AKT as locus of cancer angiogenic robustness and fragility

Angiogenesis get full robustness in metastatic cancer, relapsed leukemia or lymphoma when complex positive feedback loop signaling systems become integrative. A cancer hypoxic microenvironment generates positive loops inducing formation of the vascular functional shunts. AKT is an upstream angiogenic locus of integrative robustness and fragility activated by the positive loops. AKT controls two downstream nodes the mTOR and NOS in nodal organization of the signaling genes. AKT phosphorylation is regulated by a balance of an oxidant/antioxidant. Targeting AKT locus represents new principle to control integrative angiogenic robustness by the locus chemotherapy. J. Cell. Physiol. 228: 21–24, 2013. © 2012 Wiley Periodicals, Inc.     

Ataxia-telangiectasia: Linkage analysis in highly inbred Arab and Druze families and differentiation from an ataxia-microcephaly-cataract syndrome

Summary Ataxia-telangiectasia (A-T) is a progressive autosomal recessive disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation sensitivity and a highly increased proneness to cancer. A-T is ethnically widespread and genetically heterogeneous, as indicated by the existence of four complementation groups in this disease. Several A-T-like genetic diseases share various clinical and cellular characteristics with A-T. By using linkage analysis to study North American and Turkish A-O families, the ATA (A-T, complementation group A) gene has been mapped to chromosome 11q23. A number of Israeli Arab A-T patients coming from large, highly inbred families were assigned to group A In one of these families, an additional autosomal recessive disease was identified, characterized by ataxia, hypotonia, microcephaly and bilateral congenital cataracts. In two patients with this syndrome, normal levels of serum immunoglobulins and alpha-fetoprotein, chromosomal stability in peripheral blood lymphocytes and skin fibroblasts, and normal cellular response to treatments with X-rays and the radiomimetic drug neocarzinostatin indicated that this disease does not share, with A-T, any additional features other than ataxia. These tests also showed that another patient in this family, who is also mentally retarded, is affected with both disorders. This conclusion was further supported by linkage analysis with 11q23 markers. Lod scores between A-O and these markers, cumulated over three large Arab families, were significant and confirmed the localization of the ATA gene to aq23. However, another Druze family unassigned to a specific complementation group, showed several recombinants between A-T and the same markers, leaving the localization of the A-T gene in this family open.     

Effect of Metal-Rich Sewage Sludge Application on the Bacterial Communities of Grasslands

The effect of long-term application of heavy metal-laden sewage sludge on the total heterotrophic aerobic and the cadmium-resistant soil bacterial communities was studied. Gram-positive bacteria were completely absent from resistant communities. These findings suggest that this group is highly susceptible to Cd. Shannon's diversity indices estimated for total communities did not reveal negative effects on the communities that developed in the presence of sludge. However, Cd-resistant communities isolated from long-term sludge-amended soils were more diverse than the resistant communities from a control sample, suggesting that adaptation to Cd as a stressor had occurred in the presence of sludge constituents. This higher diversity was attributed to Cd resistance in pseudomonads and gram-negative fermenters. Resistance did not develop by dissemination of Cd resistance plasmids, because these were rarely detected in the genomes of resistant strains.     

Hyperscums and the population dynamics of Microcystis aeruginosa

Conceptual models, based on 7 years of data, are constructedto simulate the annual cycle and population dynamics of Microcystisaeruginosa in hypertrophic, warm monomictic Hartbees-poort Dam,South Africa in order to assess the role of hyperscum formation.In Hartbeespoort Dam the large summer planktonic population(mean epilimnion biovolumes of 20–50 mm³ I^–1) andthe low wind speed resulted in the formation of hyperscums (thick,crusted accumulations of floating cyanobacteria at wind-protectedsites) containing up to 50% of the total standing crop for 2–3months in 4 out of 5 years. In years in which hyperscums formedthe post-maximal summer population maintained itself throughoutautumn and into late winter before declining to the annual nadir(>1000 cells ml^–1). When hyperscums did not form, orwere artificially removed, the population fell to similarlylow levels as early as May (autumn) and remained small untilthe spring growth phase began. Microcystis cells decompose inthe upper layers of a hyperscum, but this is not a major lossto the planktonic population. Hyperscums are refuges which helpmaintain large planktonic standing crops during winter whengrowth is not possible but have no effect on the long-term (perennial)survival of Microcystis.     

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep -lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.     

Evidence for anextra-cellular function for protein kinase A

In addition to itsintra-cellular functions, cAMP-dependent protein kinase (PKA) may well have anextra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg⁺⁺; (b) In human serum, an endogenous phosphorylation of one protein (p75, Mr 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser³⁷⁸) which, at physiological pH, is buried in its two-chain form (V₆₅₊₁₀) but becomes exposed in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser³⁷⁸ in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1). Physiologically, this functional modulation may be involved in unleashing PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin.Dedicated to Edmond H. Fischer and Edwin G. Krebs, with gratitude for teaching us the right measure of thoroughness and vision in research.     

PHOTOADAPTATION AND THE “PACKAGE” EFFECT IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

In the marine unicellular chlorophyte, Dunaliella tertiolecta Butcher, the spectrally averaged m vivo absorption cross section, normalized to chlorophyll a (so-called a* values), vary two-fold in response to changes in growth irradiance. We used a kinetic approach to examine the specific factors which account for these changes in optical properties as cells photoadapt. Using Triton X-100 to solubilize membranes, we were able to differentiate between “package” effects and pigmentation effects. Our analyses suggest that 43–49% of the variability in a* is due to changes in pigmentation, whereas 51–57% is due to the “package” effect. Further analyses revealed that changes in cell sue did not significantly affect packaging, while thylakoid stacking and the transparency of thylakoid membranes were important factors. Our results suggest that thylakoid membrane protein/lipid ratios change during photoadaptation, and these changes influence the effective rate of light harvesting per unit chlorophyll a.     

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.     

Continuous representations of time-series gene expression data.

We present algorithms for time-series gene expression analysis that permit the principled estimation of unobserved time points, clustering, and dataset alignment. Each expression profile is modeled as a cubic spline (piecewise polynomial) that is estimated from the observed data and every time point influences the overall smooth expression curve. We constrain the spline coefficients of genes in the same class to have similar expression patterns, while also allowing for gene specific parameters. We show that unobserved time points can be reconstructed using our method with 10-15% less error when compared to previous best methods. Our clustering algorithm operates directly on the continuous representations of gene expression profiles, and we demonstrate that this is particularly effective when applied to nonuniformly sampled data. Our continuous alignment algorithm also avoids difficulties encountered by discrete approaches. In particular, our method allows for control of the number of degrees of freedom of the warp through the specification of parameterized functions, which helps to avoid overfitting. We demonstrate that our algorithm produces stable low-error alignments on real expression data and further show a specific application to yeast knock-out data that produces biologically meaningful results.