The novel CaMKII inhibitor GS-680 reduces diastolic SR Ca leak and prevents CaMKII-dependent pro-arrhythmic activity.

RATIONALE: Ca/calmodulin-dependent protein kinase II (CaMKII) was shown to increase diastolic sarcoplasmic reticulum (SR) Ca leak, which can result in delayed afterdepolarizations and triggered arrhythmias. Since increased CaMKII expression and activity has been mechanistically linked to arrhythmias in human heart failure (HF) and atrial fibrillation (AF), specific strategies aimed at CaMKII inhibition may have therapeutic potential. OBJECTIVE: We tested the antiarrhythmic and inotropic effects of a novel selective and ATP-competitive CaMKII inhibitor (GS-680). METHODS AND RESULTS: Trabeculae were isolated from right atrial appendage biopsies of patients undergoing cardiac surgery. Premature atrial contractions (PACs) were induced by stimulation with isoproterenol (ISO, 100 nM) at increased [Ca]o (3.5 mM). Interestingly, compared to vehicle, PACs were significantly inhibited by exposure to GS-680 (at 100 and 300 nM). GS-680 also significantly decreased early and delayed afterdepolarizations in isolated human atrial myocytes. Moreover, GS-680 (at 100 or 300 nM) significantly inhibited diastolic SR Ca leak, measured as frequency of spontaneous SR Ca release events (Ca sparks) in isolated human atrial myocytes (Fluo-4 loaded) similar to the well-established peptide CaMKII inhibitor AIP. In accordance, GS-680 significantly reduced CaMKII autophosphorylation (Western blot) but enhanced developed tension after 10 or 30 s pause of electrical stimulation (post-rest behavior). Surprisingly, we found a strong negative inotropic effect of GS-680 in atrial trabeculae at 1 Hz stimulation rate, which was not observed at 4 Hz and abolished by beta-adrenergic stimulation. In contrast, GS-680 did not impair systolic force of isolated ventricular trabeculae from explanted hearts of heart transplant recipients at 1 Hz, blunted the negative force-frequency relationship (1-3 Hz) and significantly increased the Ca transient amplitude. CONCLUSION: The novel ATP-competitive and selective CaMKII inhibitor GS-680 inhibits pro-arrhythmic activity in human atrium and improves contractility in failing human ventricle, which may have therapeutic implications.

Medical, demographical and social aspects of syphilis: the case of infected sex workers in Greece during Interwar.

The aim of this research is to present syphilis among women described as "indecent" according to the records of the Venereal Diseases Hospital "Andreas Syggros", which is located in Athens, during the period 1931-1935. In impoverished Greece of the Interwar period, factors such as criminal ignorance, or lack of information on sexually transmitted diseases (STDs) along with inadequate health controls of sex workers, resulted in a dramatic spread of syphilis, whereas "Andreas Syggros" hospital accommodated thousands of patients. The inflow of 1.300.000 Greek refugees from Asia Minor, after the Greek defeat by the Turkish army in the war of 1922, resulted in a notable change in the demographics of the country, while the combination of miserable living conditions, unemployment, economic crisis of the Interwar period, political instability and dysfunction of the State led to an increased number of illegal sex workers and syphilis outbreaks. Despite the introduction of an ad hoc Act to control STDs since 1923, the State was unable to limit the transmissibility of syphilis and to control prostitution. Unfortunately, the value of this historical paradigm is borne out by a contemporary example, i.e. the scandal of HIV seropositive sex workers in -beset by economic crisis- Greece in May 2012. It turns out that ignorance, failure to comply with the law, change in the mentality of the citizens in an economically ruined society, and most notably dysfunction of public services during periods of crisis, are all risk factors for the spread of serious infectious diseases.

Potent 2'-amino-, and 2'-fluoro-2'-deoxyribonucleotide RNA inhibitors of keratinocyte growth factor.

Reiterative in vitro selection-amplification from random oligonucleotide libraries allows the identification of molecules with specific functions such as binding to specific proteins. The therapeutic usefulness of such molecules depends on their high affinity and nuclease resistance. Libraries of RNA molecules containing 2'amino-(2'NH2)- or 2'fluoro-(2'F)-2'-deoxypyrimidines could yield ligands with similar nuclease resistance but not necessarily with similar affinities. This is because the intramolecular helices containing 2'NH2 have lower melting temperatures (Tm) compared with helices containing 2'F, giving them thermodynamically less stable structures and possibly weaker affinities. We tested these ideas by isolating high-affinity ligands to human keratinocyte growth factor from libraries containing modified RNA molecules with either 2'NH2 or 2'F pyrimidines. We demonstrated that 2'F RNA ligands have affinities (Kd approximately 0.3-3 pM) and bioactivities (Ki approximately 34 pM) superior to 2'NH2 ligands (Kd approximately 400 pM and Ki approximately 10 nM). In addition, 2'F ligands have extreme thermo-stabilities (Tm approximately 78 degrees C in low salt, and specificities).

Rapid preparation of single stranded DNA from PCR products by streptavidin induced electrophoretic mobility shift.

Streptavidin induced electrophoretic mobility shift was used to prepare single stranded (ss) DNA amplified with the polymerase chain reaction in the presence of a biotinylated and a non-biotinylated primer. A variety of denaturing conditions, including incubation at 95 degrees C in 50% formamide can be used without disrupting the streptavidin-biotinylated-ssDNA complex. Following electrophoresis, pure non-biotinylated DNA can be efficiently recovered from 7 M urea gels because it is well separated from the severely retarded streptavidin-biotinylated-ssDNA complex. Quantitative complexing of biotinylated ssDNA can occur at a streptavidin to DNA molar ratio of 1 or more.

Comparative study of magnetic resonance angiography, digital subtraction angiography, duplex ultrasound examination with surgical and histological findings of atherosclerotic carotid bifurcation disease.

This study was designed in order to determine the diagnostic accuracy in imaging of the extracranial arteries by using magnetic resonance angiography (MRA), digital subtraction angiography (DSA), B-mode duplex ultrasonic examination (DUE) in comparison with the surgical and histological findings of the specimen removed after endarterectomy. The degree of stenosis of the arterial lumen of the surgical specimen was compared with the imaging findings of MRA, DSA and DUE: a) the degree of agreement of stenosis with histologic findings was found in 89% of cases for MRA, in 93% for DSA and 88% for DUE; b) the correlation of morphology of the plaque showed agreement in 91% of the cases with MRA, in 94% with DSA and 87% with DUE; c) the constitution of the plaque was in agreement with DUE findings in 96% of cases. There is no significant difference between the three methods, as far as the estimation of degree of carotid stenosis and morphology of the atheromatous plaque in the carotid arteries. MRA findings are similar with those of DSA and DUE with a high sensitivity and specificity concerning the constitution of the plaque. The combination of MRA and DUE provides all the necessary information concerning the extracranial segments of the cerebral arteries for the preoperative evaluation of patients with carotid disease.

Molecular cloning of the pyrE gene from the extreme thermophile Thermus flavus.

Mutants of the extreme thermophile Thermus flavus in the pyrimidine biosynthetic pathway (Pyr-) were isolated by resistance to 5-fluoroorotic acid. The pyrE gene, which codes for the orotate phosphoribosyltransferase, was cloned by recombination with one of the isolated Pyr- T. flavus mutant strains. It was subcloned by complementation of an Escherichia coli pyrE mutant strain and was sequenced. The deduced polypeptide sequence extends over 183 amino acids. Several independent Pyr- mutations were mapped within the pyrE locus by recombination with fragments of the cloned gene.

Broad host range, regulated expression system utilizing bacteriophage T7 RNA polymerase and promoter.

An IPTF-regulated broad host range expression system was constructed using compatible broad host range plasmids, the T7 RNA polymerase, and T7 promoter sequences. The system is implemented by the coexistence of two plasmids. The first contains the T7 RNA polymerase gene under the control of lacl or lacl(q) genes and lacUV5 promoter. The second encodes the T7 promoter upstream of a multicloning site. IncP1 or IncP4 T7 promoter plasmids, and IncP1, IncP4 or IncW T7 RNA polymerase plasmids were constructed. The expression from the IncP1 promoter plasmids in the presence of the IncP4 polymerase plasmids was tested by in vivo lacZ fusions and vivo labeling of proteins. In this combination, the use of lac(q) improves the regulation levels in Escherichia coli, whereas, in Pseudomonas phaseolicola, a 28.5-fold regulation was obtained with lacl, Although the level of lacZ expression from the T7 promoter in P. phaseolicola is low compared with E. coli, it is similar to levels obtained with the pm promoter in Pseudomonas putida when the differences in the copy number of the expression vectors are taken into consideration.

Is cerebral arteriography necessary for decision making in carotid endarterectomy?

The development of ultrasonic diagnostic imaging technics has recently been a competitive diagnostic method in cerebral arteriography. Many vascular surgeons, based on the high specificity and sensitivity of the ultrasonic imaging technics in carotid artery disease, have been performing carotid endarterectomy without arteriography with satisfactory results. In the last four years we have performed in our Department 62 carotid endarterectomies on 57 patients without using cerebral arteriography. In this paper diagnostic ultrasonic imaging and transcranial Doppler technics are presented and the immediate results of carotid endarterectomy in the above series of patients are reported. From our and other authors' experience it is concluded that carotid endarterectomy in patients with carotid artery disease is a safe procedure. Larger series of patients are needed with a longer follow-up in order that carotid endarterectomy without arteriography be accepted by the medical profession as a safe procedure.

Genome mapping and protein coding region identification using bacteriophage Mu.

Transposons such as bacteriophage Mu provide a means to clone bacterial genes as alternatives to using standard recombinant DNA technologies. A DNA-cloning and gene-expressing system has been developed with a bacteriophage Mu (DNA capacity of 38 kb) vector that combines the Mu transposition capabilities and a specialized promoter from bacteriophage T7. Genes cloned with this vector can be identified by transcription in vivo with T7 RNA polymerase and subsequent host translation. This system, illustrated with the characterization of a 35-kb region of the Escherichia coli K-12 chromosome, is applicable to other Enterobacteriaceae, which are hosts for Mu phage, and is potentially applicable to other bacteria, including Pseudomonas aeruginosa, which have Mu-like phage, and to other organisms for which high-frequency transposons are available.

Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels.

Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription.

Minus-strand RNA synthesis by the segmented double-stranded RNA bacteriophage phi 6 requires continuous protein synthesis.

Bacteriophage phi 6 contains three dsRNA chromosomes. Strand-separating agarose gels were used to study plus- and minus-strand synthesis in vivo and the effect of protein synthesis inhibitors. Analysis of phi 6 RNA synthesis shows low levels of all three dsRNAs and ssRNAs at 10 min, increasing label uptake into all RNAs except the large message from 20 to 60 min, and a greater abundance of medium and small messages than large mRNAs at late times. Isoconformers of the small message are synthesized throughout infection. Northern analysis suggests that large messages made early may persist to direct continuing translation of L-segment-encoded transcription and replication proteins. The time course of phi 6 minus-strand RNA synthesis in vivo, in the absence of background label in host RNAs, is reported for the first time. Label in minus strands is detected only after heat denaturation of RNA samples and appears sequentially in the small, medium, and large strands beginning at 20 min. At both early and late times, chloramphenicol arrests minus-strand synthesis rapidly and all three mRNAs accumulate. The results are consistent with the reovirus asynchronous model for dsRNA viral replication: plus ssRNAs made first are used as templates for minus-strand synthesis. They also indicate that replication protein(s) acts stoichiometrically.

Vital capillaroscopy on microcirculation: pharmacodynamic activity of Daflon 500 mg in venous insufficiency.

A group of ten female patients suffering from telengiectasia of the lower limbs was treated with Daflon 500 mg administered orally. The results obtained are presented and discussed.

Generation of cDNA clones of the bacteriophage phi 6 segmented dsRNA genome: characterization and expression of L segment clones.

Bacteriophage phi 6 has three dsRNA genome segments of about 3.0, 4.0, and 6.4 kbp. More than 90% of the segmented phi 6 dsRNA genome has been cloned as subchromosomal cDNA fragments, generated by reverse transcription of denatured polyadenylated dsRNA, RNA removal, annealing, filling, size fractionation, tailing, and insertion at the PstI site of pBR322. All of the large (L) segment is represented by five overlapping fragments, 98% of the small (S) segment is present in three fragments, and 67% of the medium (M) segment is contained in two fragments. Fragments have been aligned in linear arrays by Southern blot hybridization and restriction enzyme analysis. The orientation of the ordered fragments with respect to genomic RNA and phi 6 transcriptional direction was determined by comparison of terminal DNA sequences with RNA sequences at the genomic ends of phi 6 RNA. Expression of L segment clones using both Escherichia coli minicells and T7 polymerase/promoter vectors indicate that the order of known phi 6 genes on the large chromosome is: 5'--gene 7, gene 2, gene 4, gene 1--3'. cDNA complementation of a ts mutant, ts411, has located this mutation in gene 4.