In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

Thiamine deficiency increases β-secretase activity and accumulation of β-amyloid peptides

Thiamine pyrophosphate (TPP) and the activities of thiamine-dependent enzymes are reduced in Alzheimer's disease (AD) patients. In this study, we analyzed the relationship between thiamine deficiency (TD) and amyloid precursor protein (APP) processing in both cellular and animal models of TD. In SH-SY5Y neuroblastoma cells overexpressing APP, TD promoted maturation of β-site APP cleaving enzyme 1 (BACE1) and increased β-secretase activity which resulted in elevated levels of β-amyloid (Aβ) as well as β-secretase cleaved C-terminal fragment (β-CTF). An inhibitor of β-secretase efficiently reduced TD-induced up-regulation of Aβ and β-CTF. Importantly, thiamine supplementation reversed the TD-induced alterations. Furthermore, TD treatment caused a significant accumulation of reactive oxygen species (ROS); antioxidants suppressed ROS production and maturation of BACE1, as well as TD-induced Aβ accumulation. On the other hand, exogenous Aβ_1-40 enhanced TD-induced production of ROS. A study on mice indicated that TD also caused Aβ accumulation in the brain, which was reversed by thiamine supplementation. Taken together, our study suggests that TD could enhance Aβ generation by promoting β-secretase activity, and the accumulation of Aβ subsequently exacerbated TD-induced oxidative stress.     

A crosstalk between β1 and β3 integrins controls glycine receptor and gephyrin trafficking at synapses

The regulation of glycine receptor (GlyR) number at synapses is necessary for the efficacy of inhibition and the control of neuronal excitability in the spinal cord. GlyR accumulation at synapses depends on the scaffolding molecule gephyrin and is linked to GlyR synaptic dwell time. However, the mechanisms that tune GlyR synaptic exchanges in response to different neuronal environments are unknown. Integrins are cell adhesion molecules and signaling receptors. Using single quantum dot imaging and fluorescence recovery after photobleaching, we found in rats that β1 and β3 integrins adjust synaptic strength by regulating the synaptic dwell time of both GlyRs and gephyrin. β1 and β3 integrins crosstalked via calcium/calmodulin-dependent protein kinase II and adapted GlyR lateral diffusion and gephyrin-dependent trapping at synapses. This provides a mechanism for maintaining or adjusting the steady state of postsynaptic molecule exchanges and the level of glycinergic inhibition in response to neuron- and glia-derived signals or extracellular matrix remodeling.     

Interaction of G-actin with thymosin β4 and its variants thymosin β9 and thymosin β 9 met

Summary Thymosin ₄ is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin ₉ and thymosin ₉ ^met are minor variants of thymosin ₄. The possible function of these peptides has been investigated by comparing the actin binding properties of these -thymosins. Thymosin ₉ and thymosin ₉ ^met were found to inhibit polymerization of ATP-actin with identical K _d s of 0.7–0.8 M (as compared to 2±0.3 M for thymosin ₄); like thymosin ₄, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin ₄ and thymosin ₉ ^met with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin ₄ to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin ₄ binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of 1.5 M^-1 s^-1 and 2s^-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin ₄ function in vivo is discussed.     

Expression and localization of E-cadherin and β-catenin in uterine carcinosarcoma

This study was designed to analyze the subcellular localization of E-cadherin and β-catenin both of which play a critical role in cell–cell adhesion in uterine carcinosarcoma (UCS). We performed an immunohistochemical reaction analysis of the subcellular localization of E-cadherin and β-catenin proteins in 46 cases of UCSs consisting of 28 UCSs with heterologous sarcoma and 18 UCSs with homologous sarcoma and compared their clinicopathological features. In most UCSs, membranous expression of E-cadherin and β-catenin was completely lost in sarcomatous components, but it was preserved in carcinomatous components. Nuclear β-catenin expression was observed significantly more frequently in sarcomatous components (31/46, 67.4%) than in carcinomatous components (22/46, 47.8%; P = 0.0025). In sarcomatous components, nuclear β-catenin expression was found significantly more frequently in heterologous sarcoma (23/28, 82.1%) than in homologous sarcoma (8/18, 44.4%; P = 0.0279). The stage was the only independent prognostic significant factor. These results suggest that reduced membranous expression of E-cadherin and β-catenin may contribute to the biphasic morphology of UCS. Furthermore, although the precise mechanism is unclear, nuclear β-catenin expression in sarcomatous components may also be associated with biphasic morphology and heterologous sarcomatous differentiation.     

Cystathionine β-synthase and γ-cystathionase in helminths

The activities of -cystathionase and cystathionine -synthase were investigated in a range of gastrointestinal, free-living and entomophagous nematodes. Although nermatode -cystathionase used the same range of substrates as the mammalian hepatic enzyme, its activity was extremely low and there were significant interspecies variations with respect to the relative order of active substrates. Like the mammalian liver enzyme, nematode cystathionine -synthase showed activity in the directions of both cystathionine synthesis and the for-ward and reverse l-serine sulphhydrase reactions. However, the most important feature of the survey was the widespread ability of nematode cystathionine -synthase to catalyse the non-mammalian activatedl-serine sulphhydrase reaction (l-cysteine+R-SHcysteine thioether+H₂S). Additional survey work revealed that the ability to catalyse the activatedl-serine sulphhydrase reaction was almost universal amongst nematodes. Activatedl-serine sulphhydrase activity was also demonstrated in the acanthocephalanPomphorhynchus laevis but was absent from cestodes and digeneans.     

Pattern of neurobehavioral and organ-specific toxicities of β, β’-iminodipropionitrile in mice

Introduction

β, β’-iminodipropionitrile (IDPN) is a synthetic nitrile that produces a permanent movement disorder in rodents. Although IDPN-induced vestibular pathology is well documented, the mode of IDPN interaction with other organ systems is poorly understood. We examined the behavioral signs and histopathological changes in the vestibular labyrinth, brain, liver and kidneys of mice exposed to IDPN.

Material and methods

Adult male SWR/J mice were divided into 2 groups of 6 animals each. One group of mice received normal saline (control group) and the other group was treated with IDPN (400 mg/kg, i.p.) daily for 7 days. Dyskinetic movements including vertical and horizontal head weaving, circling and backward walking were quantified on days 7, 8 and 9.

Results

We observed a direct correlation between the severity of IDPN-induced behavioral deficits and the degeneration of vestibular hair cells in the crista ampullaris of mice. The brain cortex of both groups appeared similar, whereas the kidney histopathology revealed mild nephrotoxicity in some of the IDPN-treated mice. Administration of IDPN caused severe hepatotoxicity, but the intensity of hepatic damage was not correlated with the severity of behavioral deficits.

Conclusions

Degeneration of vestibular sensory hair cells plays an important role in the development of IDPN-induced behavioral deficits in mice. Exposure to IDPN also caused severe hepatotoxicity which was independent of the behavioral symptoms. These findings could be of potential relevance to human health, particularly after the observation that IDPN not only causes a movement disorder but also produces acute liver injury.     

Plasma cortisol and β-endorphin concentrations in trained and over-trained standardbred racehorses

The effects of training and over-training on plasma cortisol and beta-endorphin (betaEP) concentrations at rest and after standardised exercise tests and the cortisol responses to adrenocorticotropin (ACTH) administration were investigated in standardbred horses. Twelve horses were divided randomly into control and over-trained (OT) groups after 17 weeks slow- and moderate-intensity treadmill training. The standardised treadmill exercise test consisted of 2 min at velocities corresponding to 30, 50, 70 and 100% of maximum O2 consumption. Over-training, defined as a significant decrease in body weight and treadmill run-time-to-fatigue in an incremental velocity test, occurred in the OT group after 32 weeks of training exercise. Peak cortisol concentrations after exercise decreased significantly in the OT group from 320+/-15.6 at week 8 to 245+/-17.0 nmol l(-1) at week 32, and mean cortisol concentrations over a 120-min period after exercise decreased from 258+/-11.7 to 192+/-16.6 nmol l(-1) (P<0.05). Mean and total cortisol and betaEP concentrations in resting horses were not significantly different after over-training. Peak cortisol concentrations after adrenocorticotropin (ACTH) administration were not significantly different in the over-trained group. Dysfunction of the hypothalamo-pituitary-adrenocortical axis occurs in over-trained horses, but this adaptation is not associated with a change in the adrenocortical responsiveness to ACTH.     

Stability and immunoreactivity of glycinin and β-conglycinin to hydrolysis in vitro

The present study was to compare the stability and immunoreactivity of glycinin and β-conglycinin to hydrolysis with pepsin, trypsin or cooperation of the two enzymes for different time intervals (0.5, 1, 15, 30, 60 and 120 min) at different ratios of enzyme/substrate (1:100, 1:10, 1:1 and 10:1) in vitro. The results showed that the immunoreactivity was positively related with stability of glycinin (r=0.776, P<0.05) and β-conglycinin (r=0.851, P<0.05). B polypeptide chain of glycinin was resistant to hydrolysis with trypsin, and β subunit of β-conlycinin was not liable to hydrolysis with pepsin. The two above proteins were little or not affected by incubation time and the enzyme/substrate ratio, while others' hydrolysed degree got higher with prolonging incubation time, and the hydrolysis accelerated with increasing enzyme/substrate ratio. Our results indicated digestive enzyme, incubation time and enzyme/substrate ratio had effects on stability and immunoreactivity of allergenic proteins. The most effective hydrolysis was cooperation of the two enzymes for glycinin and trypsin for β-conglycinin.     

Faecal carriage of extended-spectrum β-lactamase-producing and AmpC β-lactamase-producing bacteria among Danish army recruits

During May and June 2008, 84 Danish army recruits were tested for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing and AmpC β-lactamase-producing bacteria. Three ESBL-producing (CTX-M-14a) Escherichia coli isolates, two AmpC-producing (CMY-2) E. coli isolates and one AmpC-producing (CMY-34) Citrobacter freundii isolate were detected. Two of the CTX-M-14a E. coli isolates had similar pulsed-field gel electrophoresis and multilocus sequence typing profiles, indicating the same origin or transmission between the two army recruits. The bla_CTX-M-14a genes were transferable to an E. coli recipient. These commensal bacteria therefore constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria in the intestine.     

Mineral coatings modulate β-TCP stability and enable growth factor binding and release

β-Tricalcium phosphate (β-TCP) is an attractive ceramic for bone tissue repair because of its similar composition to bone mineral and its osteoconductivity. However, compared with other ceramics β-TCP has a rapid and uncontrolled rate of degradation. In the current study β-TCP granules were mineral coated with the aim of influencing the dissolution rate of β-TCP, and also to use the coating as a carrier for controlled release of the growth factors recombinant human vascular endothelial growth factor (rhVEGF), modular VEGF peptide (mVEGF), and modular bone morphogenetic protein 2 peptide (mBMP2). The biomineral coatings were formed by heterogeneous nucleation in aqueous solution using simulated body fluid solutions with varying concentrations of bicarbonate (HCO(3)). Our results demonstrate that we could coat β-TCP granules with mineral layers possessing different dissolution properties. The presence of a biomineral coating delays the dissolution rate of the β-TCP granules. As the carbonate (CO(3)(2-)) content in the coating was increased the dissolution rate of the coated β-TCP also increased, but remained slower than the dissolution of uncoated β-TCP. In addition, we showed sustained release of multiple growth factors, with release kinetics that were controllable by varying the identity of the growth factor or the CO(3)(2-) content in the mineral coating. Released rhVEGF induced human umbilical vein endothelial cell (HUVEC) proliferation, and mVEGF enhanced migration of mouse embryonic endothelial cells in a scratch wound healing assay, indicating that each released growth factor was biologically active.     

Inhaled β2 -agonists and airway responses to allergen

A series of investigations show that the regular use of inhaled β₂ -agonists will increase all aspects of the airway response to allergen. The mechanism of this effect is uncertain; however, it appears to be different from the mechanism that produces tolerance to β₂ -agonist effects. One possibility is that the regular use of β₂ -agonists might induce a mast cell β-receptor dysfunction that might make mast cells more prone to release mediators. As a result β₂ -agonist use plus allergen exposure might cause more mediator release than does allergen exposure alone. The corollary of this is that β₂ -agonist use plus allergen exposure might cause more airway inflammation than does allergen exposure alone. These hypotheses are both testable. I believe that this is a clinically important phenomenon and may well be a major reason for β₂ -agonist–induced worsened asthma control. Further investigations are indicated to identify the mechanism and the clinical relevance of the phenomenon. (J Allergy Clin Immunol 1998;102:S96-9)     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

Experimental allergic encephalomyelitis, β-adrenergic receptors and interferon gamma-secreting cells in β-adrenergic agonist-treated rats

Treatment with the β₂-adrenergic agonist terbutaline or the β-adrenergic agonist isoproterenol suppresses experimental allergic encephalomyelitis, decreases the number of IFNγ-producing splenic cells, and decreases the number of β-adrenergic receptors on splenic lymphocytes in Lewis rats. The effects of terbutaline are greater when the drug is given from the day of immunization through the acute phase of the illness or from day 15 postimmunization until recovery, than when given for the first 12 days after immunization.     

GABAA receptors involved in sleep and anaesthesia: β1- versus β3-containing assemblies

The histaminergic neurons of the posterior hypothalamus (tuberomamillary nucleus—TMN) control wakefulness, and their silencing through activation of GABA_A receptors (GABA_AR) induces sleep and is thought to mediate sedation under propofol anaesthesia. We have previously shown that the β1 subunit preferring fragrant dioxane derivatives (FDD) are highly potent modulators of GABA_AR in TMN neurons. In recombinant receptors containing the β3N265M subunit, FDD action is abolished and GABA potency is reduced. Using rat, wild-type and β3N265M mice, FDD and propofol, we explored the relative contributions of β1- and β3-containing GABA_AR to synaptic transmission from the GABAergic sleep-on ventrolateral preoptic area neurons to TMN. In β3N265M mice, GABA potency remained unchanged in TMN neurons, but it was decreased in cultured posterior hypothalamic neurons with impaired modulation of GABA_AR by propofol. Spontaneous and evoked GABAergic synaptic currents (IPSC) showed β1-type pharmacology, with the same effects achieved by 3 μM propofol and 10 μM PI24513. Propofol and the FDD PI24513 suppressed neuronal firing in the majority of neurons at 5 and 100 μM, and in all cells at 10 and 250 μM, respectively. FDD given systemically in mice induced sedation but not anaesthesia. Propofol-induced currents were abolished (1–6 μM) or significantly reduced (12 μM) in β3N265M mice, whereas gating and modulation of GABA_AR by PI24513 as well as modulation by propofol were unchanged. In conclusion, β1-containing (FDD-sensitive) GABA_AR represent the major receptor pool in TMN neurons responding to GABA, while β3-containing (FDD-insensitive) receptors are gated by low micromolar doses of propofol. Thus, sleep and anaesthesia depend on different GABA_AR types.     

O-linked β-N-acetylglucosaminidase inhibitor attenuates β-amyloid plaque and rescues memory impairment

Deposition of β-amyloid (Aβ) as senile plaques and disrupted glucose metabolism are two main characteristics of Alzheimer's disease (AD). It is unknown, however, how these two processes are related in AD. Here we examined the relationship between O-GlcNAcylation, which is a glucose level-dependent post-translational modification that adds O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins, and Aβ production in a mouse model of AD carrying 5XFAD genes. We found that 1,2-dideoxy-2′-propyl-α-d-glucopyranoso-[2,1-d]-Δ2′-thiazoline (NButGT), a specific inhibitor of O-GlcNAcase, reduces Aβ production by lowering γ-secretase activity both in vitro and in vivo. We also found that O-GlcNAcylation takes place at the S708 residue of nicastrin, which is a component of γ-secretase. Moreover, NButGT attenuated the accumulation of Aβ, neuroinflammation, and memory impairment in the 5XFAD mice. This is the first study to show the relationship between Aβ generation and O-GlcNAcylation in vivo. These results suggest that O-GlcNAcylation may be a suitable therapeutic target for the treatment of AD.     

Endotoxin-Induced Endothelial Fibrosis Is Dependent on Expression of Transforming Growth Factors β1 and β2

During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases.