4-Hydroxynonenal affects pRb/E2F pathway in HL-60 human leukemic cells

4-Hydroxynonenal (HNE), a highly reactive product of lipid peroxidation, has an antiproliferative effect in several tumor cell lines and provokes alteration of cell cycle progression in HL-60 cells. HNE down-regulates c-myc expression in K562, HL-60, and MEL cells. This prompted us to study the cascade of phenomena that, starting from the CKIs expression and the phosphorylation of pRb, arrives at the E2F binding to consensus sequence in the P2 promoter of the c-myc gene. Treatment of HL-60 cells with HNE (1 microM) causes a p53-independent increase of p21(WAF1/CIP1) expression, pRb dephosphorylation, a decrease of low molecular weight E2F complexes and an increase of high molecular weight E2F complexes bound to P2 c-myc promoter. E2F4 expression is reduced by HNE treatment as well as the amount of pRb/E2F4 complexes, whereas the amount of pRb/E2F1 complexes is increased. In conclusion, HNE can affect the pRb/E2F pathway by modifying the expression of several genes involved in the control of cell proliferation.     

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.     

Involvement of chloroplasts in the programmed death of plant cells

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN^–-induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN^–-induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN^–-induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Q_o site of the chloroplast cytochrome b ₆ f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.     

Estrogens (E2) regulate expression and response of 1,25-dihydroxyvitamin D3 receptors in bone cells: changes with aging and hormone deprivation

Studies on the effect of estrogens (E(2)) on the expression of vitamin D receptor (VDR) and its bioresponse in bone have demonstrated that E(2) modulate activity and increase the number of VDRs in vitro; however, no in vivo studies have been pursued to assess this interaction. Our study identifies the changes in the number of VDR-expressing cells in bone of C57BL/6J young and old oophorectomized mice (4 and 24 months) with and without 17beta estradiol (E(2)) replacement. A total of 36 mice were sacrificed; both tibiae and femora were isolated and VDR expression was quantified by Northern blot, immunohistochemistry, immunofluorescence, and flow cytometry. Among the intact mice there was a significant difference in the number of VDR-expressing osteoblasts between young (68%) and old (56%) (p<0.04). In young oophorectomized mice the number of VDR-expressing osteoblasts decreased from 68% to 46% after oophorectomy and recovered to 72% after E(2) administration (p<0.02), while in the group of old mice, the number of VDR-expressing osteoblasts decreased from 56% to 48% after oophorectomy (p<0.01) and recovered to 85% after E(2) administration (p<0.001). Our results show that VDR expression in bone decreases with aging and estrogen deprivation but recovers after E(2) supplementation in both young and old mice with a more significant level of response in older bone. To evaluate the level of VDR bioresponse to E(2) we assessed the effect of E(2) supplementation to human osteoblasts (N-976) in vitro. Northern blot showed a significant up-regulation of VDR expression in E(2) treated cells as compared to non-treated cells (p<0.05). We also assessed the previously known anti-apoptotic effect of vitamin D in osteoblasts in vitro after serum deprivation by using either E(2), E(2)+1,25(OH)(2)D(3), or 1,25(OH)(2)D(3) alone. We found a lower number of apoptotic cells and longer cell survival after 48 h of treatment with 1,25(OH)(2)D(3)+E(2) as compared to 1,25(OH)(2)D(3) or E(2) alone (p<0.002). In summary, our results demonstrate that E(2) increases VDR expression in bone in vivo and potentiate the bioresponse of VDR in osteoblasts in vitro.     

Protein kinase D is a downstream target of protein kinase Ctheta

Protein kinase D (PKD/PKCmu immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCtheta AE (PKCthetaAE) exhibited a marked increase in basal activity. In contrast, coexpression of constitutively active mutant of PKCzeta does not induce PKD activation in both types of cells. PKCthetaAE does not induce kinase activity in immunocomplexes of PKD kinase-deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCthetaAE signaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Our results show that PKD is a downstream target of the theta isoform of PKC in both COS-7 cells and lymphocytes. The regulation of PKD by PKCtheta reveals a new pathway in the signaling network existing between multiple members of the PKC superfamily and PKD.     

Automated multi-dimensional purification of tagged proteins

The capacity for high throughput purification (HTP) is essential in fields such as structural genomics where large numbers of protein samples are routinely characterized in, for example, studies of structural determination, functionality and drug development. Proteins required for such analysis must be pure and homogenous and available in relatively large amounts. ÄKTAT 3D system is a powerful automated protein purification system, which minimizes preparation, run-time and repetitive manual tasks. It has the capacity to purify up to 6 different His₆- or GST-tagged proteins per day and can produce 1–50 mg protein per run at >90% purity. The success of automated protein purification increases with careful experimental planning. Protocol, columns and buffers need to be chosen with the final application area for the purified protein in mind.     

Nuclear lipids: New functions for old molecules?

It is becoming increasingly evident that stimulation of nuclear lipid metabolism plays a central role in many signal transduction pathways that ultimately result in various cell responses including proliferation and differentiation. Nuclear lipid metabolism seems to be at least as complex as that existing at the plasma membrane. However, a distinctive feature of nuclear lipid biochemical pathways is their operational independence from their cell periphery counterparts. Although initially it was thought that nuclear lipids would serve as a source for second messengers, recent evidence points to the likelihood that lipids present in the nucleus also fulfil other roles. The aim of this review is to highlight the most intriguing advances made in the field over the last year, such as the production of new probes for the in situ mapping of nuclear phosphoinositides, the identification of two sources for nuclear diacylglycerol production, the emerging details about the peculiar regulation of nuclear phosphoinositide synthesizing enzymes, and the distinct possibility that nuclear lipids are involved in processes such as chromatin organization and pre-mRNA splicing.     

A new active vitamin D analog,ED-71, causes increase in bone mass with preferential effects on bone in osteoporotic patients

As a candidate for active vitamin D analogs that have selective effects on bone, 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71) has been synthesized and is currently under clinical trials. In ovariectomized rat model for osteoporosis, ED-71 caused an increase bone mass at the lumbar vertebra to a greater extent than 1alpha-hydroxyvitamin D3 (alfacalcidol), while enhancing calcium absorption and decreasing serum parathyroid hormone levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers.An early phase II clinical trial was conducted with 109 primary osteoporotic patients. The results indicate that oral daily administration of ED-71 (0.25, 0.5, 0.75, and 1.0 microgram) for 6 months increased lumbar bone mineral density in a dose-dependent manner without causing hypercalcemia and hypercalciuria. ED-71 also exhibited a dose-dependent suppression of urinary deoxypyridinoline with no significant reduction in serum osteocalcin. These results demonstrate that ED-71 has preferential effects on bone with diminished effects on intestinal calcium absorption. ED-71 offers potentially a new modality of therapy for osteoporosis with selective effects on bone.     

1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis,Jun kinase activation,and interleukin-6 production in primary human keratinocytes

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis.