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.     

Differential expression of β1, β3 and β4 integrins in sarcomas of the small,round, blue cell category

Integrins are a large and complex family of membrane spanning heterodimeric cell surface glycoproteins mediating cell/cell and cell/matrix interactions. Small, round, blue cell sarcomas (SRBCS) are a group of poorly differentiated tumours of various and in part uncertain histogenesis displaying similar cytomorphology. Among them are rhabdomyosarcomas (RMS), ganglioneuroblastomas [(G)NB], primitive peripheral neuroectodermal tumours (pPNET) and Ewing's sarcomas (ES). Thirty-two SRBCS were studied immunohistochemically for the distribution of 1, 3 and 4 integrins in situ. We found complex and to some extent differential patterns of 1, 3 and 4 integrin subunit expression in different types of SRBCS: all of the sarcomas studied were consistently 1⁺, 4^–, 2^–. Four of nine RMS were completely negative for all other integrin subunits studied while one RMS was 5⁺ throughout and three RMS were focally 5⁺. Three RMS expressed the 6 and v chains. In contrast to RMS, pPNET and ES, all of which were 1^–, 3^–, (G)NB were 3⁺ and frequently co-expressed 1. The eight pPNET and seven ES studied showed a similarily restricted integrin profile that was limited to the expression of 1 and 5 in nearly all cases. In summary, RMS were 1⁺, 1^–, 3^– and heterogeneously expressed 5 and 6. (G)NB were generally 1⁺, 1⁺, 3⁺, 5^–, 6^–. pPNET and ES were 1⁺, 1^–, 3^–, 5⁺, 6^–. The data illustrate a complex expression pattern of various integrins in SRBCS, a differential expression pattern of some of the integrin subunits among different types of SRBCS and almost identical integrin profiles in pPNET and ES.This paper is dedicated to Prof. Dr. Dres. h.c. Wilhelm Doerr on the occasion of his 80^th birthday     

Mechanical properties,electronic structure and bonding of α- and β-tricalcium phosphates with surface characterization

The mechanical properties and electronic structure of α- and β-tricalcium phosphate (TCP) crystals are studied by using two ab initio density functional methods, the Vienna Ab initio Simulation Package (VASP) and the orthogonalized linear combination of atomic orbitals method. Based on the VASP optimized crystal structures, the elastic constants of α- and β-TCP are obtained using an effective stress–strain computational scheme. From the calculated elastic constants, the bulk modulus, shear modulus, Young’s modulus and Poisson’s ratios are obtained. The results show that the mechanical properties of the two crystals are comparable and that α-TCP is somewhat softer than β-TCP. Comparison with experimental extrapolations of the elastic constants shows significant differences, which attest to the difficulty of obtaining single crystal samples. The calculated electronic structure results show that both crystals are large gap insulators with a direct band gap of 4.89 eV for α-TCP and 5.25 eV for β-TCP. Effective charge calculations show that, on average, β-TCP has slightly less charge transfer per Ca than α-TCP. The (0 1 0) ((0 0 1)) surface model for α-TCP (β-TCP) is studied using a supercell slab geometry and fully relaxed to obtain the optimized structures. The estimated surface formation energies are 0.777 and 0.842 J m^?2 for α-TCP and β-TCP, respectively. The electronic structures of the two surface models are compared with the bulk models. Charge density analysis shows that the surfaces of both TCP crystals are positively charged overall owing to the presence of Ca ions near the surfaces.     

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     

Association of CD98, integrin β1, integrin β3 and Fak with the progression and liver metastases of colorectal cancer

CD98-mediated β1 and β3 integrins activation can induce Fak phosphorylation which eventually promotes cell survival, proliferation, and migration. We evaluated the expression of CD98, integrin β1, integrin β3 and Fak in 45 cases of matched colorectal cancer (CRC) and liver metastases as well as 35 cases of CRC without liver metastases.     

Effects of glycinin and β-conglycinin on enterocyte apoptosis,proliferation and migration of piglets

Glycinin and β-conglycinin have been identified as major food/feed allergens. But effects of glycinin and β-conglycinin on enterocyte migration in piglets are scare. Fifteen weanling (7.06±0.18 kg) General No. 1 barrows, weaned at 28 days, were used. The piglets were randomly allotted to three (A, B and C) treatments with five replicates. The piglets in the A group (control group) were fed diets without ingredients originating from leguminous products, while the piglets in the B or C groups were fed the diets containing purified glycinin or β-conglycinin, which replaced protein in Group A by 4%. All the experimental periods were followed for 7 days. Five-micrometre thick sections of small intestinal tissue were stained with the TUNEL method to assess apoptotic activity, and with Ki-67 immunohistochemistry to assess cellular proliferation. The results indicated that glycinin or β-conglycinin increased proliferative index and apoptotic index in duodenum for piglets (P<0.05).     

Daily variations of epinephrine, norepinephrine, and β-adrenoceptors in the blood and lymphoid organs of intact rats

Daily variations of catecholamine concentrations in the blood and lymphoid organs in Wistar rats were revealed. Daily fluctuations of epinephrine and norepinephrine levels in the spleen and blood were synchronous. Circadian variations of epinephrine in the thymus, lymph nodes, and plasma were synphasic. A relationship between neurotransmitter concentrations and expression of β-adrenoceptors on thymic and splenic lymphocytes was noted. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 344–346, September, 1999     

Expression of E-cadherin, α-catenin, and β-catenin in tubulolobular carcinoma of the breast

Tubulolobular carcinoma (TLC) of the breast is a rare subtype of breast carcinoma categorized by Fisher et al. (Hum Pathol 8:679–683, 1977) as a tubular variant of lobular carcinoma. E-cadherin is a transmembrane glycoprotein, and complete loss of E-cadherin expression has been observed in invasive lobular carcinoma. Ductal carcinoma retains at least some expression of E-cadherin. Moreover, the adhesive function of E-cadherin is dependent on the integrity of the catenin components, which link E-cadherin to the actin filaments. In order to achieve improved categorization of TLC, we decided to investigate both E-cadherin and the catenins in TLCs and invasive lobular carcinomas. We reviewed all 1,430 cases of primary breast carcinoma that were surgically resected at Saitama Medical Center, Saitama Medical School, and at Saitama Red Cross Hospital between 1990 and 2005. Among these, 16 cases of TLC were reported retrospectively. The results were compared with those of 20 cases of invasive lobular carcinomas that were included as controls. Tumor tissue was immunostained for E-cadherin, α-catenin, and β-catenin. The presence of immunoreactivity in the TLC was seen in 12 (75%) cases for E-cadherin, in 8 (50%) cases for α-catenin, and in 10 (62.5%) cases for β-catenin. However, plasma-membrane-associated staining for E-cadherin, α-catenin, and β-catenin was completely absent in invasive lobular carcinomas. These results suggest the possibility that TLCs are not a variant of lobular carcinoma, but rather ductal carcinomas with a lobular growth pattern.     

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.     

Characteristics of Radioimmunoassays for the α- and β-Subunits of Human Luteinizing Hormone

Abstract

The binding characteristics and specificities of the National Hormone and Pituitary Program (NHPP) kits for the radioimmunoassay of the alpha- and beta-subunits of human luteinizing hormone (hLH-α and hLH-β) were studied, as well as the specificities of the anti-hLH and anti-human follicle stimulating hormone (anti-hFSH) antisera distributed by the same organization. The affinity constants of the anti-hLH-α and anti-hLH-β antisera were calculated at 157 ± 8.4 nM^?1 and 109 ± 7.4 nM^?1, respectively. Both antisera were highly specific with regard to the other subunit. However, in the homologous hLH-α RIA, native hLH cross-reacted at 21.9%, hFSH at 17.5% and hTSH at 7.9%. The alpha-subunit of the human chorionic gonadotropin, hCG-α, was equipotent with the hLH-α standard in this assay. In the homologous hLH-β RIA, hLH showed a cross-reactivity of 14.7% while the cross-reactivities of hCG-β, hFSH and hTSH were 3.5%, 1.2% and 0.6%, respectively. The anti-hFSH antiserum was highly specific, while the anti-hLH antiserum showed non parallel competition curves. With this knowledge of the specificity of each antiserum, corrections can be properly made for the assays of hLH, hLH-α and hLH-β while the hFSH RIA can be used without correction for the presence of the three other components.     

Functionalization of acrylic hydrogels with α-, β- or γ-cyclodextrin modulates protein adsorption and antifungal delivery

Poly(hydroxyethyl methacrylate) (pHEMA) hydrogels were functionalized with pendant α-, β- and γ-cyclodextrins (CD) with the aim of improving the biocompatibility and increasing the ability to host drug molecules. Pendant α-, β- and γ-CDs did not affect swelling of the hydrogels but slightly decreased the water contact angle. Protein deposition was notably dependent on the nature of the CD, due to their different affinities for hydrophobic moieties of proteins. Lysozyme and albumin sorption was hindered by γ-CD. Functionalization with β-CD also reduced protein sorption, although less so, while α-CD decreased lysozyme deposition but enhanced albumin sorption compared with control pHEMA hydrogels. Loading of the hydrogels with miconazole was carried out by immersion in drug suspension followed by autoclaving. Functionalization with γ-CD doubled the affinity of the network for the drug and resulted in the highest amount loaded (up to 170 mg g^?1). Sustained delivery was observed for several days. Some miconazole-loaded hydrogels completely prevented Candida albicans biofilm formation as assayed in an in vitro microbiological test.     

Adjustment of metabolism,catecholamines and β-adrenoceptors to 90 min of cycle ergometry

Adrenaline infusion of 0.1 g · kg^–1 · min^–1 in healthy volunteers results in an increase of hepatic glucose production, an increase of the absolute number of occupied -adrenoceptors and specific changes in metabolism. To compare these effects with the changes induced by an endogenous catecholamine release, we investigated healthy volunteers during cycle ergometry. After fasting at least 14 h seven healthy subjects exercised for 90 min at an intensity of 20% below their individual anaerobic threshold. The rate of glucose production as well as the turnover rates of alanine and leucine were calculated using stable isotope tracers. High and low affinity -adrenergic binding sites on lymphocytes were determined by an equilibrium binding assay with (–)¹²⁵ Iodocyanopindolol. After 90 min of cycling the rate of appearance of glucose increased significantly from means of 2.0 (SD 0.2) to 2.65 (SD 0.50) mg · kg^–1 · min^–1 with unchanged blood concentrations of glucose and lactate. The flux of the amino acids alanine and leucine decreased significantly from means of 0.91 (SD 0.21) to 0.62 (SD 0.14) mg · kg^–1 · min^–1 and from 0.40 (SD 0.05) to 0.32(SD 0.04) mg · kg^–1 · min^–1, respectively. The mean free fatty acid concentration increased significantly from 0.65 (SD 0.33) to 1.27 (SD 0.45) mmol · l^–1 during the endurance trial. The increase of glucose turnover and the decrease of amino acid flux point to a metabolic shift towards enhanced utilization of free fatty acids. Adrenaline and noradrenaline concentrations showed a moderate but significant increase from means of 0.61 (SD 0.20) to 0.99 (SD 0.36) nmol · l^–1 and from 2.27 (SD 0.75) to 3.46 (SD 0.38) nmol · 1^–1, respectively. The number of high affinity -adrenergic binding sites per cell (-adrenoceptors) nearly doubled from 770 (SD 130) to 1490 (SD 150) during 90 min of cycling. The observed endogenous plasma catecholamine concentrations were not sufficient to change significantly the relative receptor occupancy. This would seem to indicate that the aerobic exercise induced effects depended more on the absolute number of occupied -adrenoceptors than on their relative receptor occupancy. When compared to the results of the adrenaline infusion experiment the increases of the hepatic glucose production and the increase of -adrenoceptors were very similar in both groups despite ten times higher adrenaline plasma concentrations in the infusion group. This would seem to indicate that -adrenoceptors mediated effects do not correlate with catecholamine plasma concentrations.     

Biodistribution of synthetic thymosin β4 in the serum,urine, and major organs of mice

Thymosin β₄ (Tβ₄) is a peptide of 43 amino acids that was first isolated from the thymus gland and subsequently found to be ubiquitous in nature. Tβ₄ functions mainly as an actin-sequestering molecule in non-muscle cells, where its primary role is to maintain the large pool of unpolymerized G-actin in the cell. Studies on the pharmacokinetics of Tβ₄ in human and other mammals have not been reported so far. In the present study, we have measured Tβ₄ concentrations in serum, urine, and 10 major organs of female Swiss-Webster mice following intraperitoneal administration of 400 μg synthetic Tβ₄. Using a modified enzymatic immu-noassay, our data show a significant increase of Tβ₄ in serum starting 2 min after injection and lasting for 40 min (average: 2.34±0.54μg/ml). High concentrations were found in urine (59.3 ± 7.54μg/ml) at three different time points after injection (20 min, 40 min, and 2 h). Of the 400 μg Tβ₄ administered to mice 83 % was recovered at the end of the study, 44.6% of which corresponded to urine, 1.4% to serum, and 37.5% to the organs. In 50% of the tested organs, the wet weight concentrations of Tβ₄ increased significantly from the first 40 min to 2 h after injection in comparison to their baseline wet weight concentrations. These organs were: the brain (72 μg/g vs 42 μg/g), heart (80 μg/g vs 37 μg/g), liver (15 μg/g vs 9 μg/g), kidneys (65 μg/g vs 28 μg/g), and peritoneal fat (47 μg/g vs 13 μg/g). Wet weight concentrations increased in the thymus (196 μg/g vs 147 μg/g) and muscle (45 μg/g vs 0 μg/g) after 6 h of injection. The spleen showed an increase in wet weight concentrations at the 2 min timepoint (267 μg/g vs 161 μg/g). Ovaries had a biphasic increase at 40 min(72 μg/g vs 62 μg/g) and 24 h (92 μg/g vs 62 μg/g) after Tβ₄ administration. In lungs, the highest wet weight increase after injection (149 μg/g at timepoint 6h) was not higher than its basal wet weight concentration (153 μg/g). These phar-macokinetic studies of Tβ₄ in mice have established that high levels of Tβ₄ are found in the blood following I.P. administration and the kidney rapidly removes the peptide from the circulation. The kinetics of this response should help define the proper scheduling of administration of Tβ₄ during clinical trials in disorders, such as the acute respiratory distress syndrome (ARDS), associated with actin toxicity.     

Concentrations of α- and β-defensins in plasma of patients with inflammatory bowel disease

Background:  Impaired production/release of defensins, representative endogenous antimicrobial peptides, is associated with the pathogenesis of inflammatory bowel disease (IBD). Material and methods:  Employing in house radioimmunoassay, we examined concentrations of the major forms α-defensins, human neutrophil peptides (HNP) 1–3 and human β-defensin (HBD)-2 in plasma of 55 IBD patients consisting of 29 patients with ulcerative colitis (UC) and 26 with Crohn’s disease (CD) and 57 controls. Results:  The circulating HNP 1–3, but not HBD-2, levels in IBD patients were significantly higher than those in controls. Plasma HNP 1–3 concentrations in CD patients significantly correlated with Crohn’s disease activity index, peripheral white blood cell counts, serum CRP values and TNF-α levels. Conclusions:  Elevation of circulating α-defensins levels is suggestive of their physiopathological roles in IBD. Plasma HNP 1–3 concentrations may be an indicator for CD activity and their association with CRP and TNF-α supports a possible association with the inflammatory process. Received 2 June 2008; returned for revision 20 June 2008; received from final revision 25 June 2008; accepted by C. Kasserra 19 August 2008     

Differential expression of E-cadherin, β-catenin, and Lewis x between invasive hydatidiform moles and post-molar choriocarcinomas

Trophoblast cell adhesion and migration are carefully coordinated during normal placental development. We have compared the expression of three adhesion molecules, E-cadherin, β-catenin, and Lewis x, by immunohistochemistry during normal trophoblast differentiation, and in hydatidiform moles and choriocarcinomas. Both E-cadherin and β-catenin were expressed in normal placenta cytotrophoblast, and this expression decreased with trophoblast maturation. E-cadherin was mainly localized along the contact between cytotrophoblast and syncytiotrophoblast, which indicates its role in the differentiation of the syncytial layer. Lewis x disappeared progressively during differentiation of normal villous vessels, and was expressed in molar pregnancies. Interestingly, whereas choriocarcinomas were not, or poorly, stained, invasive hydatidiform moles (invHMs) strongly expressed Lewis x in vascular structures. This observation correlated well with E-cadherin and β-catenin expression and suggests that these three markers are associated with the invasive transformation. The presence of robust endothelial structures in invHMs could also explain their ability to maintain organized villous architecture (contrary to metastatic choriocarcinomas) during their invasion of extrauterine tissues such as the lung or the brain after dissemination through the blood flow. In our hands, Lewis x appeared to be a new, reliable marker that can be used to clearly distinguish invHMs from choriocarcinomas.     

Increased expression of dystrophin, β-dystroglycan and adhalin in denervated rat muscles

Summary To evaluate a potential regulatory role of the nerve, the distribution and expression of dystrophin, of -dystroglycan (43DAG) and adhalin (50DAG), two of the dystrophin-associated proteins and utrophin (dystrophin related protein or DRP) were studied in rat muscles after 2 weeks of denervation. We found that dystrophin, -dystroglycan and adhalin were overexpressed in denervated muscle, whereas utrophin did not increase and was found only in the post-synaptic membrane. The study of the distribution of dystrophin in the sarcolemma of single muscle fibres indicates that the molecular organization of dystrophin was maintained after denervation. Dystrophin in addition of forming a scaffold around the fibre was found around the clusters of AChR that reappeared in the extra-synaptic membrane after denervation. Also -dystroglycan colocalises at these clusters. These results suggest that the increase in dystrophin, -dystroglycan and adhalin is correlated with the reappearance of AChRs in the extra synaptic membrane.