Integrated control of proliferation and differentiation of mesenchymal stem cells.

The physiological control of cellular proliferation and differentiation is an integrated regulatory process. This conclusion is based upon observations using numerous in vivo and in vitro experimental systems of which murine BALB/c 3T3 T mesenchymal stem cells represent an excellent in vitro model. In these cells the coupling of growth arrest and differentiation occurs at a distinct biological state, and this predifferentiation arrest state is distinguishable by a variety of criteria from other restriction points, such as the growth factor deficiency arrest state and the nutrient deficiency arrest state. Most importantly, only cells at this predifferentiation arrest state acquire the potential to differentiate without undergoing DNA synthesis. From this state, differentiation can then occur as a two-step process. Cells first undergo nonterminal differentiation and, second, they terminally differentiate. Nonterminal differentiation is characterized by expression of a completely differentiated adipocyte phenotype with retention of proliferative potential. Thereafter, when nonterminally differentiated cells undergo the terminal event in differentiation, they irreversibly lose their proliferative potential. In this paper, data are reviewed which establish that the integrated control of proliferation and differentiation in 3T3 T mesenchymal stem cells is mediated both at the predifferentiation arrest state and at the state of nonterminal differentiation.

High-performance liquid chromatographic determination of cefonicid in human plasma and urine.

A high-performance liquid chromatographic assay for determination of cefonicid concentrations in human plasma and urine samples has been developed using cefazolin as an internal standard. For the analysis of plasma samples two calibration curves were utilized covering the cefonicid concentration ranges of 0.05-1.0 microgram/ml and 1.0-50.0 micrograms/ml, respectively. Coefficients of variation of 7.4% or less were obtained for cefonicid concentrations of 0.05-50.0 micrograms/ml. Mean bias was +6.0% at 0.05 micrograms/ml cefonicid and between -2.1% and +1.6% for 1.0-50.0 micrograms/ml cefonicid. Plasma samples containing 30 ng/ml cefonicid could be well distinguished from blank plasma samples. Urine samples were analysed by using a calibration curve for cefonicid concentrations between 1.0 and 50.0 micrograms/ml. ranged from 8.6% at a cefonicid concentration of 1.0 microgram/ml to 0.5% at 50.0 micrograms/ml with a mean bias between -3.0% and +0.3%.

Cell cycle and cyclic AMP-dependent phosphorylation of plasma membrane proteins p14 and p24: defects in smooth surface transformed cells.

Two proteins which are localized to the cytoplasmic surface of the plasma membrane, p14 and p24, undergo cyclic AMP-dependent phosphorylation in rapidly growing nontransformed murine embryo cells. In this cell system, growth arrest in the G1 phase of the cell cycle induced by growth factor deprivation is associated with the reversible loss in ability to phosphorylate these substrates. By contrast, Simian virus 40 and methylcholanthrene transformed cells show both defective G1 growth control and defects in their ability to phosphorylate p14 and p24 under all tested growth conditions. These data suggested a correlation between defects in the physophorylation of p14 and p24 and defects in the ability of transformed cells to G1 growth arrest. The results of the current studies by contrast show that 3T3 T proadipocytes which have been transformed by the smooth surface tumorigenesis method show different characteristics. They retain the ability to G1 growth arrest in serum-deficient medium. They show cyclic AMP-dependent phosphorylation of p14 and p24 during exponential growth. They do not, however, down regulate p14 and p24 phosphorylation in association with G1 growth arrest. These observations suggest that neoplastic transformation is not necessarily associated with absolute defects in the ability to phosphorylate p14 and p24. Rather, the results of the current study suggest that the inability to modulate the cyclic AMP-dependent phosphorylation of plasma membrane p14 and p24 proteins during the G1 phase of the cell cycle may be more tightly associated with neoplastic transformation.

Simultaneous determination of 5-aminosalicylic acid and 5-acetylaminosalicylic acid by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its main metabolite 5-acetylaminosalicylic acid (5-AcASA) in plasma, with 4-acetylaminosalicylic acid as an internal standard. Prior to extraction into ethyl acetate, 5-ASA is derivatized to 5-carbobenzyloxyaminosalicylic acid (5-CboASA). The calibration graphs for both 5-ASA and 5-CboASA are linear between 0.1 and 20.0 mumol/l. The limit of detection is 0.02 mumol/l for 5-ASA and 0.05 mumol/l for 5-AcASA. At 0.1 mumol/l, the coefficients of variation were 8.1 and 9.8% for 5-ASA and 5-AcASA, respectively. At 1.0 and 10.0 mumol/l, the coefficients of variation were 4.1% or less. The mean bias ranged from -6.2 to -2.0% for 5-ASA and from +6.0 to 0% for 5-AcASA.

Loss of alternately spliced messenger RNA of the luteinizing hormone receptor and stability of the follicle-stimulating hormone receptor messenger RNA in granulosa cell tumors of the human ovary.

OBJECTIVES: The expression of messenger RNA (mRNA) for the LH (LHR) and FSH receptors (FSHR) was examined in normal human corpora lutea and granulosa cell tumors. METHODS: Expression was examined by RT/PCR and DNA sequencing techniques. RESULTS: The full-length (FL) coding region and seven additional isoforms were identified for normal LHR mRNA. Isoform 1 had portions of exons II and III deleted, and isoform 2 had exon IX omitted. Isoform 3 also had portions of exons II and III deleted and all of exon IX deleted. Exons III through VI were missing in isoforms 4-7. Isoform 5 also had exon IX omitted, and isoform 6 also had part of exon XI missing. Isoform 7 also had exon IX and part of exon XI deleted. An aberrant migration pattern of the LHR mRNA isoforms was observed for granulosa cell tumors with FIGO Stage I-IV. Five tumor samples of Stage III-IV had many isoforms absent. Seven Stage I samples had aberrant migration patterns that depended on the size of the tumor. As the size of the tumor increased the aberrant migration pattern of the LHR mRNA isoforms was more pronounced and some isoforms were not detected. The FL and at least one additional isoform were identified for FSHR mRNA. Isoform 1 had regions of exons IV and V deleted. The FSHR mRNA isoforms had a similar migration pattern for the normal ovary and the granulosa cell tumors. CONCLUSIONS: Alternately spliced forms of mRNA for the LHR and FSHR exist for normal human ovary and granulosa cell tumors. The aberrant migration and missing LHR mRNA isoforms in granulosa cell tumors do not appear to result from general genomic instability associated with tumor progression. These findings are important to understand the role of alternate splicing in the regulation of LHR and FSHR expression in different pathological states.

Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells.

The plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP-dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP-dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP-dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP-dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.

Plasma membrane vesiculation in 3T3 and SV3T3 cells. I. Morphological and biochemical characterization.

3T3 and SV3T3 mouse embryo cells and a variety of other monolayer cell lines can be induced to form and shed plasma membrane vesicles by exposure to sulphydryl blocking agents including formaldehyde and N-ethyl malemide. Morphological studies show that multiple vesicles are formed and released from individual cells and that the vesicle membrane is continuous with the plasma membrane of the cell. Vesicles measure from o.1 to 15 micrometer in diameter and are free of detectable contamination with cytoplasmic membranes and organelles. Vesicles also show a 10-fold enrichment in the plasma membrane marker enzyme 5'-nucleotidase and are devoid of detectable NADH-cytochrome C reductase and succinic dehydrogenase activity which are marker enzymes for endoplasmic reticulum and mitochondria, respectively. Vesicles have a high cholesterol: phospholipid ratio and show enrichment in sphingomyelin content. They contain receptors for Con A and WGA, approximately 20 size class polypeptides and intramembranous particles. These results suggest that vesicles are derived from and have the general characteristics of plasma membranes.

Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states.

Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G1 phase of the cell cycle. These include the predifferentiation arrest state (GD) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (GS), and the nutrient deficiency arrest state (GN). Cells at states of reversible nonterminal differentiation (GD') and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at GD, GS and GN states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [35S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the GS, GD, GD' and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the GD' and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation.2+he identification of differential