Blockade of osteoclast-mediated bone resorption through occupancy of the integrin receptor: A potential approach to the therapy of osteoporosis

Bone resorption requires the tight attachment of the bone-resorbing cells, the osteoclasts, to the bone mineralized matric. Integrins, a class of cell surface adhesion glycoproteins, play a key role in the attachment process. Most integrins bind to their ligands via the arginyl-glycyl-aspartyl (R-G-D) tripeptide present within the ligand sequence. The interaction between integrins and ligaands results in bidirectional transfer of signals across the plasma Membrane. Tyrosine phosphorylaation occurs within cells as a result of integrin binding to ligaands and probably plays a role in the formation of the osteoclast clear zone, a specialized region of the osteoclast membraane maintained by cytoskeletal structure and involved in bone resorption. Human osteoclasts express α₂β₃ and α_vβ₃ integrins on their surface. Such signaling may also lead to “inside-out” effects, like increased expression of integrin receptors on the cell surface, or increased affinity of the integrin to its ligand. The α_vβ₃ integrin, a vitronectin receptor, plays an essential role in bone resorption. Antibodies to this integrin and short synthetic RGD-containing peptides are able to block bone resorption in vitro. Echistatin, an RGD-containing protein from a snake venom, binds to the α_vβ₃ integrin and blocks bone resssorption both in vitro and in vivo. Peptides containing the RGD motif are potential competitive “antagonists” a of the osteoclast integrins and may have utility in the blockage of bone resorption. Agonists may be identified by stimulation of intracellularsignaling. In theory, tissue spacificity can be achieved by (1) introducing specific amino acids in positions adjacent to the RGD sequence, (2) identifying non-RGD integrin binding domains, or (3) modulating the affinity of integrins for their endogenous ligands.     

In situ hybridization analysis of polyoma DNA replication in an inducible line of polyoma-transformed cells.

In situ hybridization has been used to study polyoma DNA replication in a clonal derivative of the inducible LPT line of polyoma-transformed cells designated as clone 1A. This study has shown that in clone 1A cultures maintained under normal growth conditions, 4–25 in 10,000 cells are spontaneously induced to synthesize polyoma DNA at an enhanced rate. In cultures exposed to mitomycin C (MMC), the percentage of induced cells remains approximately equal to the spontaneous level for 9 hr, and then increases for at least 24 hr up to 30–57% as more and more cells are asynchronously recruited to replicate the virus DNA.DNA reassociation kinetics and in situ hybridization have been used to determine the amount and distribution of polyoma DNA accumulated within clone 1A cells. These measurements have shown that a single induced cell in an MMCtreated culture produces 24,500 genome-equivalents of the virus DNA; second, that the average yield of virus DNA in a normally growing culture is only 41.7 genome-equivalents per cell; however, a single spontaneously induced cell in this culture produces as much virus DNA as an MMC-induced cell; third, that all the virus DNA molecules are found within the nuclei and many are clustered in aggregates containing up to 2000 genome-equivalents. We discuss the implications of these findings regarding the regulation of polyoma DNA replication in the LPT line.     

“Induced-fit”-type complex formation of the model enzyme α-cyclodextrin

On the basis of X-ray and neutron data for several α-cyclodextrin·substrate complexes it is shown that basically two different structures for α-cyclodextrin exist, one “tense”, the other “relaxed”. An “induced-fit”-like mechanism for α-cyclodextrin complex formation is proposed.     

Hematopoietic, angiogenic and eye defects in Meis1 mutant animals

Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.     

The Activity Phase of Postsynaptic Neurons in a Simplified Rhythmic Network

Many inhibitory rhythmic networks produce activity in a range of frequencies. The relative phase of activity between neurons in these networks is often a determinant of the network output. This relative phase is determined by the interaction between synaptic inputs to the neurons and their intrinsic properties. We show, in a simplified network consisting of an oscillator inhibiting a follower neuron, how the interaction between synaptic depression and a transient potassium current in the follower neuron determines the activity phase of this neuron. We derive a mathematical expression to determine at what phase of the oscillation the follower neuron becomes active. This expression can be used to understand which parameters determine the phase of activity of the follower as the frequency of the oscillator is changed. We show that in the presence of synaptic depression, there can be three distinct frequency intervals, in which the phase of the follower neuron is determined by different sets of parameters. Alternatively, when the synapse is not depressing, only one set of parameters determines the phase of activity at all frequencies.     

<Emphasis Type="Italic">CHX10</Emphasis> mutations cause non-syndromic microphthalmia/anophthalmia in Arab and Jewish kindreds

Microphthalmia/anophthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. The genetic defect underlying isolated autosomal recessive microphthalmia/anophthalmia is yet unclear. We studied four families (two of Arab origin, one of Bedouin origin, and one of Persian-Jewish origin) with autosomal recessive microphthalmia/anophthalmia and no associated eye anomalies, and one Syrian–Jewish family with associated colobomas. Assuming a founder effect in each of the families, we performed homozygosity mapping using polymorphic markers adjacent to human homologues of genes known to be associated with eye absence in various species, namely EYA1, EYA2, EYA3, SIX4, SIX6, PAX6 and CHX10. No association was found with EYA1, EYA2, EYA3, SIX6 or PAX6. In two families, linkage analysis was consistent with possible association with SIX4, but no mutations were found in the coding region of the gene or its flanking intron sequences. In three of the five families, linkage analysis followed by sequencing demonstrated that affected individuals in each family were homozygous for a different CHX10 aberration: a mutation in the CVC domain and a deletion of the homeobox domain were found in two Arab families, and a mutation in the donor-acceptor site in the first intron in the Syrian-Jewish family. There was phenotypic variation between families having different mutations, but no significant phenotypic variation within each family. It has been previously shown that mutations in a particular nucleotide in CHX10 are associated with an autosomal recessive syndrome of microphthalmia/anophthalmia with iris colobomas and cataracts in two families. We now show that different mutations in other domains of the same gene underlie isolated microphthalmia/anophthalmia.     

Modulated fatty acid desaturation via overexpression of two distinct omega-3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco cells and plants

Changes in the degree of fatty acid (FA) desaturation are implicated in plant responses to various abiotic stresses, including heat, salt and drought. However, it is still not known whether decreased levels of linolenic acid, found in many plants subjected to salt and drought stress, reflect a mechanism of defence or damage. We addressed this question by generating tobacco cells and plants ectopically overexpressing two FA desaturases: the cytosolic FAD3 or the plastidic FAD8. A remarkable increase in the ratio of total linolenic to linoleic acids resulted from overexpression of FAD3, whereas ectopic overexpression of FAD8 induced an increased ratio mainly in the plastidic lipids. Here we present evidence that overexpressing FAD8 imposes much greater heat sensitivity than does FAD3 overexpression, in both cultured cells and whole plants. Overexpression of either FAD3 or FAD8 increases tolerance to drought in tobacco plants and to osmotic stress in cultured cells. These findings suggest that a drought-induced decreased level of linolenic acid reflects damage. Our results point to the potential of exploiting FAD overexpression as a tool to ameliorate drought tolerance.     

The BsmI vitamin D receptor gene polymorphism is associated with ulcerative colitis in Jewish Ashkenazi patients

Susceptibility to inflammatory bowel disease (IBD) has a strong genetic component. The vitamin D receptor (VDR) gene maps to a region on chromosome 12 shown to be associated with IBD in some studies. In this case-control study we determined the association between the BsmI VDR gene polymorphism and IBD in patients with Crohn's disease (CD) and ulcerative colits (UC). Three hundred seventy-nine Jewish Israeli patients with IBD, 228 with CD (129 Ashkenazi and 99 non-Ashkenazi), and 151 patients with UC (72 Ashkenazi, 79 non-Ashkenazi) were studied. The control group included 495 healthy blood donors (352 non-Ashkenazi and 143 Ashkenazi). All subjects were genotyped for the BsmI VDR gene polymorphism. The frequency of the BB genotype was higher in Ashkenazi patients with UC compared to Ashkenazi controls (0.21 vs. 0.11, p = 0.042, odds ratio 2.27, 95% confidence interval [CI] 1.06-4.9). There were no differences in the prevalence of the BB genotype or the B allele between ethnically matched patients with CD and UC. Nor were there differences in the BB genotype or B allele frequencies between CD patients and ethnically matched controls. The BsmI VDR gene polymorphism is associated with increased susceptibility to UC in Israeli Ashkenazi patients with UC.     

On the regulatory role of dipeptidyl peptidase IV (=CD=adenosine deaminase complexing protein) on adenosine deaminase activity

The molecular mechanism controlling the variable activity of the malignancy marker adenosine deaminase (ADA) is enigmatic. ADA activity was found to be modulated by the membrane-bound adenosine deaminase complexing protein (CP=DPPIV=CD26). The role of lipid-protein interactions in this modulation was sought. While direct solubilization of ADA in vesicles resulted in loss of ADA activity, the binding of ADA to CP reconstituted in vesicles restored the specific activity. The activity of ADA, free or bound to CP in solution, resulted in continuous linear Arrhenius plots. However, ADA bound to reconstituted CP exhibited two breaks associated with approximately 30% increased activity, at 25 and 13 degrees C, yielding three lines with similar apparent activation energies (E(a)). Continuum solvent model calculations of the free energy of transfer of the transmembrane helix of CP from the aqueous phase into membranes of various widths show that the most favorable orientations of the helix above and below the main phase transition may be different. We suggest that the 20% change in the thickness of the bilayer below and above the main phase transition may modify the orientation of CP in the membrane, thereby affecting substrate accessibility of ADA. This could account for ADA's reduced activity associated with increased membrane fluidity in transformed vs. normal fibroblasts.