Carotid artery intima-media thickness is associated with insulin-mediated glucose disposal in nondiabetic normotensive offspring of type 2 diabetic patients

The aim of this study was to investigate whether insulin resistance is independently associated with early manifestations of atherosclerosis. To this end, 176 normotensive offspring of type 2 diabetic patients were subjected to euglycemic hyperinsulinemic clamp to assess insulin sensitivity. Early atherosclerosis was studied by ultrasonography of the common carotid artery. Of the total 176 subjects, 145 were glucose tolerant, 18 had impaired fasting glucose, and 13 had impaired glucose tolerance. Univariate correlations showed that age, body mass index, waist, blood pressure, 2-h postchallenge glucose, fasting insulin, triglycerides, interleukin-6, fibrinogen, and white blood cell count were significantly correlated with carotid intima-media thickness (IMT), whereas HDL cholesterol and glucose disposal showed a negative correlation. A stepwise multivariate regression analysis including sex, age, waist circumference, smoking status, systolic blood pressure, diastolic blood pressure, triglyceride, HDL cholesterol, 2-h postchallenge glucose, plasma IL-6, fibrinogen, white blood cell count, insulin-stimulated glucose disposal, and fasting insulin showed that the four variables that remained significantly associated with carotid IMT were waist circumference, insulin-stimulated glucose disposal, white blood cell count, and diastolic blood pressure, accounting for 33.7% of its variation. These findings support the concept that insulin sensitivity, rather than plasma insulin levels, is associated with early atherosclerosis in nondiabetic normotensive offspring of type 2 diabetic patients.     

Reduced GABAergic Inhibition in the Basolateral Amygdala and the Development of Anxiety-Like Behaviors after Mild Traumatic Brain Injury

Traumatic brain injury (TBI) is a major public health concern affecting a large number of athletes and military personnel. Individuals suffering from a TBI risk developing anxiety disorders, yet the pathophysiological alterations that result in the development of anxiety disorders have not yet been identified. One region often damaged by a TBI is the basolateral amygdala (BLA); hyperactivity within the BLA is associated with increased expression of anxiety and fear, yet the functional alterations that lead to BLA hyperexcitability after TBI have not been identified. We assessed the functional alterations in inhibitory synaptic transmission in the BLA and one mechanism that modulates excitatory synaptic transmission, the α₇ containing nicotinic acetylcholine receptor (α₇-nAChR), after mTBI, to shed light on the mechanisms that contribute to increased anxiety-like behaviors. Seven and 30 days after a mild controlled cortical impact (CCI) injury, animals displayed significantly greater anxiety-like behavior. This was associated with a significant loss of GABAergic interneurons and significant reductions in the frequency and amplitude of spontaneous and miniature GABA_A-receptor mediated inhibitory postsynaptic currents (IPSCs). Decreases in the mIPSC amplitude were associated with reduced surface expression of α1, β2, and γ2 GABA_A receptor subunits. However, significant increases in the surface expression and current mediated by α₇-nAChR, were observed, signifying increases in the excitability of principal neurons within the BLA. These results suggest that mTBI causes not only a significant reduction in inhibition in the BLA, but also an increase in neuronal excitability, which may contribute to hyperexcitability and the development of anxiety disorders.     

Genomic Characterisation of Small Cell Lung Cancer Patient-Derived Xenografts Generated from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Specimens

Patient-derived xenograft (PDX) models generated from surgical specimens are gaining popularity as preclinical models of cancer. However, establishment of PDX lines from small cell lung cancer (SCLC) patients is difficult due to very limited amount of available biopsy material. We asked whether SCLC cells obtained from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) could generate PDX lines that maintained the phenotypic and genetic characteristics of the primary tumor. Following successful EBUS-TBNA sampling for diagnostic purposes, we obtained an extra sample for cytologic analysis and implantation into the flanks of immunodeficient mice. Animals were monitored for engraftment for up to 6 months. Histopathologic and immunohistochemical analysis, and targeted next-generation re-sequencing, were then performed in both the primary sample and the derivative PDX line. A total of 12 patients were enrolled in the study. EBUS-TBNA aspirates yielded large numbers of viable tumor cells sufficient to inject between 18,750 and 1,487,000 cells per flank, and to yield microgram quantities of high-quality DNA. Of these, samples from 10 patients generated xenografts (engraftment rate 83%) with a mean latency of 104 days (range 63–188). All but one maintained a typical SCLC phenotype that closely matched the original sample. Identical mutations that are characteristic of SCLC were identified in both the primary sample and xenograft line. EBUS-TBNA has the potential to be a powerful tool in the development of new targeting strategies for SCLC patients by providing large numbers of viable tumor cells suitable for both xenografting and complex genomic analysis.     

Characterization of the Prostate-Specific Antigen (PSA) Catalytic Mechanism: A Pre-Steady-State and Steady-State Study

Prostate-specific antigen (PSA), an enzyme of 30 kDa grouped in the kallikrein family is synthesized to high levels by normal and malignant prostate epithelial cells. Therefore, it is the main biomarker currently used for early diagnosis of prostate cancer. Here, presteady-state and steady-state kinetics of the PSA-catalyzed hydrolysis of the fluorogenic substrate Mu-His-Ser-Ser-Lys-Leu-Gln-AMC (spanning from pH 6.5 to pH 9.0, at 37.0°C) are reported. Steady-state kinetics display at every pH value a peculiar feature, represented by an initial “burst” phase of the fluorescence signal before steady-state conditions are taking place. This behavior, which has been already observed in other members of the kallikrein family, suggests the occurrence of a proteolytic mechanism wherefore the acylation step is faster than the deacylation process. This feature allows to detect the acyl intermediate, where the newly formed C-terminal carboxylic acid of the cleaved substrate forms an ester bond with the -OH group of the Ser195 catalytic residue, whereas the AMC product has been already released. Therefore, the pH-dependence of the two enzymatic steps (i.e., acylation and deacylation) has been separately characterized, allowing the determination of pK_a values. On this basis, possible residues are tentatively identified in PSA, which might regulate these two steps by interacting with the two portions of the substrate.     

Inhibitors of apoptosis proteins in experimental benign prostatic hyperplasia: effects of serenoa repens,selenium and lycopene

Background

The apoptosis machinery is a promising target against benign prostatic hyperplasia (BPH). Inhibitors of apoptosis proteins (IAPs) modulate apoptosis by direct inhibition of caspases. Serenoa Repens (SeR) may be combined with other natural compounds such as Lycopene (Ly) and Selenium (Se) to maximize its therapeutic activity in BPH. We investigated the effects of SeR, Se and Ly, alone or in association, on the expression of four IAPs, cIAP-1, cIAP-2, NAIP and survivin in rats with experimental testosterone-dependent BPH. Moreover, caspase-3, interleukin-6 (IL-6) and prostate specific membrane antigen (PSMA) have been evaluated.Rats were administered, daily, with testosterone propionate (3 mg/kg/sc) or its vehicle for 14 days. Testosterone injected animals (BPH) were randomized to receive vehicle, SeR (25 mg/kg/sc), Se (3 mg/kg/sc), Ly (1 mg/kg/sc) or the SeR-Se-Ly association for 14 days. Animals were sacrificed and prostate removed for analysis.

Results

BPH animals treated with vehicle showed unchanged expression of cIAP-1 and cIAP-2 and increased expression of NAIP, survivin, caspase-3, IL-6 and PSMA levels when compared with sham animals. Immunofluorescence studies confirmed the enhanced expression of NAIP and survivin with a characteristic pattern of cellular localization. SeR-Se-Ly association showed the highest efficacy in reawakening apoptosis; additionally, this therapeutic cocktail significantly reduced IL-6 and PSMA levels. The administration of SeR, Se and Ly significantly blunted prostate overweight and growth; moreover, the SeR-Se-Ly association was most effective in reducing prostate enlargement and growth by 43.3% in treated animals.

Conclusions

The results indicate that IAPs may represent interesting targets for drug therapy of BPH.     

Contrasting effects of habitat area and connectivity on evenness of pollinator communities

Losses of both habitat area and connectivity have been identified as important drivers of species richness declines, but little theoretical and empirical work exists that addresses the effect of fragmentation on relative commonness of highly mobile species such as pollinating insects. With a large dataset of wild bee and butterfly abundances collected across Europe, we first tested the effect of habitat area and connectivity on evenness in pollinator communities using a large array of indexes that give different weight to dominance and rarity. Second, we tested if traits related to mobility and diet breadth could explain the observed evenness patterns. We found a clear negative effect of area and a weaker, but positive effect of connectivity on evenness. Communities in small habitat fragments were mainly composed of mobile and generalist species. The higher evenness in small fragments could thereby be generated by highly mobile species that maintain local populations with frequent inter‐fragment movements. Trait analysis suggested an increasing importance of dispersal over local recruitment, as we move from large to small fragments and from less to more connected fragments. Species richness and evenness were negatively correlated indicating that the two variables responded differently to habitat area and connectivity, although the mechanisms underlying the observed patterns are difficult to isolate. Even though habitat area and connectivity often decrease simultaneously due to habitat fragmentation, an interesting practical implication of the contrasting effect of the two variables is that the resulting community composition will depend on the relative strength of these two processes.     

Increased sensitivity of the neuronal nicotinic receptor alpha 2 subunit causes familial epilepsy with nocturnal wandering and ictal fear

Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the alpha 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor alpha 2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic alpha 2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior.     

Chaperone-like activity of alpha-crystallin toward aldose reductase oxidatively stressed by copper ion

The protective action of alpha-crystallin against copper-induced protein stress is studied using bovine lens aldose reductase (ALR2) as protein model. The oxidative inactivation of ALR2 induced by CuCl2 at the stoichiometric Cu2+/ALR2 ratio of 2/1 [I. Cecconi, M. Moroni, P.G. Vilardo, M. Dal Monte, P. Borella, G. Rastelli, L. Costantino, D. Garland, D. Carper, J.M. Petrash, A. Del Corso, U. Mura, Biochemistry 37 (1998) 14167-14174] is accompanied by protein aggregation phenomena when the metal ion concentration is increased (Cu2+/ALR2>3). Protein oxidation precedes protein precipitation. Both inactivation and precipitation of ALR2 are prevented by alpha-crystallin in a concentration-dependent manner. The rationale for the stabilization of ALR2 exerted by alpha-crystallin at low metal concentration is given on the basis of the ability of alpha-crystallin to chelate copper. However, the overall protective action exerted by alpha-crystallin at higher copper concentration may be explained invoking the contribution of the special features of alpha-crystallin to easily interact with target proteins undergoing structural rearrangement.     

Sequential events of apoptosis involving docetaxel, a microtubule-interfering agent: A cytometric study

Background  

Despite the great advances in the understanding of programmed cell death, little attention has been paid to the sequence of the events that characterise it. In particular, the course of apoptotic events induced by microtubule-interfering agents such as taxanes is poorly understood. In order to increase such knowledge, we studied a number of independent biochemical and cytological modifications using cytometric methods in a bladder cancer cell line treated with the second generation taxane, docetaxel.