Tachycardia dependent early repolarisation pattern in subarachnoid haemorrhage related takotsubo syndrome.

We present a case of a patient who suffered subarachnoid haemorrhage (SAH), complicated by takotsubo syndrome, paroxysmal atrial fibrillation and ECG repolarisation abnormality, compatible with Brugada phenocopy. The early repolarisation morphology showed a paradox association with the cardiac cycle length; a relationship not yet documented in SAH. Our observation also sheds light on the genesis of the "spiked helmet" ECG sign.

Neuroprotective effect of oxaloacetate in a focal brain ischemic model in the rat.

During an ischemic event, the well-regulated glutamate (Glu) homeostasis is disturbed, which gives rise to extremely high levels of this excitatory neurotransmitter in the brain tissues. It was earlier reported that the administration of oxaloacetate (OxAc) as a Glu scavenger reduces the Glu level in the brain by enhancing the brain-to-blood Glu efflux. Here, we studied the neuroprotective effect of OxAc administration in a new focal ischemic model in rats. Occlusion of the middle cerebral artery resulted in immediate reduction of the somatosensory-evoked responses (SERs), and the amplitudes remained at the reduced level throughout the whole ischemic period. On reperfusion, the SERs started to increase, but never reached the control level. OxAc proved to be protective, since the amplitudes started to recover even during the ischemia, and finally fully regained the control level. The findings of the histological measurements were in accordance with the electrophysiological data. After Fluoro Jade C staining, significantly fewer labeled cells were detected in the OxAc-treated group relative to the control. These results provide new evidence of the neuroprotective effect of OxAc against ischemic injury, which strengthens the likelihood of its future applicability as a novel neuroprotective agent for the treatment of ischemic stroke patients.

Acetyl-L-carnitine and oxaloacetate in post-treatment against LTP impairment in a rat ischemia model. An in vitro electrophysiological study.

A high proportion of research relating to cerebral ischemia focuses on neuroprotection. The application of compounds normally present in the organism is popular, because they do not greatly influence the synaptic activity by receptor modulation, and can be administered without serious side effects. Oxaloacetate (OxAc) and acetyl-L-carnitine (ALC) are such favorable endogenous molecules. ALC can exert a protective effect by improving the energy state of the neurons under ischemic conditions. A promising neuroprotective strategy is glutamate scavenging, which can be achieved by the intravenous administration of OxAc. This study involved the possible protective effects of ALC and OxAc in different post-treatment protocols against long-term potentiation (LTP) impairment. Ischemia was induced in rats by 2-vessel occlusion, which led to a decreased LTP relative to the control group. High-dose (200 mg/kg) ALC or OxAc post-treatment resulted in a higher potentiation relative to the 2VO group, but it did not reach the control level, whereas low-dose ALC (100 mg/kg) in combination with OxAc completely restored the LTP function. Many previous studies have concluded that ALC can be protective only as pretreatment. The strategy described here reveals that ALC can also be neuroprotective when utilized as post-treatment against ischemia.

Molecular ABO phenotyping in cynomolgus macaques using real-time quantitative PCR.

Macaques are commonly used in biomedical research as animal models of human disease. The ABO phenotype of donors and recipients plays an important role in the success of transplantation and stem cell research of both human and macaque tissue. Traditional serological methods for ABO phenotyping can be time consuming, provide ambiguous results and/or require tissue that is unavailable or unsuitable. We developed a novel method to detect the A, B, and AB phenotypes of macaques using real-time quantitative polymerase chain reaction. This method enables the simple and rapid screening of these phenotypes in macaques without the need for fresh blood or saliva. This study reports the distribution of the A, B, and AB phenotypes of captive cynomolgus macaques that, while regionally variable, closely resembles that of rhesus macaques. Blood group B, as in rhesus macaques, predominates in cynomolgus macaques and its frequency distribution leads to a probability of major incompatibility of 41%. No silencing mutations have been identified in exon 6 or 7 in macaques that could be responsible for the O phenotype, that, although rare, have been reported. The excess homozygosity of rhesus and cynomolgus macaque genotypes in this study, that assumes the absence of the O allele, suggests the possibility of some mechanism preventing the expression of the A and B transferases.

Synthesis and biological effects of some kynurenic acid analogs.

The overactivation of excitatory amino acid receptors plays a key role in the pathomechanism of several neurodegenerative disorders and in ischemic and post-ischemic events. Kynurenic acid (KYNA) is an endogenous product of the tryptophan metabolism and, as a broad-spectrum antagonist of excitatory amino acid receptors, may serve as a protective agent in neurological disorders. The use of KYNA is excluded, however, because it hardly crosses the blood-brain barrier. Accordingly, new KYNA analogs which can readily cross this barrier and exert their complex anti-excitatory activity are generally needed. During the past 6 years, we have developed several KYNA derivatives, among others KYNA amides. These new analogs included one, N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride (KYNA-1), that has proved to be neuroprotective in several models. This paper reports on the synthesis of 10 new KYNA amides (KYNA-1-KYNA-10) and on the effectiveness of these molecules as inhibitors of excitatory synaptic transmission in the CA1 region of the hippocampus. The molecular structure and functional effects of KYNA-1 are compared with those of other KYNA amides. Behavioral studies with these KYNA amides demonstrated that they do not exert significant nonspecific general side-effects. KYNA-1 may therefore be considered a promising candidate for clinical studies.

Real-time PCR-based pathotyping of Newcastle disease virus by use of TaqMan minor groove binder probes.

A real-time reverse-transcription PCR was developed to detect and pathotype Newcastle disease viruses (NDV) in clinical samples. Degenerate oligonucleotide primers and TaqMan probes with nonfluorescent minor groove binder (MGB) quencher amplified and hybridized to a region in the fusion protein (F) gene that corresponds to the cleavage site of the F0 precursor, which is a key determinant of NDV pathogenicity. The application of degenerate primers and TaqMan MGB probes provided high specificity to the assay, as was shown by the successful and rapid pathotype determination of 39 NDV strains representing all the known genotypes (I to VIII) and pathotypes (lentogens/mesogens/velogens). The PCR assays specific for lentogenic and velogenic/mesogenic strains had high analytical sensitivity, detecting approximately 10 and 20 copies of the target molecule per reaction, respectively. The detection limit was also determined in terms of 50% egg infective dose (EID(50)) by using dilution series of virus stock solutions to be approximately 10(1.0) and 10(-1.3) EID(50)/ml for lentogens and velogens/mesogens, respectively. Organ, swab, and stool specimens from experimentally infected animals were tested to prove the clinical suitability of the method. The results of this study suggest that the described real-time PCR assay has the potential to be used for the rapid detection/pathotyping of NDV isolates and qualitative/quantitative measurement of the virus load.

Intramolecular repression of mouse heat shock factor 1.

The pathway leading to transcriptional activation of heat shock genes involves a step of heat shock factor 1 (HSF1) trimerization required for high-affinity binding of this activator protein to heat shock elements (HSEs) in the promoters. Previous studies have shown that in vivo the trimerization is negatively regulated at physiological temperatures by a mechanism that requires multiple hydrophobic heptad repeats (HRs) which may form a coiled coil in the monomer. To investigate the minimal requirements for negative regulation, in this work we have examined mouse HSF1 translated in rabbit reticulocyte lysate or extracted from Escherichia coli after limited expression. We show that under these conditions HSF1 behaves as a monomer which can be induced by increases in temperature to form active HSE-binding trimers and that mutations of either HR region cause activation in both systems. Furthermore, temperature elevations and acidic buffers activate purified HSF1, and mild proteolysis excises fragments which form HSE-binding oligomers. These results suggest that oligomerization can be repressed in the monomer, as previously proposed, and that repression can be relieved in the apparent absence of regulatory proteins. An intramolecular mechanism may be central for the regulation of this transcription factor in mammalian cells, although not necessarily sufficient.

Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux.

Macroautophagy (autophagy) is a dynamic intracellular degradation pathway. Monitoring the flux through the autophagy pathway is experimentally challenging but obviously a prerequisite for the proper investigation of the process. Here, we present an indirect autophagy flux assay based on monitoring the degradation of an autophagosome-associated fusion protein Rluc-LC3 by luminescence detection. The method is suitable for screening purposes with a high number of parallel samples and can be used for measurements in cell lysates as well as in living cells. The Rluc-LC3 assay has proven useful for the identification of genes, miRNAs, and small molecules that regulate autophagy flux in mammalian cells.

Application of real-time RT-PCR utilising lux (light upon extension) fluorogenic primer for the rapid detection of avian influenza viruses.

A real-time RT-PCR assay utilising light upon extension fluorogenic primer (LUX RT-PCR) was developed for the rapid and efficient detection of avian influenza viruses (AIV). The assay detected each of the AIV isolates tested (16/16) and gave negative results with heterologous pathogens (17/17). The detection limit of the assay proved to be 10(-0.5) EID50/0.2 ml and 10(1.5) EID50/0.2 ml in allantoic fluid of virus-infected embryonated chicken eggs and in spiked chicken faeces samples, respectively. Based on its specificity, sensitivity and relative simplicity, the LUX RT-PCR assay provides a novel, rapid and cost-effective diagnostic tool for avian influenza surveillance and monitoring programs.

Self-regulation of the lipid content of membranes by non-bilayer lipids: a hypothesis.

Many biological membranes contain lipids that do not form a lamellar phase but the roles of these lipids are not well understood. An artificial membrane assembled from the main non-bilayer lipid and the major integral protein of pea thylakoids revealed that the protein spatially inhibits the formation of non-bilayer structures in the lamellae. Without this inhibition, excess lipids are secreted, creating lipid reservoirs for metabolism and/or later uptake. This determines the protein:lipid ratio in the membrane and hence the balance between structural flexibility and the stability of the key constituents that participate in cooperative interactions.

Spin label EPR study of lipid solvation of supramolecular photosynthetic protein complexes in thylakoids.

Lipid-protein association in the chloroplast membrane and its various thylakoid fractions from higher plants, namely pea and maize, rich in Photosystem I (PSI) and Photosystem II (PSII), respectively, were studied using EPR spectroscopy of spin-labelled lipid molecules. All the EPR spectra consisted of two spectral components corresponding to bulk fluid lipids and solvation lipids motionally restricted at the hydrophobic surface of membrane proteins. Spin-labelled stearic acid and phosphatidylglycerol exhibited marked selectivity towards the supramolecular protein complexes of both PSI and PSII although to different extent. In addition, lipid-protein titration experiments are described for partially delipidated PSII-enriched membrane fractions of pea chloroplasts, incorporating unlabelled egg phosphatidylcholine prior to or after the incorporation of spin-labelled lipids. Two sets of solvation sites were resolved by timed labelling experiments and a significant result of these studies was that a well-defined population of solvation sites (approx. 100 mol lipids/820 kDa protein) was rapidly exchanged by laterally diffusing membrane lipids, while other solvation sites (approx. 50 mol lipids/820 kDa protein) were exchanged much slower or not exchanged at all.

Regional brain glucose metabolism in chronic schizophrenia. A positron emission transaxial tomographic study.

Thirteen diagnosed schizophrenics and 11 normal controls were studied with a method using the PETT III positron emission tomograph (PET) and fluorodeoxyglucose labeled with fluorine 18. Each subject also had a computed tomographic (CT) scan. For each subject, two brain levels, one through the basal ganglia and one through the semioval center, were analyzed for the mean regional metabolic glucose rate. Specifically, relationships between frontal and posterior regions were evaluated. The CT scans of matching levels were superimposed on the functional PET images to provide anatomic criteria for region of interest selection. While no whole-slice metabolic differences were apparent between groups, schizophrenics had significantly lower activity in the frontal lobes, relative to posterior regions. The medicated and drug-free groups did not differ from one another in these regards. Trait v state dependency of the phenomenon was analyzed, and several technological limitations were considered.

Noninvasive continuous arterial pressure measurements in the assessment of acute, severe central hypovolemia.

UNLABELLED: Acute, severe hypovolemia is a medical emergency. Traditional vital sign parameters allow no optimal triage. High predictive power of finger plethysmography-based stroke volume (SV) and pulse pressure (PP) was recently suggested. To assess the performance of the PP and SV parameters, lower body negative pressure of -40 mmHg, than -60 mmHg - corresponding to moderate and severe central hypovolemia - was applied in 22 healthy males (age 35 ± 7 years). Slow breathing induced fluctuations in the above indices, characterized by stroke volume variability (SVV), and pulse pressure variability (PPV), were assessed. Responses in heart rate (HR) and shock index (SI) were also studied. Discriminative capacity of these parameters was characterized by the area under the ROC (receiver operating characteristic) curves (AUC). RESULTS: In comparison of baseline to severe central hypovolemia SV, PP, HR, and SI showed good discriminating capacity (AUC 99%, 88%, 87%, and 93%, respectively). The discriminating capacity of SVV and PPV was poor (77% and 70%, respectively). In comparison of moderate and severe hypovolemia, the discriminating capacity of the studied parameters was uniformly limited. CONCLUSIONS: Plethysmography-based SV and PP parameters can be used to detect acute severe volume loss. Sensitive parameters discriminating moderate and severe central hypovolemia are still lacking.

L-tryptophan in depression.

L-tryptophan, the amino acid precursor of serotonin, was administered to 16 depressive patients in a double-blind study of its potential antidepressant efficacy. Antidepressant responses were observed in one of ten unipolar patients and in three of six bipolar patients. These results are discussed in the context of possible interactions of amines with electrolyte systems in the etiology of affective illness.

Platelet membrane fluidity and plasma malondialdehyde levels in Alzheimer's demented patients with and without family history of dementia.

Platelet membrane fluidity (PMF) was measured with three different fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 3-(p-phenyl-1,3,5-hexatrienyl)phenyl-propionic acid (DPH-PA), which labeled different parts of the bilayer (the hydrophobic core and the positively and negatively charged regions, respectively) in Alzheimer's disease (AD) patients with and without a family history of dementia, and in a control group. In support of earlier findings in the literature, significantly increased PMF was found by the application of DPH in both groups with AD. The use of the fluorescence probe TMA-DPH, however, revealed no differences between the groups. In contrast, significant rigidification was observed with DPH-PA, but only in the AD group with a positive family history of dementia. The plasma malondialdehyde levels appeared to be similar in each group. Our findings are discussed in light of the controversies regarding the value of PMF measurements in AD.

Positron emission tomography in the study of aging and senile dementia.

(18)F-2-deoxy-2-fluoro-D-glucose ((18)FDG) is a positron emitting tracer for rate of glucose utilization in brain. When used in conjunction with positron emission tomography (PET), the PET-FDG technique permits in vivo quantitation of regional brain metabolism in man. We have applied this technique to the study of regional brain function in normal aging and senile dementia. Preliminary results for 7 patients with senile dementia of the Alzheimer's type (SDAT) and 3 elderly normal subjects indicated a large, statistically significant (p < 0.01) diminution in rate of glucose utilization in SDAT. Furthermore, the degree of diminution in metabolic activity in SDAT was highly correlated with objective measures of degree of cognitive impairment. These results demonstrate the feasibility and potential utility of the PET-FDG technique for studying regional brain function in normal aging and dementia.

Treatement of excessive weight gain in patients taking lithium.

Six female primary affective disorder patients who had gained an average of 9.5 kg while taking lithium lost an average of 2.9 kg on a 10-day 900 calorie a day hospital diet containing 100 mEq of sodium per day. No evidence of lithium toxicity was observed on this regimen. There was no evidence that fluid retention played a major role in the weight gain.

The construction and characterization of an effective transpositional system based on IS30.

We constructed an in vivo system to detect transpositional rearrangements induced by the insertion sequence IS30. The transposase protein expressed from the transposase producer plasmids catalyzed rearrangements on different target sequences presented in trans. High yields, up to 83%, of transpositional frequencies were observed. The frequency of rearrangements correlated with the amount of transposase protein produced and the attractivity of the target sequences. Alteration in the frequency of transposition was observed in the recA- E. coli strains JM109 and TG2. Remarkable structural and functional analogy was found with site-specific recombination systems.

Mutations in the carboxy-terminal part of IS30 transposase affect the formation and dissolution of (IS30)2 dimer.

The transposase of IS30 catalyses different transpositional rearrangements via the dimer (IS30)2 intermediate structure. Mutation analysis provides evidence that the C-terminal part of IS30 transposase is required for the formation and dissolution of (IS30)2 dimer. C-terminal mutants are also defective in transpositional fusion; however, this deficiency can be 'suppressed' by addition of the final product of site-specific dimerisation, the core (IS30)2 intermediate structure. The transposase part studied shows significant homologies in three highly conserved regions to proteins of IS30-related mobile elements.

Phospholipase C and phospholipase A2 are involved in the antiviral activity of human interferon-alpha.

Treatment of human amniotic cells (UAC) with human interferon-alpha (Hu-IFN alpha) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C (PK-C) activity from the cytosol fraction to that of the membranes. Analysis of 32P incorporation into phospholipid fractions and studies of alterations in fatty acid content for the major phospholipids of IFN-treated cells suggest that phospholipases C and A2 are activated by Hu-IFN alpha. Addition of neomycin (an inhibitor of phospholipase C), as well as mepacrine (an inhibitor of phospholipase A2) to IFN-treated cells inhibited the antiviral activity of Hu-IFN alpha in the vesicular stomatitis virus (VSV)-UAC system used. These observations indicate that (i) activation of PK-C and (ii) diacylglycerol formation, arachidonic acid and/or lysophosphatidylcholine release are important steps in the mechanism of action of IFN.