Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.     

Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.     

Involvement of glutathione in the inhibition of sea urchin egg mitosis by phenyl glyoxal

Cell division in fertilized sea urchin eggs was reversibly inhibited when the ketoaldehyde phenyl glyoxal (PG) at a concentration of 0.1 mM was added to eggs for ten minutes prior to the formation of the mitotic spindle. We investigated whether inhibition of mitosis was due to PG binding to the cell surface (as previously suggested by Stein and Berestecky, '74) or to some intracellular effect. When 14C-PG was added to eggs, label was readily taken up into the egg cytoplasm; very little label was associated with the egg surface. In the cytoplasm PG combined with equimolar amounts of reduced glutathione (GSH), decreasing the levels of cellular GSH to less than 15% of normal and accounting for at least 50% of the PG taken up by eggs. The concentrations of oxidized and protein-bound glutathione were unaffected by PG treatment. We showed that glyoxalase enzymes were present in sea urchin eggs and were capable of metabolizing the PG-GSH complex, thereby restoring GSH to normal levels after PG was removed from the sea water. Though some other effect of PG cannot be ruled out, the major fate of PG in eggs was to combine with GSH, and the transient decrease in GSH which resulted could lead to inhibition of mitosis. While other reports (Nath and Rebhun, '76; Oliver et al., '76) have shown that reagents which oxidize GSH disrupt microtubule-related events, our results showed that such inhibition could be caused by decreased GSH levels alone.     

Alterations in high-affinity binding characteristics and levels of opioids in invertebrate ganglia during aging: Evidence for an opioid compensatory mechanism

In Mytilus and Leucophaea the high-affinity binding site density is significantly lower in old animals than in young animals, whereas the low-affinity site density remains unchanged. In Mytilus the estimated met-enkephalin and met-enkephalin-Arg6-Phe7 levels are significantly higher in old than in young animals. In Leucophaea only the met-enkephalin level can be determined, and it is also higher in old animals. The decrease in the high-affinity binding site density and the corresponding increase in endogenous enkephalin levels suggest the existence of an opioid compensatory mechanism associated with the aging process. In Mytilus there is a demonstrated decrease with age in intraganglionic dopamine levels in response to applied opiates. In addition, the inhibition of dopamine-stimulated adenylate cyclase activity by opiates also decreases in older animals. In Leucophaea the sex difference in opioid binding densities diminishes with age.     

Novel methoxy-carotenoids from the burgundy-colored plumage of the Pompadour Cotinga Xipholena punicea

Recent advances in the fields of chromatography, mass spectrometry, and chemical analysis have greatly improved the efficiency with which carotenoids can be extracted and analyzed from avian plumage. Prior to these technological developments, Brush (1968) [1] concluded that the burgundy-colored plumage of the male pompadour Cotinga Xipholena punicea is produced by a combination of blue structural color and red carotenoids, including astaxanthin, canthaxanthin, isozeaxanthin, and a fourth unidentified, polar carotenoid. However, X. punicea does not in fact exhibit any structural coloration. This work aims to elucidate the carotenoid pigments of the burgundy color of X. punicea plumage using advanced analytical methodology. Feathers were collected from two burgundy male specimens and from a third aberrant orange-colored specimen. Pigments were extracted using a previously published technique (McGraw et al. (2005) [2]), separated by high-performance liquid chromatography (HPLC), and analyzed by UV/Vis absorption spectroscopy, chemical analysis, mass spectrometry, nuclear magnetic resonance (NMR), and comparison with direct synthetic products. Our investigation revealed the presence of eight ketocarotenoids, including astaxanthin and canthaxanthin as reported previously by Brush (1968) [1]. Six of the ketocarotenoids contained methoxyl groups, which is rare for naturally-occurring carotenoids and a novel finding in birds. Interestingly, the carotenoid composition was the same in both the burgundy and orange feathers, indicating that feather coloration in X. punicea is determined not only by the presence of carotenoids, but also by interactions between the bound carotenoid pigments and their protein environment in the barb rami and barbules. This paper presents the first evidence of metabolically-derived methoxy-carotenoids in birds.     

Stimulation of the protein synthetic process by adenosine 3':5'-monophosphate and hexose phosphates in gel-filtered rabbit reticulocyte lysates.

The addition of 0.167 to 4.0 mM cAMP to gel-filtered rabbit reticulocyte lysates stimulates the initial rate and the extent of polypeptide synthesis. The stimulation is at the initiation step of polypeptide synthesis as measured by the (i) increased dipeptide, methionyl-valine, accumulation in the presence of the specific initiation inhibitor, pactamycin, and (ii) increased formation of the 40 S and 80 S initiation complex when gel-filtered lysates are incubated with [35S]Met-tRNAFMet. Furthermore, a synergistic stimulation of protein synthesis is observed when cAMP and hexose phosphates (which alone elicit a 1.8-fold stimulation of protein synthesis) are added simultaneously to gel-filtered rabbit reticulocyte lysates. These results indicate that cAMP and hexose phosphates are both essential to maintain the high rate of initiation.     

Wiring diagrams of MAPK regulation by MEKK1, 2, and 3.

Mitogen-activated protein kinase (MAPK) pathways are activated by a plethora of stimuli. The literature is filled with papers describing the activation of different MAPKs by almost any stimulus or insult imaginable to cells. In this review, we use signal transduction wiring diagrams to illustrate putative upstream regulators for the MAPK kinase kinases, MEKK1, 2, and 3. Targeted gene disruption of MEKK1, 2, or 3 defined phenotypes for each MEKK associated with loss of specific MAPK regulation. Genetic analysis of MEKK function clearly defines specific components of the wiring diagram that require MEKK1, 2, or 3 for physiological responses. We propose that signal transduction network wiring diagrams are valuable tools for hypothesis building and filtering physiologically relevant phenotypic responses from less connected protein relations in the regulation of MAPK pathways.