Inhibition of Cdk2 Activation by Selected Tyrphostins Causes Cell Cycle Arrest at Late G1 and S Phase

We have previously reported that certain tyrphostins which block EGF-R phosphorylation in cell-free systems fail to do so in intact cells. Nevertheless, we found that this family of tyrphostins inhibits both EGF- and calf serum-induced cell growth and DNA synthesis [Osherov, N.A., Gazit, C., Gilon, and Levitzki, A. (1993). Selective inhibition of the EGF and HER2/Neu receptors by Tyrphostins.J. Biol. Chem.268, 11134–11142.] Now we show that these tyrphostins exert their inhibitory activity even when added at a time when the cells have already passed their restriction point and receptor activation is no longer necessary. AG555 and AG556 arrest 85% of the cells at late G1, whereas AG490 and AG494 cause cells to arrest at late G1 and during S phase. No arrest occurs during G2 or M phase. Further analysis revealed that these tyrphostins act by inhibiting the activation of the enzyme Cdk2 without affecting its levels or its intrinsic kinase activity. Furthermore, they do not alter the association of Cdk2 to cyclin E or cyclin A or to the inhibitory proteins p21 and p27. These compounds also have no effect on the activating phosphorylation of Cdk2 by Cdk2 activating kinase (CAK) and no effect on the catalytic domain of cdc25 phosphatase. These compounds lead to the accumulation of phosphorylated Cdk2 on tyrosine 15 which is most probably the cause for its inhibition leading to cell cycle arrest at G1/S. A structure–activity relationship study defines a very precise pharmacophore, suggesting a unique molecular target not yet identified and which is most probably involved in the regulation of the tyrosine-phosphorylated state of Cdk2. These compounds represent a new class of cell proliferation blockers whose target is Cdk2 activation.     

Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease

Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation (delta F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cystic fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57 (60%) are of Ashkenazi origin. Together with the delta F508 (23% in this group), G542X, N1303K, and 1717-1G----A mutations, the identification of 92% of cystic fibrosis chromosomes of Ashkenazi origin becomes possible. Patients homozygous for the W1282X mutation (n = 16) and patients heterozygous for the delta F508 and W1282X mutations (n = 22) had similarly severe disease, reflected by pancreatic insufficiency, high incidence of meconium ileus (37% and 27%, respectively), early age at diagnosis, poor nutritional status, and variable pulmonary function. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease.     

The effect of blue light on energy levels in epidermal strips

Red light applied together with blue enhanced stomatal opening in epidermal strips of Commelina communis L. more than red light alone. In red light, stomatal opening was enhanced by exogenously applied ATP and was inhibited by 3-(3,4-dichlorophe-nyl)-l,l-dimethylurea (DCMU), while in the presence of blue light external ATP was almost without effect, and DCMU stimulated stomatal opening. Blue light increased the ATP levels in the epidermal strips. DCMU diminished the amount of ATP in both red light and red + blue light treatments, but did not abolish the stimulatory effect of blue light. Blue light also stimulated the respiration rate of the epidermal strips. Rotenone, which inhibited stomatal opening and respiration rate, abolished the effect of blue light in both processes. These results imply that blue light increases the ATP levels by stimulation of oxidative phosphorylation.     

Scanning electron microscopic analysis of intramyocellular lipid droplets in an animal model of type 2 diabetes

Objective: To evaluate the accumulation pattern of intramyocellular lipids (IMCLs) in striated muscle during the development and progression of diabetes, using a novel scanning electron microscopic method. Methods and Procedures: Hyperglycemia was induced by feeding diabetes‐prone (DP) Psammomys obesus a high‐energy (HE) diet. Lipid accumulation within gastrocnemius muscle fibers was assessed in formalin‐fixed muscle samples during the development of hyperglycemia using high resolution imaging in a scanning electron microscope. We evaluated the temporal relationship between changes in IMCL quantity and morphology and the altered glucose metabolism and assessed the effect of reversal of hyperglycemia on IMCL level and morphology. Diabetes‐resistant (DR) P. obesus served as controls. Results: Lipid accumulation in the muscle fibers of DP animals was increased with the development of hyperglycemia. This was characterized by increased lipid density as well as by an abundance of large lipid droplets. Reversal of the phenotype resulted in the disappearance of large lipid droplets. The IMCL level and the distribution of lipid droplet size were similar in muscles of both the normoglycemic DR and DP animals, with an abundance of small lipid droplets. This profile was changed following a HE diet only in the DP animals. Discussion: Lipid accumulation in the muscle of P. obesus during the development of hyperglycemia is characterized by increased quantity and accumulation of large lipid droplets. These changes were reversible upon normalization of blood glucose. The evaluated methodology is a useful tool for the study of the dynamics of lipid accumulation in different metabolic conditions.     

Ubiquitin-proteasome-dependent degradation of a mitofusin, a critical regulator of mitochondrial fusion

The mitochondrion is a dynamic membranous network whose morphology is conditioned by the equilibrium between ongoing fusion and fission of mitochondrial membranes. In the budding yeast, Saccharomyces cerevisiae, the transmembrane GTPase Fzo1p controls fusion of mitochondrial outer membranes. Deletion or overexpression of Fzo1p have both been shown to alter the mitochondrial fusion process indicating that maintenance of steady-state levels of Fzo1p are required for efficient mitochondrial fusion. Cellular levels of Fzo1p are regulated through degradation of Fzo1p by the F-box protein Mdm30p. How Mdm30p promotes degradation of Fzo1p is currently unknown. We have now determined that during vegetative growth Mdm30p mediates ubiquitylation of Fzo1p and that degradation of Fzo1p is an ubiquitin-proteasome-dependent process. In vivo, Mdm30p associates through its F-box motif with other core components of Skp1-Cullin-F-box (SCF) ubiquitin ligases. We show that the resulting SCF(Mdm30p) ligase promotes ubiquitylation of Fzo1p at mitochondria and its subsequent degradation by the 26S proteasome. These results provide the first demonstration that a cytosolic ubiquitin ligase targets a critical regulatory molecule at the mitochondrial outer membrane. This study provides a framework for developing an understanding of the function of Mdm30p-mediated Fzo1p degradation in the multistep process of mitochondrial fusion.     

MicroRNAs accurately identify cancer tissue origin

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.     

Growth inhibition of myeloma cells by anti-idiotype antibodies in the absence of membrane-bound immunoglobulin

Immunoglobulins are expressed as membrane-bound or secreted forms. Plasma cells produce little or no membrane immunoglobulin but secrete immunoglobulin molecules in large amounts. Immunoglobulin idiotypes of malignant B cells are tumor-specific antigens that may be targeted for immunotherapy. Thus, idiotype vaccination is being evaluated in clinical trials to control residual disease in multiple myeloma and non-Hodgkin's lymphoma. It is traditionally considered that anti-idiotype antibodies are not effective against plasma cell tumors, because the large amounts of immunoglobulin molecules secreted by the tumors block anti-idiotype antibodies, and because the absence of membrane immunoglobulin on the surface of these tumor cells renders them resistant to the effect of anti-idiotype antibodies. While the obstacle of abundant circulating idiotype may be obviated by reducing tumor burden to minimal residual disease, the absence of membrane immunoglobulin has been considered as a limiting factor that prevents tumor eradication by anti-idiotype antibodies. We demonstrate here that murine plasmacytoma cells can produce small amounts of membrane immunoglobulin M (IgM) heavy chains. However, the latter are precursor molecules that do not reach the cell surface. Although membrane-bound IgM is absent, the cells stain positively for surface IgM, reflecting molecules of the secreted form in the process of secretion. In spite of the relatively low levels of secreted immunoglobulin on the cell surface, anti-idiotype antibodies are effective in retardation of tumor growth in vivo. Thus, while there is no doubt that idiotype-specific cell-mediated responses are very important, myeloma patients in complete remission may additionally benefit from idiotype-specific humoral responses.     

Electrostatic contributions drive the interaction between Staphylococcus aureus protein Efb-C and its complement target C3d

The C3-inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate subdomain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. In order to characterize more completely the physical and chemical driving forces behind this important interaction, we employed in this study a combination of structural, biophysical, and computational methods to analyze the interaction of C3d with Efb-C and the single-point mutants R131A and N138A. Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.     

Conjugates of gonadotropin releasing hormone (GnRH) with carminic acid: Synthesis, generation of reactive oxygen species (ROS) and biological evaluation

We synthesized two carminic acid (7-alpha-d-glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxo-2-anthracene carboxlic acid, CA)-GnRH conjugates to be used as a model for potential photoactive targeted compounds. CA was conjugated to the epsilon-amino group of [d-Lys(6)]GnRH through its carboxylic moiety or via a beta-alanine spacer (beta-ala). Redox potentials of CA and its conjugates were determined. We used electron spin resonance (ESR) and spin trapping techniques to study the light-stimulated redox properties of CA and its CA-GnRH conjugates. Upon irradiation, the compounds stimulated the formation of reactive oxygen species (ROS), that is, singlet oxygen ((1)O(2)) and oxygen radicals (O(2)(-*) and OH(*)). Both conjugates exhibited higher ROS production than the non-conjugated CA. The bioactivity properties of the CA conjugates and the parent peptide, [d-Lys(6)]GnRH, were tested on primary rat pituitary cells. We found that the conjugates preserved the bioactivity of GnRH as illustrated by their capability to induce ERK phosphorylation and LH release.