Functional analysis and intracellular localization of the human menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1)

The Menkes protein (MNK or ATP7A) is an important component of the mammalian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure and function of this protein and to elucidate its role in cellular copper homeostasis, a cDNA construct encoding the full-length MNK protein was cloned into a mammalian expression vector under the control of the CMV promoter. Transfection of this plasmid construct into CHO-K1 cells yielded clones that expressed MNK at varying levels. Detailed characterization of four clones showed that an increase in MNK protein expression led to a corresponding increase in the level of copper resistance of the cells. Subcellular localization studies showed that in the parental CHO-K1 and the transfected cell lines, MNK was located in a post-Golgi compartment which, based on immunogold electron microscopic analyses, most likely represented the trans -Golgi network (TGN). When the extracellular copper concentration was increased, MNK in the clones as well as in CHO-K1 cells was redistributed to the cytoplasm and plasma membrane, but returned to the TGN under basal, low copper conditions. This report presents the first ultrastructural evidence for the association of MNK with vesicles within the cell and with the TGN and plasma membrane. It also demonstrates the stable expression of a functional MNK protein from a cDNA construct in mammalian cells, as well as the copper-induced redistribution of MNK in a cell line (CHO-K1) that was not selected for copper resistance or overexpression of MNK.      

Thromboxane A2 causes feedback amplification involving extensive thromboxane A2 formation on close contact of human platelets in media with a low concentration of ionized calcium

Close platelet-to-platelet contact induced by weak agonists in a medium with a low concentration of Ca2+ leads to thromboxane A2 (TXA2) formation, release of granule contents, and secondary aggregation. These responses do not occur in a medium containing Ca2+ in the physiological range (1 to 2 mmol/L). Experiments were done to determine whether feedback amplification is required to generate amounts of TXA2 that are sufficient to cause secondary aggregation and the reactions associated with it, or whether close platelet-to-platelet contact alone is sufficient to generate enough TXA2 to produce these responses. Platelets were washed and resuspended in a modified Tyrode solution to which no calcium salt was added that contained 0.35% albumin and apyrase. This medium contains 20 mumol/L Ca2+ and 1 mmol/L Mg2+. Platelets were aggregated with adenosine diphosphate (ADP) in the presence of fibrinogen, agglutinated with polylysine, or after pretreatment with chymotrypsin, aggregated with fibrinogen. In the low- Ca2+ medium, all these agonists caused platelets to adhere to each other, followed by secondary aggregation with TXA2 formation and release of granule contents. When Ca2+ (1 to 2 mmol/L), aspirin, or the thromboxane receptor blocker BM 13.177 was present, the secondary responses did not occur; dazoxiben decreased thromboxane formation, but did not prevent secondary aggregation or release. Aspirin-treated platelets were less responsive to ADP, U46619, or TXA2 in the low-Ca2+ medium, which indicated that the secondary responses of untreated platelets were not caused by a generalized increase in sensitivity. The reactions that result from close platelet-to-platelet contact in a low- Ca2+ medium can be caused by a wide variety of weak agonists; the secondary aggregation response and release of granule contents are dependent on TXA2 formation and on feedback amplification by TXA2 or the prostaglandin endoperoxides. The secondary responses caused by weak agonists in citrated platelet-rich plasma (which has a concentration of Ca2+ similar to the low-Ca2+ medium used in the present studies) do not occur at the concentration of Ca2+ in circulating blood and thus may have little biologic relevance.     

Selective treatment of SCID mice bearing methotrexate-transport- resistant human acute lymphoblastic leukemia tumors with trimetrexate and leucovorin protection

Impaired transport of methotrexate (MTX) is a common resistance mechanism of tumor cells to this drug. Trimetrexate (TMTX), a second- generation folate antagonist, is still active against MTX-transport- resistant cells because it enters cells by passive diffusion and does not use the reduced folate transport system for cell entry. Therefore, although leucovorin (LV) protects MTX-sensitive cells from TMTX toxicity, MTX-transport defective cells are poorly rescued by LV. Severe combined immunodeficiency mice bearing MTX-transport-resistant CCRF-CEM acute lymphoblastic leukemia tumors were treated with TMTX alone or with the combination of TMTX and LV, with tumor regressions in both groups (P < .001) and without significant toxicity. These results indicate that TMTX with LV protection may be a useful therapeutic regimen for patients with MTX-transport-defective acute lymphoblastic leukemia. Furthermore, resistance to TMTX plus LV may result in reversion to MTX sensitivity.     

The effect of EDTA, cations, and various buffers on the morphology of erythrocyte membranes: an electron-microscopic study

Membranes of human erythrocytes were prepared by stepwise osmotic hemolysis in Ca2+-free solutions. Examination with the electron microscope after negative staining showed some short, conelike protuberances on the surface of about 20 percent of the ghosts, while 80 percent were round, intact spheres. After Ca2+ treatment, all membranes were round and intact. After exposure to ethylenediaminetetraacetic acid (EDTA) (1.0 mM, pH 7.4), the entire ghost surface was covered with long, thin extrusions called stromalytic forms (about 460 per cell). Their sizes, shapes, and fine structure are described. Exposure to ionic calcium (1.4 times 10-minus 4M) abolished the EDTA-induced stromalytic forms. A second exposure to EDTA reversed this Ca2+ effect. ATP, like EDTA, produced stromalytic forms. EDTA- induced stromalytic forms were also abolished by Zn2+, La3+, and Nd3+ at concentrations of 1-5 times 10-minus 4 M. Mg2+ at 10-minus 2 M was ineffective. Ghosts were prepared by graded lysis in various buffers. Those prepared in phosphate were the most stable and provided consistent EDTA effects and Ca2+ reversal. Ghosts in Tris-HCl showed breakdown unless salt was added. Moderately satisfactory ghosts were also obtained in Hepes-NaOH buffer and salt.     

The cDNA and derived amino acid sequence of porcine factor VIII

The cDNA corresponding to 137 bp of the 5' untranslated region, the signal peptide, and the A1, A3, C1, and C2 domains of porcine factor VIII (fVIII) have been cloned and sequenced. Along with previously determined sequences of the porcine fVIII B domain and the A2 domain, this completes the sequence determination of the cDNA corresponding to the translated protein. Alignments of the derived amino acid sequence of porcine fVIII with human and murine fVIII indicate that the A1, A2, A3, C1, and C2 domains are more conserved than the B domains or the proteolytic cleavage peptides corresponding to residues 337-372 and 1649-1689. The knowledge of the porcine fVIII cDNA may be useful to understand functional and immunological differences between human and porcine fVIII and may lead to improved fVIII replacement products for hemophilia. A patients through the development of recombinant porcine fVIII or hybrid human/porcine fVIII derivatives.     

Individual Differences in Cue-Induced Motivation and Striatal Systems in Rats Susceptible to Diet-Induced Obesity

Pavlovian cues associated with junk-foods (caloric, highly sweet, and/or fatty foods), like the smell of brownies, can elicit craving to eat and increase the amount of food consumed. People who are more susceptible to these motivational effects of food cues may have a higher risk for becoming obese. Further, overconsumption of junk-foods leading to the development of obesity may itself heighten attraction to food cues. Here, we used a model of individual susceptibility to junk-foods diet-induced obesity to determine whether there are pre-existing and/or diet-induced increases in attraction to and motivation for sucrose-paired cues (ie, incentive salience or ‘wanting’). We also assessed diet- vs obesity-associated alterations in mesolimbic function and receptor expression. We found that rats susceptible to diet-induced obesity displayed heightened conditioned approach prior to the development of obesity. In addition, after junk-food diet exposure, those rats that developed obesity also showed increased willingness to gain access to a sucrose cue. Heightened ‘wanting’ was not due to individual differences in the hedonic impact (‘liking’) of sucrose. Neurobiologically, Mu opioid receptor mRNA expression was lower in striatal ‘hot-spots’ that generate eating or hedonic impact only in those rats that became obese. In contrast, prolonged exposure to junk-food resulted in cross-sensitization to amphetamine-induced locomotion and downregulation of striatal D2R mRNA regardless of the development of obesity. Together these data shed light on individual differences in behavioral and neurobiological consequences of exposure to junk-food diets and the potential contribution of incentive sensitization in susceptible individuals to greater food cue-triggered motivation.