Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.     

Nuclear wavepacket motion between P and P(+)B(A)(-) potential surfaces with subsequent electron transfer to H(A) in bacterial reaction centers. 1. Room temperature

Formation and coherent propagation of nuclear wavepackets on potential energy surfaces of the excited state of the primary electron donor P and of the charge transfer states P(+)B(A)(-) and P(+)H(A)(-) were studied in native and pheophytin-modified Rhodobacter sphaeroides R-26 reaction centers (RCs) induced by 25 fs excitation (where B(A) and H(A) are the primary and secondary electron acceptors, respectively). The processes were monitored by measuring coherent oscillations in kinetics of the time evolution of the stimulated emission band of P at 935 nm, of the absorption band of B(A)(-) at 1020 nm, and of the bleaching band of H(A) at 760 nm. It was found that the nuclear wavepacket motion on the 130-140 cm(-1) surface of P is directly induced by light absorption in P. When the wavepacket approaches the intersection between P and P(+)B(A)(-) surfaces at 120 and 380 fs delays, the formation of intermediate mixed-state emitting light at 935 nm (P) and absorbing light at 1020 nm (P(+)B(A)(-)) takes place. At the latter time, the wavepacket is transferred to the 32 cm(-1) mode which can belong to the P hypersurface effectively transferring the wavepacket to the P(+)B(A)(-) surface or can represent a diabatic surface which is formed by the states P and P(+)B(A)(-). The wavepacket motion on the P(+)B(A)(-) surface or on the P(+)B(A)(-) part of the mixing surface is accompanied by irreversible electron transfer to H(A). This process is monitored by the kinetics of 1020 nm band development and 760 nm band bleaching (delayed with respect to 1020 nm band development) which both have the enhanced 32 cm(-1) mode in Fourier transform (FT) spectra. The mechanism of wavepacket transfer from the 130-140 cm(-1) to the 32 cm(-1) mode is discussed.     

Nuclear wave packet motion between P* and P(+)B(A)(-) potential surfaces with a subsequent electron transfer to H(A) in bacterial reaction centers at 90 K. Electron transfer pathway

In Rhodobacter sphaeroides R-26 reaction centers (RCs) the nuclear wave packet induced by 25 fs excitation at 90 K moves on the primary electron donor P* potential energy hypersurface with initial frequency at approximately 130 cm(-1) (monitored by stimulated emission measurement). At the long-wavelength side of P* stimulated emission at 935 nm the wave packet is transferred to the surface with P(+)B(A)(-) character at 120, 380, 1.2 fs, etc. delays (monitored by measurement of the primary electron acceptor B(A)(-) band at 1020 nm). However, only beginning from 380 fs delay and later the relative stabilization of the state P(+)B(A)(-) is observed. This is accompanied by the electron transfer to bacteriopheophytin H(A) (monitored by H(A) band measurement at 760 nm). The most active mode of 32 cm(-1) in the electron transfer and its overtones up to the seventh were found in the Fourier transform spectrum of the oscillatory part of the kinetics of the P* stimulated emission and of the P(+)B(A)(-) and P(+)H(A)(-) formation. This mode and its overtones are apparently populated via the 130 cm(-1) vibrational mode. The deuteration of the sample shifts the fundamental frequency (32 cm(-1)) and all overtones by the same factor of approximately 1.3. This mode and its overtones are suppressed by a factor of approximately 4.7 in the dry film of RCs. The results obtained indicate that the 32 cm(-1) mode might be related to a rotation of hydrogen-containing groups (possibly the water molecule) participating in the modulation of the primary electron transfer from P* to B(A)(-) in at least 35% of RCs. The Brookhaven Protein Data Bank (1PRC) displays the water molecule located at the position HOH302 between His M200 (axial ligand for P(B)) and the oxygen of ring V of B(A) which might be a part (approximately 35%) of the molecular pathway for electron transfer from P* to B(A).     

Spatiotemporal dynamics of contact activation factors of blood coagulation

A new in vitro model is proposed for studying the spatiotemporal distributions of activated clotting factors, in which clotting is activated in a thin layer of non-stirred plasma supplemented with a fluorogenic substrate and is monitored by fluorescence from its cleavage product. Analysis of the spatiotemporal dynamics of factor XIa and kallikrein in glass-activated human plasma provides evidence that both contact factors remain restricted to the glass surface and possibly a narrow boundary zone (<0.1 mm). The kinetics of factor XIa and kallikrein studied by a new method (in non-stirred plasma) coincided with those studied fluorimetrically with full stirring: their concentrations rapidly rose for the first few minutes after activation and then slowly declined. Factor XI and prekallikrein activation is likely to be restricted by the limited number of sites available for binding to the surface. The maximum concentration of the active factors was estimated at 2 x 10(8) molecules per mm(2) at the glass surface (irrespective of stirring). At the plastic surface, this value was 15--30 times lower.     

Bioinformatics classification and functional analysis of PhoH homologs

PhoH protein is a putative ATPase belonging to the phosphate regulon in Escherichia coli. EC-PhoH homologs are present in different organisms, but it is not clear if they are functionally related, besides nothing is known about their regulation. To distinguish true functional orthologs of EC-PhoH in different classes of bacteria and to identify their functional role in bacterial metabolic network we performed phylogenetic analysis of these proteins and comparative study of position and regulation of the related genes. Three groups of proteins were identified. Proteins of the first group (BS-PhoH orthologs) are present in most of bacteria and are proposed to be functionally linked to phospholipid metabolism and RNA modification. Proteins of the second group (BS-YlaK orthologs) are present in most of aerobes and Actinobacterial YlaK orthologs are shown to be members of a fatty acid beta-oxidation regulons. EC-PhoH orthologs are classified in a third group, specific for Enterobacteria. Functional role of PhoH homologs in the lipid and RNA metabolism and proposed interrelation of PhoH paralogs in one organism are discussed.     

Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris

Cell adhesion and communication are interdependent aspects of cell behavior that are critical for morphogenesis and tissue architecture. In the skin, epidermal adhesion is mediated in part by specialized cell-cell junctions known as desmosomes, which are characterized by the presence of desmosomal cadherins, known as desmogleins and desmocollins. We identified a cadherin family member, desmoglein 4, which is expressed in the suprabasal epidermis and hair follicle. The essential role of desmoglein 4 in skin was established by identifying mutations in families with inherited hypotrichosis, as well as in the lanceolate hair mouse. We also show that DSG4 is an autoantigen in pemphigus vulgaris. Characterization of the phenotype of naturally occurring mutant mice revealed disruption of desmosomal adhesion and perturbations in keratinocyte behavior. We provide evidence that desmoglein 4 is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation.     

Peripheral neuropathy and light-preliminary report indicating prevalence of nanobacteria in HIV

Peripheral neuropathy is a common condition in HIV-positive patients and is often experienced in diabetes mellitus. The primary mechanism of the disease, which can considerably aggravate the patient's state, is unknown. The perineurium of patients with peripheral neuropathy is frequently enveloped by apatite. Nanobacteria (NB) are protectd by a mineral shell consisting of apatite. Light has been shown to elevate the vitality level of cells, and was predicted to inhibit deposition of stressed NB in the cardiovascular system. Results indicate that light can durably restore the condition of patients with severe peripheral neuropathy.     

Sealing porous nanovesicles--solutions inspired by primordial biosystems

Microcapsules designed for slow drug release have preferably some porosity. There are, however, applications in which a hermetical sealing of the microcapsules is desired. Sealing is not a trivial problem and could be necessary to durably encapsulate toxic compounds which cannot be eliminated from the body, or to encapsulate harmful substances stored in the atmosphere. Nature may have one solution: Nanobacteria have developed surprisingly simple mechanisms to access and use primal energies, and to survive arid periods by sealing their surface.     

Telomerase as a growth-promoting factor

Beyond its role in telomere maintenance, telomerase provides additional functions in tumorigenesis, DNA repair, and cell survival. Telomerase protects cells from apoptosis and necrosis, and stimulates growth in adverse conditions. Furthermore, gross overexpression of the catalytic subunit of telomerase (hTERT) may act as a hyperproliferative signal to induce a senescence-like phenotype in normal fibroblasts, which is similar to the senescence induced by overexpression of oncogenes. As some of these functions can be dissociated from telomere lengthening, the question arises as to how the mere presence of telomerase can serve as a survival and growth-promoting factor.