Replication of Bacteriophage φX174 DNA in a Temperature-Sensitive dnaC Mutant of Escherichia coli C

Bacteriophage phiX174 cannot grow in a temperature-sensitive dnaC mutant of Escherichia coli C at the nonpermissive temperature. The inability to grow is the result of inhibition of virus DNA synthesis. Parental replicative form synthesis is not temperature sensitive. Single-stranded virus DNA continues to be synthesized for at least 45 min after shifting to the nonpermissive temperature late in infection. In contrast, the replication of the replicative form terminates within 5 min after shifting to the nonpermissive temperature.     

Combined metformin-salicylate treatment provides improved anti-tumor activity and enhanced radiotherapy response in prostate cancer; drug synergy at clinically relevant doses

BackgroundThere is need for well-tolerated therapies for prostate cancer (PrCa) secondary prevention and to improve response to radiotherapy (RT). The anti-diabetic agent metformin (MET) and the aspirin metabolite salicylate (SAL) are shown to activate AMP-activated protein kinase (AMPK), suppress de novo lipogenesis (DNL), the mammalian target of rapamycin (mTOR) pathway and reduce PrCa proliferation in-vitro. The purpose of this study was to examine whether combined MET+SAL treatment could provide enhanced PrCa tumor suppression and improve response to RT.MethodsAndrogen-sensitive (22RV1) and resistant (PC3, DU-145) PrCa cells and PC3 xenografts were used to examine whether combined treatment with MET+SAL can provide improved anti-tumor activity compared to each agent alone in non-irradiated and irradiated PrCa cells and tumors. Mechanisms of action were investigated with analysis of signaling events, mitochondria respiration and DNL activity assays.ResultsWe observed that PrCa cells are resistant to clinically relevant doses of MET. Combined MET + SAL treatment provides synergistic anti-proliferative activity at clinically relevant doses and enhances the anti-proliferative effects of RT. This was associated with suppression of oxygen consumption rate (OCR), activation of AMPK, suppression of acetyl-CoA carboxylase (ACC)-DNL and mTOR-p70^s6k/4EBP1 and HIF1α pathways. MET + SAL reduced tumor growth in non-irradiated tumors and enhanced the effects of RT.ConclusionMET+SAL treatment suppresses PrCa cell proliferation and tumor growth and enhances responses to RT at clinically relevant doses. Since MET and SAL are safe, widely-used and inexpensive agents, these data support the investigation of MET+SAL in PrCa clinical trials alone and in combination with RT.     

Simultaneous bilateral posteromedial tibial epiphysis stress fractures in a healthy young man: A case report

We present a compelling case of simultaneous, bilateral tibial stress fractures occurring in a unique epiphyseal and posterior location, with unclear aetiology. An overweight, Caucasian male in his late 20s developed synchronous bilateral medial knee pain following an intense 10-day training regimen. His radiographies were normal, but MRI revealed almost identical bilateral stress fracture lines in the posteromedial tibial epiphyses. Bone mineral densitometry and a full metabolic and hormonal panel were performed to further investigate potential underlying metabolic bone disease. He was found to have normal bone mineral densitometry and low Vitamin D serum values. Symptomatology greatly improved with activity modification. There were no further complaints and complications at 12 months’ follow-up. Diagnosis can be challenging and the treating physician should be acquainted with the basic science of stress fractures and main discriminating clinical, biochemical and radiological characteristics from insufficiency fractures, to avoid pitfalls in treatment decision.     

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.     

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL

Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, “Stefanidis medium” from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.     

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

Peptide aptamer-mediated inhibition of target proteins by sequestration into aggresomes

Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.     

Seasonal vertical distribution and diel migration of zooplankton in a temperate stratified lake

The investigation of the vertical distribution of the zooplankton community in the temperate Lake Trichonis during four seasons in 2005, showed the existence of vertical segregation among species, ontogenetic stages and sexes within and between the major groups. In each season, the two or three more abundant rotifer species distributed at separate depth layers, while this feature was maintained during the entire 24 h period, since no diel vertical migrations (DVM) were performed. In contrast, the crustacean community, comprised mainly by the calanoid copepod Eudiaptomus drieschi and the cladoceran Diaphanosoma orghidani, showed various patterns of DVM, being more pronounced in spring and summer. Females of E. drieschi distributed deeper than males, while the copepod nauplii were found mainly in the surface layer in all four seasons. Temperature was the most important abiotic factor affecting directly and indirectly the vertical distribution and migration of various species. During stratification, the metalimnion was the most productive layer in Lake Trichonis, having maximum values of dissolved oxygen and low transparency due to high concentration of organic matter and phytoplankton. The DVM patterns of the crustaceans indicate that the metalimnion acts probably as a daylight refuge against predation by Atherina boyeri, which is the dominant planktivorous fish in the lake.     

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor. This designation, however, is controversial as a result of recent studies in which lysophosphatidylinositol (LPI) was identified as a GPR55 agonist. Defining a biological role for GPR55 requires GPR55 selective ligands that have been unavailable. From a β-arrestin, high-throughput, high-content screen of 300000 compounds run in collaboration with the Molecular Libraries Probe Production Centers Network initiative (PubChem AID1965), we identified potent GPR55 selective agonists. By modeling of the GPR55 activated state, we compared the GPR55 binding conformations of three of the novel agonists obtained from the screen, CID1792197, CID1172084, and CID2440433 (PubChem Compound IDs), with that of LPI. Our modeling indicates the molecular shapes and electrostatic potential distributions of these agonists mimic those of LPI; the GPR55 binding site accommodates ligands that have inverted-L or T shapes with long, thin profiles that can fit vertically deep in the receptor binding pocket while their broad head regions occupy a horizontal binding pocket near the GPR55 extracellular loops. Our results will allow the optimization and design of second-generation GPR55 ligands and provide a means for distinguishing GPR55 selective ligands from those interacting with cannabinoid receptors.     

Genome sequence of the Chlamydophila abortus variant strain LLG

Chlamydophila abortus is a common cause of ruminant abortion. Here we report the genome sequence of strain LLG, which differs genotypically and phenotypically from the wild-type strain S26/3. Genome sequencing revealed differences between LLG and S26/3 to occur in pseudogene content, in transmembrane head/inc family proteins, and in biotin biosynthesis genes.