In the process of endochondral ossification, chondrocytes progress through a series of maturational changes, including division and hypertrophy, that culminate in chondrocyte loss and cartilage resorption. From an investigation of morphology, DNA fragmentation, and collagen synthesis in the developing chick sterna we have characterized chondrocytes death in this process. Light microscopy of resorbing sterna demonstrated chondrocyte condensation at the interface with the invading vasculature and electron microscopy demonstrated a range of chondrocyte morphologies, including retraction from the pericellular matrix, cytoplasmic and nuclear condensation, and vesiculation suggestive of sequential changes characteristic of apoptosis.Isolation and end-labeling of DNA from chick primary ossification centers demonstrated fragmentation to nucleosome sized units, only in primary ossification centers exhibiting active resorption, and in situ detection of DNA fragmentation showed a restriction to chondrocytes at the interface with invading blood. We conclude that terminal differentiation of chondrocytes results in death by an apoptotic process prior to resorption of the tissue and invasion by blood vessels. The extent of DNA fragmentation correlated closely with the proportion of cells displaying a condensed phenotype in contralateral primary ossification centers and peaked at an early stage of resorption, suggesting that chondrocyte apoptosis may be an initiating event in tissue resorption and vascular invasion. Comparison of DNA fragmentation with expression of the hypertrophic chondrocyte phenotype, as indicated by type X collagen synthesis, suggested that DNA fragmentation was a late event in the process of chondrocyte hypertrophy and probably corresponded with chondrocyte condensation. o 1995 Wiley-Liss, Inc.

We report here the tissue-specific expression and gabapentin-binding properties of calcium channel alpha2delta subunits. Northern blot analysis demonstrated that human alpha2delta-1, -2, and -3 mRNA all had high levels of expression in brain, heart and skeletal muscle. However, the highest expression of human alpha2delta-2 mRNA was found in lung. Human alpha2delta-1, -2, and -3 mRNAs were detected in all portions of brain tested. Western blotting revealed that alpha2delta-2 protein was predominantly expressed in cerebellar cortex (brain) and undetectable in lung. The dissociation between mRNA and protein levels of human alpha2delta-2 in lung suggests possible post-transcriptional regulation. Although mouse alpha2delta-1 proteins exhibited a similar tissue distribution profile as that of human, tissue distribution of mouse alpha2delta-2 and -3 mRNA revealed a different profile. Mouse alpha2delta-3 mRNA was restricted to brain and mouse alpha2delta-2 mRNA was not detectable in lung. Gel electrophoresis under a reduced condition resulted in a mobility shift of both alpha2delta-1 and alpha2delta-2 proteins, suggesting that alpha2 and delta of alpha2delta-2 protein are linked by disulfide bond as are alpha2 and delta of alpha2delta-1. Scatchard plots revealed a single population of gabapentin binding sites for human alpha2delta-2 with the KD value twofold higher than that of porcine alpha2delta-1 (156 +/- 25 nm vs. 72 +/- 9 nm). Inhibition of gabapentin binding to alpha2delta-2 by selected amino acids and gabapentin analogs produced a binding profile similar, but not identical to that of alpha2delta-1.

The Colorado Plateau is a large crustal block in the southwestern United States that has been raised intact nearly 2 km above sea level since Cretaceous marine sediments were deposited on its surface. Controversy exists concerning the thickness of the plateau crust and the source of its buoyancy. Interpretations of seismic data collected on the plateau vary as to whether the crust is closer to 40 or 50 km thick. A thick crust could support the observed topography of the Colorado Plateau isostatically, while a thinner crust would indicate the presence of an underlying low‐density mantle. This paper reports results on long‐offset seismic data collected during the 1989 segment of the U.S. Geological Survey Pacific to Arizona Crustal Experiment that extended from the Transition Zone into the Colorado Plateau in northwest Arizona. We apply two new methods to analyze long‐offset data that employ finite difference travel time calculations: (1) a first‐arrival time inverter to find upper crustal velocity structure and (2) a forward‐modeling technique that allows the direct use of the inverted upper crustal solution in modeling secondary reflected arrivals. We find that the crustal thickness increases from 30 km beneath the metamorphic core complexes in the southern Basin and Range province to about 42 km beneath the northern Transition Zone and southern Colorado Plateau margin. We observe some crustal thinning (to ∼37 km thick) and slightly higher lower crustal velocities farther inboard; beneath the Kaibab uplift on the north rim of the Grand Canyon the crust thickens to a maximum of 48 km. We observe a nonuniform crustal thickness beneath the Colorado Plateau that varies by ∼15% and corresponds approximately to variations in topography with the thickest crust underlying the highest elevations. Crustal compositions (as inferred from seismic velocities) appear to be the same beneath the Colorado Plateau as those in the Basin and Range province to the southwest, implying that the plateau crust represents an unextended version of the Basin and Range. Some of the variability in crustal structure appears to correspond to preserved lithospheric discontinuities that date back to the Proterozoic Era.

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Two variant sublines of murine L1210 leukemia cells (L1210A and L1210JF) overexpress the cell surface folate receptor (FR). The membrane bound FR in L1210A cells exhibited significantly (up to 17-fold) greater relative affinities for (6S)-N5-methyltetrahydrofolate, (6S)-N5-formyltetrahydrofolate and methotrexate compared to the FR in L1210JF cells. Furthermore, receptor-mediated transport of [3H]-(6S)-N5-methyltetrahydrofolate was much more efficient in L1210A cells compared to L1210JF cells. When solubilized with Triton X-100, the ligand binding characteristics of FR from both sublines resembled those of the receptor associated with L1210 JF cell membranes. N-terminal amino acid sequence analysis as well as RT-PCR analysis of the entire coding region revealed a single species of FR in both cells, identical to murine FR-alpha. The FR in L1210JF cells was sensitive to phosphatidylinositol specific phospholipase C (PI-PLC) indicating the presence of a glycosyl-phosphatidylinositol (GPI) membrane anchor while the FR in L1210A cells was resistant to PI-PLC; however, the FR in L1210A cells was released from plasma membranes by nitrous acid, as expected for GPI and its PI-PLC resistant structural variants. Treatment of L1210A cell membranes with mild base rendered the protein PI-PLC sensitive as expected for GPI anchors acylated in the inositol ring and also decreased the affinities of the membrane associated FR for reduced folates. When the cDNA for murine FR-alpha was expressed in parental L1210 cells the protein was PI-PLC resistant but was sensitive to PI-PLC when the cDNA was expressed in human 293 fibroblasts. In L1210JF, L1210A, and parental L1210 cells, several cell surface proteins, including FR, incorporated [3H]ethanolamine, a component of the GPI membrane anchor; however, the labeled proteins were released by PI-PLC only in L1210JF cells. The above results preclude any peculiarity of the FR polypeptide in either L1210 subline as the basis for the observed differences in PI-PLC sensitivity and membrane-associated functions of FR. Partial deglycosylation of membrane associated FR from either cell with N-glycanase did not influence its ligand binding characteristics. The results of this study lead to the hypothesis that variant GPI structures may modulate the function of a protein by influencing its conformation/topography in the membrane. Such effects may be identified by their disappearance/reduction upon detergent solubilization or mild base treatment of the membrane.

Accumulating evidence suggests that prothymosin alpha has an as yet undefined intracellular, perhaps intranuclear, function related to cell proliferation. Prothymosin alpha mRNA and/or peptide levels increase when cells are stimulated to proliferate. Because proliferation and differentiation events are often inversely correlated, we examined prothymosin alpha gene expression during proliferation and differentiation of HL-60 myeloid leukemia cells. Steady-state levels of prothymosin alpha mRNA, which are high in exponentially growing HL-60, decrease within hours after induction of HL-60 to differentiate along the neutrophil pathway with dimethylsulfoxide (DMSO) or along the macrophage lineage with either tetradecanoylphorbol acetate (TPA) or bryostatin 1. The decline in prothymosin alpha mRNA in response to these differentiation signals parallels that of c-myc mRNA under the same conditions. We then determined whether the downregulation of prothymosin alpha and c-myc mRNA were due to differentiation or cessation or proliferation. Recombinant human gamma-interferon induces monocytic differentiation of HL-60, but permits continued proliferation, and, under these conditions, expression of prothymosin alpha, as well as of c-myc, mRNA remains elevated. We conclude that prothymosin alpha and c-myc expression are coregulated in differentiating HL-60 and that their expression correlates with the proliferative state of HL-60 cells, rather than with the differentiated state.

Species differ greatly in their rates of aging. Among mammalian species life span ranges from 2 to over 60 years. Here, we test the hypothesis that skin-derived fibroblasts from long-lived species of animals differ from those of short-lived animals in their defenses against protein damage. In parallel studies of rodents, nonhuman primates, birds, and species from the Laurasiatheria superorder (bats, carnivores, shrews, and ungulates), we find associations between species longevity and resistance of proteins to oxidative stress after exposure to H(2)O(2) or paraquat. In addition, baseline levels of protein carbonyl appear to be higher in cells from shorter-lived mammals compared with longer-lived mammals. Thus, resistance to protein oxidation is associated with species maximal life span in independent clades of mammals, suggesting that this cellular property may be required for evolution of longevity. Evaluation of the properties of primary fibroblast cell lines can provide insights into the factors that regulate the pace of aging across species of mammals.

Analogues of methotrexate (MTX) and aminopterin (AMT) with aminophosphonoalkanoic, aminoalkanesulfonic, and aminoalkanephosphonic acid side chains in place of glutamate were synthesized and tested as inhibitors of folylpolyglutamate synthetase (FPGS) from mouse liver. The aminophosphonoalkanoic acid analogues were also tested as inhibitors of dihydrofolate reductase (DHFR) from L1210 murine leukemia cells and as inhibitors of the growth of MTX-sensitive (L1210) and MTX-resistant (L1210/R81) cells in culture. The optimal number of CH2 groups in aminophosphonoalkanoic acid analogues of AMT was found to be two for both enzyme inhibition and cell growth inhibition but was especially critical for activity against FPGS. Deletion of the alpha-carboxyl also led to diminished anti-FPGS activity in comparison with previously studied homocysteic acid and 2-amino-4-phosphonobutyric acid analogues. In the aminoalkanesulfonic acid analogues of MTX without an alpha-carboxyl, anti-FPGS activity was low and showed minimal variation as the number of CH2 groups between the carboxamide and sulfonate moieties was changed from one to four. In similar aminoalkanephosphonic acid analogues of MTX, anti-FPGS activity was also low, was comparable for two and three CH2 groups between the carboxamide and phosphonate moieties, and was diminished by monoesterification of the phosphonate group. These effects demonstrate that the alpha-carboxyl group of folate analogues is involved in binding to the active site of FPGS, and that an alpha-carboxyl group should be retained as part of the structure of FPGS inhibitors.