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Biomedical subjects

R G Ham

Publications and source records attributed to R G Ham.

At least 19 recordsLinked to original sources
PubMed1995

Expression of adhesion molecules in cultured human pulmonary microvascular endothelial cells.

HPMECs were successfully isolated by differential trypsinization from peripheral lung lobes. The cells proliferated rapidly in EGM-MV with 10% FBS and were serially cultivated for more than 20 passages (1:4 split ratio) in vitro. Cells were characterized as endothelial based upon their cobblestone morphology, the presence of factor VIII-related antigen, incorporation of DiI-Ac-LDL, tubule-like structure formation in Matrigel, and positive staining for ACE. Adhesion molecules were tested at passage 3 and passage 12. Cells demonstrated intense staining for PECAM-1 both unstimulated and stimulated with TNF-alpha (20 ng/ml). The adhesion molecules ICAM-1, VCAM-1, ELAM-1, and P-selectin differed in expression on unstimulated cells. ICAM-1 was constitutively expressed on unstimulated cells and the expression was increased by TNF-alpha stimulation (20 hr). In contrast, VCAM-1, ELAM-1, and P-selectin were not detected on unstimulated cells but were detected after stimulation with TNF-alpha. The inducibility of adhesion molecules was different. VCAM-1 (10 hr) and ELAM-1 (4 hr) were expressed more strongly than P-selectin (minutes to 4 hr). The adhesion molecule profile found on passage 12 was the same as on passage 3. CD36 was not detected on both unstimulated and stimulated (4 and 8 hr) cells. The peak of adhesion of HL-60 cells to TNF-alpha activated HPMEC monolayers was around 8 hr. The results indicate that HPMEC can be continuously grown in vitro for many passages without losing their adhesion molecule expression. This expression of adhesion molecules confirms that HPMECs might be a good in vitro model in the understanding of various aspects of pulmonary microvascular endothelial cell function and may be useful as the basis for studies of adhesion molecule targeted therapies of pulmonary inflammatory diseases.

Cell Adhesion Molecules↗
PubMed1992

Improved medium with EGF and BSA for differentiated human skeletal muscle cells.

Epidermal growth factor (EGF) and bovine serum albumin (BSA) are both required for serum-free clonal growth of human muscle satellite cells (HMSC). However, neither inhibits differentiation of HMSC, and when both are added to a minimal serum-free differentiation medium, they enhance survival and maintenance of human myotubes. A combination of 10 ng/mL EGF and 0.5 mg/mL BSA, added to MCDB 120 plus 10 micrograms/mL insulin, increases both total protein per dish and total creatine kinase activity, and keeps the myotubes in good condition for a longer period of time. The myotubes become cross-striated and exhibit frequent spontaneous twitching. Substantial amounts of neonatal myosin heavy chain and the MM isozyme of creatine kinase are expressed, together with detectable amounts of adult fast myosin heavy chain. With regular feeding, these cultures can be maintained for at least 3 weeks with no overgrowth by mononucleate cells, and with far less degeneration than with insulin as the only supplement.

Adolescent↗
PubMed1992

Regional localization of human beta-casein gene (CSN2) to 4pter-q21.

Milk proteins are crucial for the development of all newborn mammals. Caseins that constitute the bulk of the protein in mammalian milk have been shown to be members of a multigene family in at least two species. They are among the most rapidly diverging groups of proteins, and their numbers vary widely among species. beta- and kappa-Caseins are the only caseins present in human milk. Using polymerase chain reaction on genomic DNA from somatic cell hybrids, we have localized the human beta-casein gene (CSN2) to 4pter----q21.

Blotting, Southern↗
PubMed1992

Exon skipping in human beta-casein.

Earlier amino acid alignments of mature beta-caseins showed that the human protein was shifted in alignment relative to other species, with amino acid deletions in the N-terminal region and others inserted in the C-terminal region. Our alignment, based on cDNA sequences and their translation products, has shown that the amino acid deletions correspond exactly to exon 3 in the other species. Cloning and sequencing of a segment of the human beta-casein gene between exons 2 and 4 revealed the presence of an intact exon 3 sequence in the gene. An interruption of the polypyrimidine tract adjacent to the 5' end of exon 3 sequence may account for the omission of the exon from human beta-casein mRNA.

Amino Acid Sequence↗
PubMed1991

Separation of growth-promoting activity for human muscle cells from fetuin.

Whether the growth-promoting activity of Pedersen fetuin is due to fetuin itself or to a contaminant(s) has been a long-standing puzzle. The possibility that the growth-promoting activity of Pedersen fetuin for human muscle satellite cells (HMSC) could be caused by some other component of fetal bovine serum (FBS) that remained in the fetuin as a contaminant has been investigated. One liter of FBS was first precipitated with 50% saturated ammonium sulfate, which leaves the serum albumin in solution, and then with 25% polyethylene glycol, which leaves the fetuin in solution, to generate a fraction 50 PEG 2x that was enriched 11-fold in growth-promoting activity for HMSC, with 68% recovery of total activity. Further purification with FPLC anion exchange chromatography achieved 99-fold enrichment of the activity with 30% overall recovery. The activity is heat labile and pH sensitive, suggesting that it is of protein nature, and the size of the activity is above 70 kDa. SDS-PAGE of the most active fractions shows that they are virtually free of fetuin. Thus, although the active fractions are not homogeneous, these studies demonstrate that the growth-promoting activity for HMSC can be fully separated from fetuin.

Animals↗
PubMed1989

Growth of rat mammary tumor line 64-24 in liposome-supplemented defined medium. I. Effect of liposome B components on colony growth.

An improved serum-free medium has been developed that supports growth of rat mammary tumor line 64-24 with far less protein supplementation and with a much smaller inoculum than previously possible. An initial survey showed that MCDB 202 supported clonal growth with 1% dialyzed serum. The remaining serum was then replaced with 5 micrograms/ml insulin, 10 ng/ml epidermal growth factor (EGF), 1 micrograms/ml hydrocortisone, 50 ng/ml ovine prolactin, and 5 micrograms/ml liposome B (a mixture of soy lecithin, sphingomyelin, cholesterol, vitamin E, and vitamin E acetate in liposome form). Insulin and EGF are required and growth is improved by hydrocortisone and prolactin. Estradiol is stimulatory in the absence of liposome B. With adequate iron supplementation, transferrin has no effect. Liposome B increases growth rate substantially. Most of the growth stimulation can be replaced with phosphatidylethanolamine or sphingomyelin.

Animals↗
PubMed1989

Growth of rat mammary tumor line 64-24 in liposome-supplemented defined medium. II. Effect of liposome B and prolactin on colony forming efficiency.

During studies on serum-free clonal growth of rat mammary tumor line 64-24, we observed that liposome B causes a major increase in colony-forming efficiency. This phenomenon was studied with a short-term assay based on phase contrast microscopic observation of the effects of liposome B on recently plated, serum-depleted cells. In the serum-free medium, colony forming efficiency is not determined primarily by viability (measured by dye exclusion) or cell attachment. Instead, most of the effect is determined by the extent of degeneration that occurs during cell elongation and the first division. Factors that prevent degeneration, such as liposome B, restore colony forming efficiency. Prolactin alone stimulates cell elongation but does not prevent degeneration. However, the combination of prolactin and liposome B increases colony formation to levels observed in the presence of serum.

Animals↗
PubMed1988

Improved media for normal human muscle satellite cells: serum-free clonal growth and enhanced growth with low serum.

We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium, MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells.

Cell Differentiation↗
PubMed1987

Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum.

An optimized basal nutrient medium, MCDB 131, has been developed that supports clonal growth of human microvascular endothelial cells (HMVEC) with as little as 0.7% dialyzed fetal bovine serum (dFBS) when also supplemented with 10 ng/ml epidermal growth factor (EGF) and 1 microgram/ml hydrocortisone. An extensive initial survey of available media showed that MCDB 402, a medium optimized for low-serum growth of Swiss 3T3 cells, supported the best clonal growth of HMVEC with 10% dFBS. Quantitative adjustment of the composition of MCDB 402 for improved clonal growth of HMVEC with reduced amounts of dFBS resulted in development of MCDB 131. Although many different adjustments contributed to the optimal properties of MCDB 131 for growth of HMVEC, the most unusual feature of this medium is its high magnesium concentration. A major benefit was achieved by increasing Mg2+ from 0.8 mM in MCDB 402 to 10.0 mM in MCDB 131. In the absence of defined supplements, MCDB 131 supports good clonal growth of HMVEC with 2% dFBS. This can be reduced to 0.7% by adding EGF and hydrocortisone, which act synergistically to improve growth with low levels of dFBS.

Animals↗
PubMed1984

Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract.

A serum-free medium with bovine pituitary extract as the only undefined supplement has been developed for long-term culture of human mammary epithelial cells. This medium supports serial subculture of normal cells for 10-20 passages (1:10 splits) without conditioning or special substrates, and it supports rapid clonal growth with plating efficiencies up to 35%. It consists of an optimized basal nutrient medium, MCDB 170, supplemented with insulin, hydrocortisone, epidermal growth factor, ethanolamine, phosphoethanolamine, and bovine pituitary extract. Replacement of pituitary extract with prostaglandin E1 and ovine prolactin yields a defined medium that supports rapid clonal growth and serial subculture for three or four passages. Cultures initiated in these media from normal reduction mammoplasty tissue remain diploid and maintain normal epithelial morphology, distribution of cell-associated fibronectin, expression of keratin fibrils, and a low level of expression of milk fat globule antigen. Large cell populations can now be generated and stored frozen, permitting multiple experiments over a period of time with cells from a single donor. These media greatly extend the range of experiments that can be performed both conveniently and reproducibly with cultured normal and tumor-derived human mammary epithelial cells.

Animals↗
PubMed1983

Growth and adipose differentiation of sheep preadipocyte fibroblasts in serum-free medium.

Fibroblasts from ovine skin, and from the perirenal and subcutaneous adipose tissues of sheep were grown at clonal densities in medium MCDB 202 supplemented with 1 microgram/ml bovine insulin, 1 microM dexamethasone, 100 ng/ml fibroblast growth factor and 20 micrograms/ml of the lipid preparation described by Bettger, W. J., Boyce, S. T., Walthall, B. J. and Ham, R. G. [(1981) Proc. Natl Acad. Sci, USA, 78, 5588-5592]. When maintained as a confluent monolayer in this medium, the fibroblasts from the adipose tissues spontaneously underwent an adipose differentiation. This was accelerated by substituting medium F12 for medium MCDB 202, and by raising the CO2 tension from 2% to 7.5% in air over the cultures. The differentiation was inhibited by deleting FGF from the growth medium, or by coating the culture surface with fibronectin or poly-D-lysine. Differentiation also failed to occur when the defined supplements were replaced with fetal bovine serum. The synthesis of triacylglycerol by the cells, as seen by the increased specific activity of [14C]acetate incorporated into this lipid class, was accompanied by an increase in the specific activity of glycerol-3-phosphate dehydrogenase.

Adipose Tissue↗
PubMed1983

Clonal growth of primary cultures of rabbit ear chondrocytes in a lipid-supplemented defined medium.

Clonal growth of primary cultures of rabbit ear chondrocytes in a defined medium without serum or other undefined additives has been achieved. The clonal inoculum is a suspension of fully differentiated chondrocytes prepared by collagenase digestion of rabbit ear cartilage and used with no prior adaptation or selection in culture. When inoculated into medium MCDB 104 supplemented with 100 ng/ml fibroblast growth factor (FGF), 1 microgram/ml insulin, and 5 micrograms/ml of a lipid supplement previously developed for human fibroblasts, the isolated chondrocytes undergo clonal multiplication to form large colonies of epithelial-like cells. Colonies grown in the defined medium for 14 days accumulate at their centers refractile cartilage-like matrix that is stained by acidified Alcian green, although the amount is significantly less than with undefined additives. This system opens the way for detailed studies, in a defined background medium, of factors that regulate phenotypic expression of cartilage-like differentiated properties.

Animals↗
PubMed1983

Multiplication of Swiss 3T3 cells in a serum-free medium.

Gently trypsinized Swiss 3T3 cells inoculated into medium MCDB 402 attach readily to polylysine-coated surfaces and remain viable for several days in the absence of exogenously added protein. Short-term multiplication under defined conditions can be obtained by supplementing the MCDB 402 with fibroblast growth factor (FGF), insulin (INS), and dexamethasone (DEX). Addition of bovine plasma fibronectin further improves attachment and viability. This system does not require initial plating in serum or the addition of poorly defined extracts for cellular attachment or for multiplication. In the complete system minus FGF, cells plated at a low density attach to the culture surface and become quiescent. The addition of FGF or PDGF 48-72 h after plating stimulates a high level of DNA synthesis during the following 24 h. EGF also stimulates DNA synthesis in these cells, but to a lesser extent. Insulin and dexamethasone are not needed for the initial DNA synthesis response to FGF, but are needed for continuing multiplication over a period of several days. This system provides a means for studying the effects of specific mitogens on Swiss 3T3 cells in the absence of undefined supplements, and without complications due to density-dependent inhibition.

Animals↗