Workplace violence among healthcare workers, a multicenter study in Italy.

OBJECTIVES: The purpose of this study is to assess the prevalence and determinants of workplace violence and the sociodemographic risk factors associated. STUDY DESIGN: This was a multicenter cross-sectional study. METHODS: The study was performed using self-compiled Italian version of the World Health Organization's questionnaire on workplace violence online by filling in a Google form. The survey was opened from May 2018 to March 2020 and lasted 5-10 min. RESULTS: The sample consists of 3659 healthcare workers, of which 2525 (69%) are females, 1446 (39.5%) are nurses, and 2029 (55.5%) are health workers from northern Italy. The most frequent age group of the sample is 50-54 years (16.7%). A total of 366 (10%) healthcare workers are victims of physical aggression at work in the last 12 months, of which 6.3% with a weapon. The risk of being a victim of physical aggression at work in the last 12 months is significantly associated with the following independent variables: male gender (odds ratio [OR] 1.72, 95% confidence interval [CI]: 1.36-2.17), work in southern Italy (OR 1.59, 95% CI: 1.10-2.28), and being a nurse (OR 2.56, 95% CI: 2.01-3.25). The risk of being a victim of physical aggression at work with a weapon in the last 12 months is significantly associated with work in southern Italy (OR 9.33, 95% CI: 3.83-22.73). A total of 1723 (47.1%) of healthcare workers declare to be a victim of verbal aggression at work in the last 12 months. The risk of being a victim of verbal aggression at work in the last 12 months is significantly associated with the following independent variables: work in northern Italy (adjusted OR [aOR] 1.54, 95% CI: 1.32-1.81), work in southern Italy (aOR 3.68, 95% CI: 2.90-4.68), and be more than 55 years old (aOR 0.73, 95% CI: 0.63-0.85). CONCLUSIONS: The study underlines that the problem of verbal and physical aggression against healthcare workers is still central and is a further starting point for research. The prevalence of violence is difficult to assess because violent incidents are underreported or unreported. The results of the study suggest that increased awareness is needed to develop effective control strategies at the individual, hospital, and national levels to prevent aggression and improve the conditions of victims.

Fractional Quantum Hall Effect in Weyl Semimetals.

Weyl semimetal may be thought of as a gapless topological phase protected by the chiral anomaly, where the symmetries involved in the anomaly are the U(1) charge conservation and the crystal translational symmetry. The absence of a band gap in a weakly interacting Weyl semimetal is mandated by the electronic structure topology and is guaranteed as long as the symmetries and the anomaly are intact. The nontrivial topology also manifests in the Fermi arc surface states and topological response, in particular taking the form of an anomalous Hall effect in magnetic Weyl semimetals, whose magnitude is only determined by the location of the Weyl nodes in the Brillouin zone. Here we consider the situation when the interactions are not weak and ask whether it is possible to open a gap in a magnetic Weyl semimetal while preserving its nontrivial electronic structure topology along with the translational and the charge conservation symmetries. Surprisingly, the answer turns out to be yes. The resulting topologically ordered state provides a nontrivial realization of the fractional quantum Hall effect in three spatial dimensions in the absence of an external magnetic field, which cannot be viewed as a stack of two dimensional states. Our state contains loop excitations with nontrivial braiding statistics when linked with lattice dislocations.

NCCT Markers of Intracerebral Hemorrhage Expansion Using Revised Criteria: An External Validation of Their Predictive Accuracy.

BACKGROUND AND PURPOSE: Several NCCT expansion markers have been proposed to improve the prediction of hematoma expansion. We retrospectively evaluated the predictive accuracy of 9 expansion markers. MATERIALS AND METHODS: Patients admitted for intracerebral hemorrhage within 24 hours of last seen well were retrospectively included from April 2016 to April 2020. The primary outcome was revised hematoma expansion, defined as any of a ≥6-mL or ≥33% increase in intracerebral hemorrhage volume, a ≥ 1-mL increase in intraventricular hemorrhage volume, or de novo intraventricular hemorrhage. We assessed the predictive accuracy of expansion markers and determined their association with revised hematoma expansion. RESULTS: We included 124 patients, of whom 51 (41%) developed revised hematoma expansion. The sensitivity of each marker for the prediction of revised hematoma expansion ranged from 4% to 78%; the specificity, 37%-97%; the positive likelihood ratio, 0.41-7.16; and the negative likelihood ratio, 0.49-1.06. By means of univariable logistic regressions, 5 markers were significantly associated with revised hematoma expansion: black hole (OR = 8.66; 95% CI, 2.15-58.14; P = .007), hypodensity (OR = 3.18; 95% CI, 1.49-6.93; P = .003), blend (OR = 2.90; 95% CI, 1.08-8.38; P = .04), satellite (OR = 2.84; 95% CI, 1.29-6.61; P = .01), and Barras shape (OR = 2.41, 95% CI; 1.17-5.10; P = .02). In multivariable models, only the black hole marker remained independently associated with revised hematoma expansion (adjusted OR = 5.62; 95% CI, 1.23-40.23; P = .03). CONCLUSIONS: No single NCCT expansion marker had both high sensitivity and specificity for the prediction of revised hematoma expansion. Improved image-based analysis is needed to tackle limitations associated with current NCCT-based expansion markers.

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection.

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 µg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85% and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.

Successful Reperfusion is Associated with Favorable Functional Outcome despite Vessel Perforation during Thrombectomy: A Case Series and Systematic Review.

BACKGROUND: Arterial perforation is a potentially serious complication during endovascular thrombectomy. PURPOSE: Our aim was to describe interventional approaches after arterial perforation during endovascular thrombectomy and to determine whether reperfusion remains associated with favorable outcome despite this complication. DATA SOURCES: Data from consecutive patients with acute stroke undergoing endovascular thrombectomy were retrospectively collected between 2015 to 2020 from a single-center cohort, and a systematic review was performed using PubMed, EMBASE, and Ovid MEDLINE up to June 2020. STUDY SELECTION: Articles reporting functional outcome after arterial perforation during endovascular thrombectomy were selected. DATA ANALYSIS: Functional outcomes of patients achieving successful reperfusion (TICI 2b/3) were compared with outcomes of those with unsuccessful reperfusion in our single-center cohort. We then summarized the literature review to describe interventional approaches and outcomes after arterial perforation during endovascular thrombectomy. DATA SYNTHESIS: In our single-center cohort, 1419 patients underwent endovascular thrombectomy, among whom 32 (2.3%) had vessel perforation and were included in the analysis. The most common hemostatic strategy was watchful waiting (71% of cases). Patients with successful reperfusion had a higher proportion of favorable 90-day mRS scores (60% versus 12.5%; P = .006) and a lower mortality rate (13.3% versus 56.3%, P = .01) than patients without successful reperfusion. Thirteen articles were included in the systematic review. Successful reperfusion also appeared to be associated with better outcomes. LIMITATIONS: Given the low number of published reports, we performed only a descriptive analysis. CONCLUSIONS: Arterial perforation during endovascular thrombectomy is rare but is associated with high mortality rates and poor outcome. However, successful reperfusion remains correlated with favorable outcome in these patients.

Cloning and characterization of a new isoform of the interleukin 1 receptor antagonist.

By reverse transcriptase polymerase chain reaction on messenger RNA from human polymorphonuclear cells, we have isolated a sequence identical to the cDNA coding for intracellular interleukin 1 receptor antagonist (icIL-1ra), but containing an additional in-frame 63-bp sequence located three codons downstream of the translation start of icIL-1ra. This additional sequence is inserted between the first and second exon of the intracellular form, the latter of which is colinear with part of the first exon of the secreted form of IL-1ra. The additional sequence is coded by an extra exon located 2 kb downstream the first icIL-1ra-specific exon. The complementary DNA sequence of the alternatively spliced form of icIL-1ra shows that the predicted protein differs from classical icIL-1ra in the NH2 terminus by insertion of a leaderless sequence of 21 amino acids rich in glycine and glutamic acid residues. Transcripts coding for this new form of icIL-1ra were detected in activated fibroblasts, keratinocytes, and at low levels in myelomonocytic cells. The recombinant protein expressed in COS cells had an apparent molecular mass in sodium dodecyl sulfate polyacrylamide gel electrophoresis of 25 kD compared to 22 kD of classical icIL-1ra, and was mostly intracellular. The ability of this new form of icIL-1ra to inhibit IL-1 activity, in terms of induction of E-selectin and human immunodeficiency virus replication, was comparable to that of classical icIL-1ra. We propose to refer to this new form of icIL-1ra as icIL-1ra type II.

Exophthalmos following mechanical thrombectomy for anterior circulation stroke: A retrospective study and review of literature.

BACKGROUND: Anecdotal cases of exophthalmos after acute mechanical thrombectomy have been described. We sought to estimate the incidence in a large cohort of patients with acute anterior circulation stroke treated with mechanical thrombectomy. Secondarily, we aimed to evaluate the underlying mechanism and to differentiate it on imaging from other pathology with similar clinical orbital features. METHODS: Between November 2016 and November 2018, we performed a retrospective single-center study of 250 patients who underwent anterior circulation mechanical thrombectomy. Development of exophthalmos was independently evaluated by two readers on preprocedure and 24-h postprocedure non-contrast cerebral CT. RESULTS: In the mechanical thrombectomy cohort, six individuals (2.4%) developed interval ipsilateral exophthalmos at 24 h. Of these, at least two patients developed clinical symptoms. There was almost perfect agreement between assessments of the two readers (Cohen's kappa = 0.907 (95% confidence interval: 0.726, 1.000)). In two patients, there was delayed ophthalmic artery filling on digital subtraction angiography. None of the patients had features of a direct carotid-cavernous fistula. CONCLUSIONS: Exophthalmos is not uncommon after mechanical thrombectomy (2.4%). The underlying mechanism is difficult to confirm, but it is most likely due to orbital ischemia from hypoperfusion or distal emboli.

Fusion as the result of sperm-somatic cell interaction.

The research has been designed to investigate whether acrosome-reacted spermatozoa can fuse with somatic cells and to check whether this event may involve the molecular machinery implicated in the sperm-egg fusion. Boar spermatozoa were capacitated in vitro and then treated with A23187 to induce acrosome reaction and activate their fusogenic potential. Reacted spermatozoa, loaded with the membrane-permeant fluorescent dye calcein AM, were incubated with plated granulosa cells or cells derived from stable cell lines: CRFK, VERO, and ESK4. The fusion between spermatozoa and somatic cells was revealed by the diffusion of the fluorescent dye from the sperm to the cell as membrane fusion and cytoplasmic continuity between the two cells were established. The involvement of integrin alpha6 and tetraspanin CD9 in the process of fusion was assessed by carrying out the experiment in the presence of antibodies against these molecules. Moreover, the incidence of fusion displayed by the different cell types used was analyzed in relation to their content in the above molecules assessed by western blot and immunostaining. The role of CD9 was additionally investigated by using CD9-negative cells. The data presented demonstrate that boar spermatozoa can fuse with different somatic cell types derived from different species and the process requires the combined presence of both integrin and tetraspanin molecules on the cell plasma membrane.

ETHE1 mutations are specific to ethylmalonic encephalopathy.

Mutations in ETHE1, a gene located at chromosome 19q13, have recently been identified in patients affected by ethylmalonic encephalopathy (EE). EE is a devastating infantile metabolic disorder, characterised by widespread lesions in the brain, hyperlactic acidaemia, petechiae, orthostatic acrocyanosis, and high levels of ethylmalonic acid in body fluids. To investigate to what extent ETHE1 is responsible for EE, we analysed this gene in 29 patients with typical EE and in 11 patients presenting with early onset progressive encephalopathy with ethylmalonic aciduria (non-EE EMA). Frameshift, stop, splice site, and missense mutations of ETHE1 were detected in all the typical EE patients analysed. Western blot analysis of the ETHE1 protein indicated that some of the missense mutations are associated with the presence of the protein, suggesting that the corresponding wild type amino acid residues have a catalytic function. No ETHE1 mutations were identified in non-EE EMA patients. Experiments based on two dimensional blue native electrophoresis indicated that ETHE1 protein works as a supramolecular, presumably homodimeric, complex, and a three dimensional model of the protein suggests that it is likely to be a mitochondrial matrix thioesterase acting on a still unknown substrate. Finally, the 625G-->A single nucleotide polymorphism in the gene encoding the short chain acyl-coenzyme A dehydrogenase (SCAD) was previously proposed as a co-factor in the aetiology of EE and other EMA syndromes. SNP analysis in our patients ruled out a pathogenic role of SCAD variants in EE, but did show a highly significant prevalence of the 625A alleles in non-EE EMA patients.

Giant reed growth and effects on soil biological fertility in assisted phytoremediation of an industrial polluted soil.

Phytoremediation is a cost-effective "green technology" that uses plants to improve the soil properties of polluted sites, preventing the dispersion of pollutants and reducing the mobility of potentially toxic elements (PTEs) through their adsorption and accumulation by roots or precipitation within the root zone. Being highly tolerant to pollutants and other abiotic stresses, giant reed (Arundo donax L.) is a suitable biomass crop for phytoremediation of contaminated soils. We report the results of a two-year open-air lysimeter study aimed at assessing the adaptability of giant reed to grow on industrial substrates polluted by Pb and Zn and at testing commercial humic acids from leonardite as improvers of plant performance. We evaluated giant reed potential for: 1) biomass production for energy or biomaterial recovery; 2) PTE phytoextraction and 3) soil fertility restoration. Chemical fertility was monitored by measuring soil C while soil biological fertility was estimated by quantifying the abundance of bacterial functional genes regulating nitrogen fixation (nifH) and nitrification (amoA). Giant reed above-ground growth on the polluted soils was slightly lower (-16%) than on a non-polluted soil, with a preferential storage of biomass in the rhizome acting as a survival strategy in limiting growing conditions. Humic acids improved plant stress tolerance and production levels. As aerial biomass (shoots) did not accumulate PTEs, the plant in question can be used for bioenergy or biopolymer production. In contrast, below-ground biomass (rhizomes) accumulated PTEs, and can thus be harvested and removed from soil to improve phytoremediation protocols and also used as industrial biofuel. Giant reed growth increased the abundance of N-cycling bacteria and soil C in the rhizospheric soil, as well as reduced soil Pb and Zn EDTA extractable fraction.

Spatio-temporal analysis of vascular endothelial growth factor expression and blood vessel remodelling in pig ovarian follicles during the periovulatory period.

Vascular endothelial growth factor (VEGF) expression pattern and blood vessel remodelling were evaluated during the transition from the preovulatory follicle to the corpus luteum (CL). To this end, prepubertal gilts were treated with equine chorionic gonadotrophin (eCG) to collect preovulatory follicles (60 h after eCG) and with human chorionic gonadotrophin (hCG) to obtain periovulatory follicles 18 h and 36 h later. The VEGF mRNA content was analysed by in situ hybridization, while protein localization in follicular fluid (FF) and in granulosa and theca compartments was evaluated by ELISA, immunohistochemistry or western blot. Blood vessel architecture and vascular area (VA) were investigated using immunohistochemistry for von Willenbrand Factor, a specific endothelial marker. Vascular remodelling was finally tested using Ki-67 immunocytochemistry as a proliferation marker, or alpha-smooth muscle actin (alpha-SMA) as a specific mural cell marker. eCG-treated follicles showed high VEGF levels and two concentric blood vessel networks composed of proliferating endothelial cells without any association with mural components. hCG injection inhibited VEGF synthesis in the granulosa compartment and, as a consequence, the protein fell within the FF. In parallel, endothelial cell proliferation stopped and the VA decreased. Close to ovulation, VEGF production restarted in both follicular compartments and VEGF mRNA content significantly increased in the theca layer. Changes in follicular VEGF secretion were observed; the protein disappeared from FF and was observed in the extracellular matrix. An active angiogenesis characterized the follicle; endothelial cell proliferation was associated with a recruitment of alpha-SMA-positive mural cells. The data presented in this work showed that, in the phases preceding ovulation, a complete vascular remodelling occurs, characterized by both an evident neovascularization and the appearance of blood vessels presenting smooth musculature which could be involved in CL formation after ovulation.

Synthesis and biological activity of Akt/PI3K inhibitors.

Phosphatidylinositol 3-kinase (PI3K) and serine/threonine protein kinase B (PKB or Akt) pathways regulate important cellular processes and are related to a number of human pathologies, such as cancer. The development of kinase inhibitors, with particular attention to small molecule analogues of natural phosphoinositides for pathway interruption and therapeutic applications will be reviewed.

Cerebral Perfusion Pressure is Maintained in Acute Intracerebral Hemorrhage: A CT Perfusion Study.

BACKGROUND AND PURPOSE: Although blood pressure reduction has been postulated to result in a fall in cerebral perfusion pressure in patients with intracerebral hemorrhage, the latter is rarely measured. We assessed regional cerebral perfusion pressure in patients with intracerebral hemorrhage by using CT perfusion source data. MATERIALS AND METHODS: Patients with acute primary intracerebral hemorrhage were randomized to target systolic blood pressures of <150 mm Hg (n = 37) or <180 mm Hg (n = 36). Regional maps of cerebral blood flow, cerebral perfusion pressure, and cerebrovascular resistance were generated by using CT perfusion source data, obtained 2 hours after randomization. RESULTS: Perihematoma cerebral blood flow (38.7 ± 11.9 mL/100 g/min) was reduced relative to contralateral regions (44.1 ± 11.1 mL/100 g/min, P = .001), but cerebral perfusion pressure was not (14.4 ± 4.6 minutes(-1) versus 14.3 ± 4.8 minutes(-1), P = .93). Perihematoma cerebrovascular resistance (0.34 ± 0.11 g/mL) was higher than that in the contralateral region (0.30 ± 0.10 g/mL, P < .001). Ipsilateral and contralateral cerebral perfusion pressure in the external (15.0 ± 4.6 versus 15.6 ± 5.3 minutes(-1), P = .15) and internal (15.0 ± 4.8 versus 15.0 ± 4.8 minutes(-1), P = .90) borderzone regions were all similar. Borderzone cerebral perfusion pressure was similar to mean global cerebral perfusion pressure (14.7 ± 4.7 minutes(-1), P ≥ .29). Perihematoma cerebral perfusion pressure did not differ between blood pressure treatment groups (13.9 ± 5.5 minutes(-1) versus 14.8 ± 3.4 minutes(-1), P = .38) or vary with mean arterial pressure (r = -0.08, [-0.10, 0.05]). CONCLUSIONS: Perihematoma cerebral perfusion pressure is maintained despite increased cerebrovascular resistance and reduced cerebral blood flow. Aggressive antihypertensive therapy does not affect perihematoma or borderzone cerebral perfusion pressure. Maintenance of cerebral perfusion pressure provides physiologic support for the safety of blood pressure reduction in intracerebral hemorrhage.

Tetracycline affects abnormal properties of synthetic PrP peptides and PrP(Sc) in vitro.

Prion diseases are characterized by the accumulation of altered forms of the prion protein (termed PrP(Sc)) in the brain. Unlike the normal protein, PrP(Sc) isoforms have a high content of beta-sheet secondary structure, are protease-resistant, and form insoluble aggregates and amyloid fibrils. Evidence indicates that they are responsible for neuropathological changes (i.e. nerve cell degeneration and glial cell activation) and transmissibility of the disease process. Here, we show that the antibiotic tetracycline: (i) binds to amyloid fibrils generated by synthetic peptides corresponding to residues 106-126 and 82-146 of human PrP; (ii) hinders assembly of these peptides into amyloid fibrils; (iii) reverts the protease resistance of PrP peptide aggregates and PrP(Sc) extracted from brain tissue of patients with Creutzfeldt-Jakob disease; (iv) prevents neuronal death and astrocyte proliferation induced by PrP peptides in vitro. NMR spectroscopy revealed several through-space interactions between aromatic protons of tetracycline and side-chain protons of Ala(117-119), Val(121-122) and Leu(125) of PrP 106-126. These properties make tetracycline a prototype of compounds with the potential of inactivating the pathogenic forms of PrP.

[[(Arylpiperazinyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives as high-affinity, selective 5-HT1A receptor ligands.

A series of 2-[[(4-aryl-1-piperazinyl)alkyl]thio]thieno[2,3-d]pyrimidin-4 (1H)-one and 3-substituted 2-[[(4-aryl-1-piperazinyl)alky]thio]thieno[2,3-d]pyrimidin-4 (3H)-one derivatives was prepared and evaluated for in vitro 5-HT1A receptor affinity by radioligand binding assays; the selectivity for 5-HT1A receptors rather than alpha 1-adrenoceptors was also examined (ratio of the IC50 alpha 1 to IC50 5-HT1A). The binding tests gave indications about the best features of the [(arylpiperazinyl)alkyl]thio moiety and of the substituents on the thiophene and pyrimidinone rings for efficacious and selective 5-HT1A ligands. The most effective derivative for displacing [3H]-8-OH-DPAT from rat hippocampal membranes was the 3-amino-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl] propyl]thio]-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (70) (IC50 = 0.3 nM) with selectivity of 24 for the 5-HT1A over the alpha 1-adrenoceptor. Compound 73, where the 2-methoxyphenyl on the N4 piperazine ring was replaced with a pyrimidine group, showed the best selectivity, with a ratio of 74, while its affinity IC50 for 5-HT1A was 6.8 nM. These results, compared to those for compounds 46 (IC50 24 nM; selectivity 2) and 49 (IC50 226 nM; selectivity 5), N3 unsubstituted analogues of derivatives 70 and 73, show the importance of an amino group in position 3 of the thienopyrimidine system for the interaction with 5-HT1A receptor binding sites, although this fragment can affect the affinity and selectivity only if linked to the (arylpiperazinyl)alkyl moiety. The better selectivity of piperidine 74 (IC50 0.8; selectivity 45) compared to the analogous piperazine 70 is also noteworthy. Twenty of the 30 molecules used for determining the binding affinity to 5-HT1A and alpha 1-adrenergic receptors were selected for QSAR analysis using a series of molecular descriptors and calculated with the TSAR software.

Activation of protein kinase A and protein kinase C mediates the depolarising effect of LH in ovine cumulus-corona cells.

We have studied the changes in membrane potential induced by LH in cumulus and granulosa cells isolated from sheep antral follicles. The investigation was carried out by using a non-invasive technique based on the use of a membrane potential sensitive probe, bis-oxonol. The membrane potential of mural granulosa cells was totally unaffected by LH, while that of cumulus or corona cells showed a marked depolarisation, starting 2-3 min after the addition of the hormone and plateauing after 5-10 min. None of the cells tested reacted to FSH. In the second part of the experiment the role of protein kinase A (PKA) and protein kinase C (PKC) in mediating the effect of LH was studied. The selective activation of PKA or PKC induced in cumulus-corona cells a rapid hyperpolarisation due to increased Cl and K conductance respectively. By contrast, the simultaneous activation of the two kinases induced a rapid membrane depolarisation due to the progressive decrease in K conductance. The activation of each kinase or their combined stimulation did not induce any change in the membrane potential of mural granulosa cells. These data demonstrated that LH has a depolarising effect regionally circumscribed to cumulus-corona cells and that this depolarisation depends on a reduction of K conductance caused by the activation of PKA and PKC.

PCR-based apolipoprotein E genotype analysis from archival fixed brain.

A technique is described for determining the apolipoprotein E genotype (apo E; alleles epsilon2, epsilon3, or epsilon4) from tissues which have been fixed with 4-10% formaldehyde and archived. The procedure requires efficient extraction and exhaustive purification of DNA from the fixed tissue. Because the fixation process renders the DNA largely crosslinked and/or sheared (therefore unsuitable for traditional analysis), a nested polymerase chain reaction (PCR) is employed (using two apo E gene specific primer pairs) to specifically amplify the polymorphic region of the gene. The genotype was then determined using previously reported HhaI polymorphisms that occur as a direct result of the variant codons responsible for the three alleles. This protocol permitted the successful genotyping of 90% (34 out of 38) of the archived brain samples from Alzheimer's disease (AD) patients. These samples included such extremes as a sample that had been stored for 12 years in formalin. This procedure permits the retrospective analysis of samples that had been processed and stored well before the original characterization of apo E alleles as risk factors in AD. Finally, this approach is readily adapted to the analysis of any gene of interest, whether by restriction fragment length polymorphism or direct amplicon DNA sequencing. It is also a very robust assay for less stringent conditions such as DNA isolated from whole blood or frozen tissue.

A cocaine analog, 2beta-propanoyl-3beta-(4-tolyl)-tropane (PTT), reduces tyrosine hydroxylase in the mesolimbic dopamine pathway.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Previously published results have established that chronic cocaine administration (30-45 mg/kg per day, 10-14 days) resulted in an upregulation of TH gene expression in dopaminergic pathways of rats. The present studies tested the effects of a tropane analog, PTT (2beta-propanoyl-3beta-(4-tolyl)-tropane), on TH expression. This drug has similar actions to cocaine, but possesses markedly different pharmacokinetics (20 times more potent at binding the dopamine transporter, markedly increased metabolic stability, and 10-20 times more potent in behavioral measures). Moreover, PTT demonstrates an increased selectivity for the dopamine (DA) and norepinephrine (NE) transporters compared with cocaine. In direct contrast to the previously reported effects of cocaine, 10 days of PTT administration (3.0 mg/kg per day, i.p.) produced a uniform downregulation of TH protein and activity gene expression. TH activity and immunoreactive protein where decreased by 54 and 69%, respectively in the nucleus accumbens. Within the ventral tegmental area, TH activity and protein were decreased by 33 and 19%, respectively. The underlying mechanisms for these fundamental differences are unclear, but likely reflect varying and selective affinities and lengths of occupancy at biogenic amine transporters.

Covalently modified microperoxidases as heme-peptide models for peroxidases.

Microperoxidase-8 (MP8) and microperoxidase-9 (MP9) have been covalently modified by attachment of proline-containing residues to the amino terminal peptide chain in order to obtain new peroxidase model systems. The catalytic activities of these derivatives in the oxidation of p-cresol by hydrogen peroxide have been compared to that of MP8. The presence of steric hindrance above the heme reduces the formation rate of the catalytically active species, while the reactivity is increased when the amino group of a proline residue is close to the iron. The modification of the catalyst affects the rate of degradation processes undergone by the heme group during catalysis. A bulky aromatic group on the distal side decreases the stability of the complex because it reduces the mobility of a phenoxy radical species formed during catalysis, while the presence of proline residues increases the number of turnovers of the heme catalysts before degradation. The complex Pro2-MP8 obtained by addition of two proline residues to MP8 exhibits the best catalytic performance in terms of activity and chemical stability.

Spectroscopic and binding studies on the interaction of inorganic anions with lactoperoxidase.

The interaction of several inorganic species (SCN-, I-, Br-, Cl-, F-, NO2-, N3-, CN-) with bovine lactoperoxidase was investigated through kinetic and binding studies by using UV-Vis spectroscopy. The above ligands form 1:1 complexes with the protein and can be assigned to three different groups, on the basis of the dissociation constant values (KD) of the adducts: (1) SCN-, I-, Br-, and Cl- (KD increases along the series); (2) F- (which shows a singular behavior); (3) NO2-, N3-, and CN- (that bind at the iron site). KD values for the LPO/SCN- adduct appeared to be modified in the presence of other inorganic species; a strong competition between this substrate and all other anions (with the exception of F-) was evidentiated. Binding investigations on the natural substrates SCN- and I-, at varying pH and temperature, showed that their interaction with lactoperoxidase involves the protonation of a common site in proximity of the iron (possibly distal histidine). Michaelis-Menten constants for SCN-, I-, and Br- followed roughly the same trend as KD; KM for hydrogen peroxide is strongly dependent on the cosubstrate. Computer-assisted docking simulations showed that all ligands can penetrate inside the heme pocket.