Gene expression of D-beta-hydroxybutyrate dehydrogenase. III. Molecular cloning of a DNAc

In order to continue the molecular studies of D-beta-hydroxybutyrate dehydrogenase (BDH) undertaken in our laboratory for several years, we have initiated a genetic approach which consists in the BDH cDNA cloning from a rat liver cDNA library. The immunoscreening method allowed to isolate a clone which exhibits a DNA insert shorter than the expected full length BDH cDNA.     

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability.

Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51- to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability.     

Behavior of plasma insulin and GIP in obese patients subjected to biliopancreatic bypass

Biliopancreatic bypass for obesity entails a 2/3 distal gastrectomy with Roux-en-Y reconstruction, being the small bowel transected at its midpoint and the enteroenteroanastomosis placed 50 cm proximal to the ileocecal valve. Insulin and GIP fasting and meal-stimulated plasma concentrations were determined in 13 nonobese healthy volunteers, in 13 nonoperated obese patients, in 11 subjects within two months, in 12 subjects four to twelve months, and in 7 subjects fifteen to twenty months after operation. Insulin in the obese patients was significantly higher than in the control group. Postoperatively these patients showed a sharp reduction in basal and postprandial values. Plasma insulin levels, both basally and following the test meal, were very similar in the 15-20 month and the control group. Plasma GIP fasting level, meal-stimulated peak and integrated response in the obese group were higher than in control group. Due to the extreme variability among subjects in the obese group, the difference was significant only for the mean peak response. All values were greatly reduced after surgery. The mean fasting level in the 15-20 month group was very similar to that in the control group, and both peak and integrated responses were significantly lower than in the preoperative and control groups.     

Evolution of complex modular biological networks

Biological networks have evolved to be highly functional within uncertain environments while remaining extremely adaptable. One of the main contributors to the robustness and evolvability of biological networks is believed to be their modularity of function, with modules defined as sets of genes that are strongly interconnected but whose function is separable from those of other modules. Here, we investigate the in silico evolution of modularity and robustness in complex artificial metabolic networks that encode an increasing amount of information about their environment while acquiring ubiquitous features of biological, social, and engineering networks, such as scale-free edge distribution, small-world property, and fault-tolerance. These networks evolve in environments that differ in their predictability, and allow us to study modularity from topological, information-theoretic, and gene-epistatic points of view using new tools that do not depend on any preconceived notion of modularity. We find that for our evolved complex networks as well as for the yeast protein–protein interaction network, synthetic lethal gene pairs consist mostly of redundant genes that lie close to each other and therefore within modules, while knockdown suppressor gene pairs are farther apart and often straddle modules, suggesting that knockdown rescue is mediated by alternative pathways or modules. The combination of network modularity tools together with genetic interaction data constitutes a powerful approach to study and dissect the role of modularity in the evolution and function of biological networks.     

Immunological study of the tissue expression of D-beta-hydroxybutyrate dehydrogenase, a ketone body-converting enzyme

In rats, as in most mammal, ketone bodies are mainly produced in liver while they are metabolized in extrahepatic tissues. The expression of mitochondrial membrane-bound D-beta-hydroxybutyrate dehydrogenase (BDH), a ketone body-converting enzyme, has been estimated by two immunological techniques: immunohistofluorescence and Western blotting. The in situ labeling with anti-BDH antibody shows that the enzyme is expressed differently among the organs. Furthermore, within a given organ there are strong differences according to the cell type. The quantification of the enzyme by immunoblotting reveals that liver mitochondria have the highest content (more than 3% in protein mass). This content is 3,5 and 10 times lower in kidney, heart and brain mitochondria, respectively. Parallel D-beta-hydroxybutyrate dehydrogenase activity measurements on isolated mitochondria show differences in molecular activity of this enzyme according to the tissue origin. Due to the phospholipid requirement of this enzyme these differences in molecular activity are related to specific membrane lipid composition.     

Rhodamine phalloidin F-actin: critical concentration versus tensile strength.

The mechanic and elastic properties of rhodamine phalloidin F-actin were investigated as a function of the ionic strength and in the absence of Mg2+. By increasing ionic strength from 3 to 19 mM, critical concentration decreased from 146 to 36 nM and the yield strength increased from 5.6 pN to 28.6 pN. At the ionic strength of 12-13 mM, the elastic modulus by stretching increased by 330-430 kP. nm-1 up to the break point, where it was 38-44.2 MP. The work required to break the filament, 403-439 kJ.M-1 provides an estimate of the free energy of annealing of rhodamine phalloidin F-actin, the annealing constant being 2.8 x 1074 M-1.     

Synthesis,antiproliferative and mitochondrial impairment activities of bis-alkyl-amino transplatinum complexes

A convenient synthetic route and the characterization of complexes trans-[PtCl₂(L)(PPh₃)] (L = Et₂NH (2), (PhCH₂)₂NH (3), (HOCH₂CH₂)₂NH) (4) are reported. The antiproliferative activity was evaluated on three human tumor cell lines. The investigation on the mechanism of action highlighted for the most active complex 4 the capacity to affect mitochondrial functions. In particular, both the induction of the mitochondrial permeability transition phenomenon and an aspecific membrane damage occurred, depending on concentration.     

Inhibition of protein kinase CK2 with the clinical-grade small ATP-competitive compound CX-4945 or by RNA interference unveils its role in acute myeloid leukemia cell survival,p53-dependent apoptosis and daunorubicin-induced cytotoxicity

Background

The involvement of protein kinase CK2 in sustaining cancer cell survival could have implications also in the resistance to conventional and unconventional therapies. Moreover, CK2 role in blood tumors is rapidly emerging and this kinase has been recognized as a potential therapeutic target. Phase I clinical trials with the oral small ATP-competitive CK2 inhibitor CX-4945 are currently ongoing in solid tumors and multiple myeloma.

Methods

We have analyzed the expression of CK2 in acute myeloid leukemia and its function in cell growth and in the response to the chemotherapeutic agent daunorubicin We employed acute myeloid leukemia cell lines and primary blasts from patients grouped according to the European LeukemiaNet risk classification. Cell survival, apoptosis and sensitivity to daunorubicin were assessed by different means. p53-dependent CK2-inhibition-induced apoptosis was investigated in p53 wild-type and mutant cells.

Results

CK2α was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34⁺ hematopoietic and bone marrow cells. Inhibition of CK2 with CX-4945, K27 or siRNAs caused a p53-dependent acute myeloid leukemia cell apoptosis. CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin. Baseline and daunorubicin-induced STAT3 activation was hampered upon CK2 blockade.

Conclusions

These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival. CK2 negative regulation of the protein levels of tumor suppressor p53 and activation of the STAT3 anti-apoptotic pathway might antagonize apoptosis and could be involved in acute myeloid leukemia cell resistance to daunorubicin.