part of the beta sequence (482 bp) was targeted for amplification. Despite the presence of Hp mRNA, Hp protein was not secreted by U-87MG cells as compared to the hepatoma cell line, HepG2, where Hp protein (approximately 46 kDa) was detected in the media. The results suggest the expression of Hp protein by glioblastoma cells is possible since the Hp mRNA transcript exist, but whether or not Hp mRNA is contained in a storage pool requiring a specific signal for translation or is transiently expressed remains to be uncovered in future studies.

Generation, characterization and utilization of anti-human growth hormone 1–43, (hGH1–43), monoclonal antibodies in an ELISA

The major isoform of hGH is a polypeptide of 191 amino acids. Human GH1-43 is an amino terminal segment of hGH1-191 which comprises the first 43 amino acids. This peptide is a potent regulator of glucose homeostasis. To facilitate our understanding of the physiological regulation of hGH1-43 an assay to measure its levels in biological fluids and extracts is needed. This communication describes the development of anti-hGH1-43 monoclonal antibodies and their use in the development of an indirect competitive ELISA for the quantification of hGH1-43. Hybridomas were produced by the fusion of FOX-NY myeloma cells with spleen cells taken from a mouse immunized with hGH1-43. The hybridomas were screened for production of antibodies to hGH1-43 by antibody capture ELISA. Hybridomas which produced antibodies reactive to hGH1-43 were cloned by limiting dilution. Three monoclonal hybridomas, CCL-1, CCL-2, and CCL-3 were subsequently obtained. These hybridomas secreted antibodies that were highly reactive towards hGH1-43 but minimally reactive towards hGH1-191. The isotypes of the mAbs secreted by CCL-1, CCL-2 and CCL-3 were all IgG1 kappa as shown by isotype specific antibody capture analysis. An indirect competitive ELISA with a detection limit that ranged from 1 to 10 ng/ml was developed using mAbs from monoclonal hybridoma CCL-3. Dose-response curves for competing hGH1-191, hPRL, and hPL indicated minimal cross-reactivity of mAbs with these hormones and conversely, a high degree of specificity for hGH1-43. Dose-response curves for dilutions of human serum and pituitary extract were parallel to the standard. The availability of a sensitive assay for the measurement of hGH1-43 will help us answer questions regarding the biosynthesis, regulation of secretion, and role of hGH1-43 in the control of glucose homeostasis.

Increased uptake and retention of rhodamine 123 by mitochondria of old human fibroblasts

Binding of the fluorescent dye R123 by a variety of mammalian cells has been shown to be dependent on the high transmembrane potential maintained in functional mitochondria. Recent studies in our laboratory have shown that old human fibroblasts (HF) bind and retain more R123 than young HF. In an effort to determine whether this difference in R123 uptake indeed reflected a difference in mitochondrial transmembrane potential, drugs known to disrupt the transmembrane potential of mitochondria were used to monitor the R123-mitochondria interaction of young and old HF. Distinct differences indicating that old HF maintain a higher mitochondrial transmembrane potential were observed. More significantly, perhaps this difference reflects an age-related change(s) in the structure and/or function of mitochondria.