Expression of intermediate filaments (IF) in tissues and cultured cells

Intermediate filaments are found in most nucleated cells as part of their cytoskeleton. Intermediate filaments are formed by different proteins in cells of major tissues types. Therefore, antibodies against intermediate filaments can be used in tissue typing, in the analysis of cell lineages during development and in the elucidation of the origin of unknown tumors.     

Dolichos biflorus agglutinin (DBA) reacts selectively with mast cells in human connective tissues

Dolichos biflorus agglutinin (DBA) binds to N-acetyl-D-galactosamine (GalNAc) residues in glycoconjugates and agglutinates erythrocytes carrying blood group antigen A. In cryostat sections of various tissues from blood group-specified humans, fluorochrome-coupled DBA bound preferentially to fusiform connective tissue cells and to certain epithelial cells. The connective tissue cells were identified as mast cells by their typical metachromasia in consecutive staining with toluidine blue. Double labeling with DBA and conjugated avidin revealed two distinct populations of mast cells. In several tissues the DBA-reactive cells likewise displayed uniform avidin reactivity. In intestinal mucosa, however, morphologically distinct DBA-binding mast cells were found, which were labeled with the avidin conjugates only in specially fixed paraffin sections. DBA did not bind to vascular endothelial cells, which could be identified by double staining with antibodies to factor VIII-related antigen. Labeling with Helix pomatia agglutinin (HPA), another blood group A-reactive lectin, resulted in distinct blood group-dependent fluorescence of the endothelia. Sophora japonica agglutinin (SJA), a blood group B-reactive lectin, labeled vascular endothelial cells in tissues from blood group A, AB, and B donors. HPA and SJA reacted with small mast cells in the gastrointestinal mucosa but failed to label large mast cells in any of the tissues. These results indicate that the blood group reactivity of lectins, as determined by erythroagglutination, is not necessarily consistent with their reactivity with blood group determinants in tissue sections. Moreover, DBA conjugates appear to be a reliable probe for detection of mast cells in various human connective tissues.     

Involvement of an "Antizyme'' in the Inactivation of Ornithine Decarboxylase

DL-Allylglycine causes a marked increase in mouse brain ornithine decarboxylase (ODC) activity. The amount of immunoreactive enzyme protein increases concomitantly with the activity, but the enzyme protein decreases more slowly than that of the activity. The amount of immunoreactive ODC in brain is many hundred times that of the catalytically active enzyme. The fact that mouse brain cytosol contains high amounts of dissociable antizyme (an inactivating protein) indicates the existence of an inactive, immunoreactive ODC-antizyme pool. The total antizyme content does not change markedly, but instead there are significant changes in different antizyme pools. Putrescine concentrations start to increase 8 h after treatment with allylglycine and concomitantly with this increase, antizyme is released to inhibit enzyme activity. These results indicate the involvement of antizyme in the inactivation process of ODC.     

Psophocarpus tetragonolobus agglutinin reveals N-acetyl galactosaminyl residues confined to endothelial cells and some epithelial cells in human tissues

We studied the binding of Psophocarpus tetragonolobus agglutinin (PTA) conjugates to human adult tissues. In all kidney specimens studied, PTA bound in a blood group-independent way to endothelia in glomerular and intertubular capillaries as well as in larger vessels. In addition, a heterogeneous binding to collecting duct cells was seen. In specimens of human smooth, cardiac, and skeletal muscle, cerebellum, lung, thyroid gland, liver, proliferative endometrium, and placenta, PTA bound only to endothelial of capillaries and larger vessels. In epidermis and gingiva, PTA conjugates additionally revealed reactivity with keratinocytes. Similarly, in salivary gland, urinary bladder, gastrointestinal tract, mammary gland, and renal pelvis, PTA reacted with some epithelial cell layers. The PTA conjugates gave an even cell surface membrane staining of cultured umbilical vein endothelial cells. Lectin-affinity binding of radioactively surface-labeled endothelial cells showed that PTA and Ulex europaeus I agglutinin (UEA-I) recognized related major cell surface glycoproteins. The results with PTA conjugates show that certain N-acetyl galactosaminyl residues are, in addition to some epithelial cells, confined to endothelial cells in human tissues.     

Cholinergic signaling in the rat pineal gland

Summary 1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotoninN-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion.2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland.3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs).4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.     

Amniotic fluid zinc in risk pregnancies

The zinc concentration of amniotic fluid (AF) of 129 pregnant women was analyzed by the flame atomic absorption spectrometry. This prospective study was performed in order to find out whether the determination of the AF zinc concentration can be used to monitor the growth and development of the fetus. There were two groups of patients: early stage (15th–19th gestation wk) in which the amniocentesis was performed as a prenatal genetic examination, and late stage (26th–40th wk) in which the amniocentesis was performed due to obstetric reasons. The average AF zinc concentrations were 1.2 and 1.0 μmol/L in the early and late gestation group, respectively. The AF zinc concentration did not correlate with the weight, height, or Apgar scores of the newborn nor with the maternal diseases, age, or parity. The AF zinc concentration in the late gestation group was significantly lower if the fetus was male than if it was female. If the AF was greenish the zinc concentration was elevated. One malformation, congenital nephrosis, with an exceptionally high zinc concentration (9.0 μmol/L), was found.     

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

Background  

Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).     

Exhaled pentane as a possible marker for survival and lipid peroxidation during radiotherapy for lung cancer—a pilot study

To examine lipid peroxidation during radiotherapy (RT), exhaled pentane samples were collected from 11 lung cancer patients before RT and 30 and 120 min after the start of RT on days 1, 4 and 5 and at 30 and 40 Grays, if possible. Exhaled pentane samples were collected once from 30 healthy controls. Serum thiobarbituric-acid-reactive substances (TBARS) and conjugated dienes (CD) were obtained from patients on each exhaled air collection day. Lung cancer patients had higher exhaled pentane levels than controls (1.73 ng/L vs 0.83 ng/L, p=0.017). Exhaled pentane levels tended to decrease during the first RT day (p=0.075) and levels of CD decreased during the first week of RT (p=0.014). Higher pre-treatment pentane levels predicted better survival (p=0.003). Elevated exhaled pentane levels before RT may be due to the lipid peroxidation burden associated with cancer. The decrease of lipid peroxidation markers during RT may be attributable to enhanced antioxidant defense mechanisms.     

The effect of selenium on the hepatic drug metabolism and inducibility in rat

1. Rats were fed either a normal or selenium-deficient diet for 4 weeks. The subgroup on selenium deficient diet had selenium supplementation as 3 ppm Se in the drinking water. Benzo(a)pyrene was given intraperitoneally as an inducer. 2. Se deficiency decreased glutathione peroxidase and cytochrome c-reductase activities while other activities were unchanged as compared to normal diet. 3. Selenium deficiency was a prerequisite for the induction of glutathione peroxidase, S-reductase and S-transferase enzymes. 4. Benzo(a)pyrene increased hepatic microsomal cytochrome P-450 content in rats on normal and selenium supplemented diet but not in the selenium deficient group. 5. The 7-ethoxyresorufin and 7-ethoxycoumarin deethylase, aryl hydrocarbon hydroxylase and cytochrome c-reductase activities were increased by benzo(a)pyrene in all the dietary groups. 6. The UDP-glucuronosyltransferase activity was also increased by benzo(a)pyrene in all the experimental groups and this was true with p-nitrophenol and phenolphthalein as aglycons.     

Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

Background

α/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design.

Methodology/Principal Findings

Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors.

Conclusions/Significance

We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore model of ABHD12 inhibitors. This model should pave the way for further discovery of novel lead structures for ABHD12 selective inhibitors.