Impaired PBPC collection in patients with myeloma after high-dose melphalan

BACKGROUND: Tandem stem cell transplantation is an important treatment option for patients with myeloma and some additional tumors. In an attempt to reduce the contamination of the stem cell graft with tumor cells, patients with myeloma who entered complete remission after the first transplant underwent a second episode of mobilization to obtain progenitor cells for the second transplant. METHODS: Twenty-two patients with myeloma participated in the study. The first mobilization utilized CY, etoposide and filgrastim. The second mobilization used the same regimen, but seven patients received only filgrastim. The interval between the two collection periods was 6 months (median; range 4-9 months). The preparative regimen for the first transplant consisted of melphalan 200 mg/m(2). RESULTS: The number of total white cells collected during the two collection episodes was similar: 10.8+/-1.6 x 10(8)/kg white cells vs. 11.8+/-1.7 x 10(8)/kg white cells (P=0.63). The collected CD34(+) cell dose was much larger during the first collection: 45.2+/-8.4 x 10(6)/kg vs. 6.9+/-2.7 x 10(6)/kg (P<0.001). Similarly, the collected colony-forming unit (CFU)-GM dose was much larger during the first collection: 295.4+/-59.3 x 10(4)/kg vs. 67.3+/-21.6x10(4)/kg (P<0.001). While the CD34(+) cells collected during the two collection episodes correlated significantly (r=0.55, P<0.01); the first dose was a median of 14.9-fold larger. DISCUSSION: No laboratory parameter was able reliably to predict the results of the second collection. A second mobilization/collection episode as part of a tandem transplant approach carries a considerable risk of failing to obtain sufficient progenitor cells.     

Redox state and carbonic anhydrase isozyme IX expression in human renal cell carcinoma: biochemical and morphological investigations

Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis (p < 0.05). Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression (p = 0.057). The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC.     

Effect of water on the molecular mobility of elastin

Purified and hydrated elastin is studied by both thermal and dielectric techniques to have insight into the chain dynamics of this protein. By differential scanning calorimetry, the glassy behavior of elastin is highlighted; the glass transition temperature (T(g)) of elastin is found to be widely dependent on hydration, falling from 200 degrees C in the dehydrated state to 30 degrees C for 30% hydration. A limit of T(g) at around 0 degrees C is found when crystallizable water is present in the system, that is, when the formation of ice prevents motions of some 10 nm along the polypeptidic chains. The technique of thermally stimulated currents, carried out in the -180 to 0 degrees C temperature range, is useful to detect localized motions. In this case, too, the localized motions vary considerably according to hydration: a first relaxation mode is observed at -145 degrees C and it is associated with the reorientation of crystallizable water in ice I; a second relaxation mode, more complex and cooperative, occurs at around -80 degrees C and could be attributed to the complex constituted by the dipolar groups of the polypeptidic chain and noncrystallizable water, behaving as a glassy system.     

New semidominant mutations that affect mouse development

Dominantly acting mutations that produce visible phenotypes are frequently recovered, either during routine maintenance of colonies or from mutagenesis experiments. We have studied 12 dominant mouse mutations that cause a tail dysmorphology, a coat spotting phenotype, or a combination of these. The majority of these mutations act in a semidominant manner with the homozygous state associated with embryonic lethality and a visible phenotype at or before midgestation. The homozygous phenotypes include axis truncation and neural crest cell defects, as may be expected from the heterozygous phenotypes. The majority of mutations, however, also produced other phenotypes that include neural tube closure defects and aberrant heart looping. In one coat spotting mutant the homozygous condition is lethal before neural crest cell production commences. The mutated genes often function in processes additional to those alluded to by the heterozygous phenotype.     

Benzimidazole derivatives. Part 5: design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT(1A) and 5-HT(3) receptors. Compounds 1-11 were synthesized and evaluated for binding affinity at both serotoninergic receptors, all of them exhibiting high 5-HT(3)R affinity (K(i)=10-62nM), and derivatives with an o-alkoxy group in the arylpiperazine ring showing nanomolar affinity for the 5-HT(1A)R (K(i)=18-150nM). Additionally, all the synthesized compounds were selective over alpha(1)-adrenergic and dopamine D(2) receptors (K(i)>1000-10,000nM). Compound 3 was selected for further pharmacological characterization due to its interesting binding profile as mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both receptors (5-HT(1A): K(i)=18.0nM, 5-HT(3): K(i)=27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial agonist at 5-HT(1A)Rs and as a 5-HT(3)R antagonist. This novel mixed 5-HT(1A)/5-HT(3) ligand was also effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the treatment of cognitive dysfunction.     

Expression, purification, and characterization of authentic monoferric and apo-human serum transferrins

Transferrin is a bilobal protein with the ability to bind iron in two binding sites situated at the bottom of a cleft in each lobe. We have previously described the production of recombinant non-glycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at the amino-terminus. Constructs in this background that contain strategic mutations to completely prevent iron binding in each lobe or in both lobes have now been produced. These monoferric hTFs will allow dissection of the contribution of each lobe to transferrin function. In addition, the construct completely lacking in the ability to bind iron in either lobe provides an opportunity to assess whether hTF has any other functions in addition to iron transport. Following insertion of the His-tagged hTF molecules into the pNUT vector, transfection into baby hamster kidney cells and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium and characterized with regard to their iron binding properties. Significant improvements over our previous protocol include: (1) addition of butyric acid at a level of 1mM which leads to a substantial increase in protein production (as much as a 65% increase compared to control cells); and (2) elimination of an anion exchange column prior to isolation on a Qiagen Ni-NTA column which makes purification of the His-tagged constructs faster and therefore more efficient. These improvements should be applicable to expression of other recombinant proteins in mammalian cells.     

Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP

We have determined a 2.1 A crystal structure for human mitochondrial ClpP (hClpP), the proteolytic component of the ATP-dependent ClpXP protease. HClpP has a structure similar to that of the bacterial enzyme, with the proteolytic active sites sequestered within an aqueous chamber formed by face-to-face assembly of the two heptameric rings. The hydrophobic N-terminal peptides of the subunits are bound within the narrow (12 A) axial channel, positioned to interact with unfolded substrates translocated there by the associated ClpX chaperone. Mutation or deletion of these residues causes a drastic decrease in ClpX-mediated protein and peptide degradation. Residues 8-16 form a mobile loop that extends above the ring surface and is also required for activity. The 28 amino acid C-terminal domain, a unique feature of mammalian ClpP proteins, lies on the periphery of the ring, with its proximal portion forming a loop that extends out from the ring surface. Residues at the start of the C-terminal domain impinge on subunit interfaces within the ring and affect heptamer assembly and stability. We propose that the N-terminal peptide of ClpP is a structural component of the substrate translocation channel and may play an important functional role as well.     

Phosphoinositides,ezrin/moesin,and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.     

Src SH3/2 domain-mediated peripheral accumulation of Src and phospho-myosin is linked to deregulation of E-cadherin and the epithelial-mesenchymal transition

Elevated Src kinase in epithelial cancer cells induces adhesion changes that are associated with a mesenchymal-like state. We recently showed that Src induces dynamic integrin adhesions in KM12C colon cancer cells, whereas E-cadherin-dependent cell-cell contacts become disorganized. This promotes a fibroblastic-like morphology and expression of the mesenchymal marker vimentin. Furthermore, Src-induced deregulation of E-cadherin, and the associated mesenchymal transition, is dependent on integrin signaling (Avizienyte et al., Nat. Cell Biol. 2002, 4, 632-638), although the nature of downstream signals that mediate these Src- and integrin-dependent effects are unknown. Here we show that the SH2 and SH3 domains of Src mediate peripheral accumulation of phospho-myosin, leading to integrin adhesion complex assembly, whereas loss of SH2 or SH3 function restores normal regulation of E-cadherin and inhibits vimentin expression. Inhibitors of MEK, ROCK, or MLCK also suppress peripheral accumulation of phospho-myosin and Src-induced formation of integrin-dependent adhesions, whereas at the same time restoring E-cadherin redistribution to regions of cell-cell contact. Our data therefore implicate peripheral phospho-myosin activity as a point of convergence for upstream signals that regulate integrin- and E-cadherin-mediated adhesions. This further implicates spatially regulated contractile force as a determinant of epithelial cell plasticity, particularly in cancer cells that can switch between epithelial and mesenchymal-like states.