Exquisite binding specificity of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> lectin toward TF-related O-linked mucin-type glycans

Sclerotium rolfsii lectin (SRL), a secretory protein from the soil borne phytopathogenic fungus Sclerotium rolfsii, has shown in our previous studies to bind strongly to the oncofetal Thomson-Friedenreich carbohydrate (Galβ1-3GalNAc-ser/thr, T or TF) antigen. TF antigen is widely expressed in many types of human cancers and the strong binding of SRL toward such a cancer-associated carbohydrate structure led us to characterize the carbohydrate binding specificity of SRL. Glycan array analysis, which included 285 glycans, shows exclusive binding of SRL to the O-linked mucin type but not N-linked glycans and amongst the mucin type O-glycans, lectin recognizes only mucin core 1, core 2 and weakly core 8 but not to other mucin core structures. It binds with high specificity to “α-anomers” but not the “β-anomers” of the TF structure. The axial C4-OH group of GalNAc and C2-OH group of Gal is both essential for SRL interaction with TF disaccharide, and substitution on C3 of galactose by sulfate or sialic acid or N-acetylglucosamine, significantly enhances the avidity of the lectin. SRL differs in its binding to TF structures compared to other known TF-binding lectins such as the Arachis hypogea (peanut) agglutinin, Agaricus bisporus (mushroom) lectin, Jackfruit, Artocarpus integrifolia (jacalin) and Amaranthus caudatus (Amaranthin) lectin. Thus, SRL has unique carbohydrate-binding specificity toward TF-related O-linked carbohydrate structures. Such a binding specificity will make this lectin a very useful tool in future structural as well as functional analysis of the cellular glycans in cancer studies.     

PACAP-38 is a chemorepellent and an agonist for the lysozyme receptor in Tetrahymena thermophila

Pituitary adenylate cyclase activating peptide (PACAP-38) is a peptide hormone which functions in many mammalian systems, including the nervous and digestive systems. Using in vivo behavioral studies, we have found that this hormone functions as a chemorepellent in Tetrahymena thermophila with an EC₅₀ of 10 nM. Cells previously adapted to PACAP-38 were found to be adapted to lysozyme, and vice versa. Furthermore, the in vivo behavioral activity of PACAP-38 was blocked by addition of the anti-lysozyme receptor antibody, 5545. Chemorepellent activity of PACAP-38 was also inhibited by the addition of neomycin sulfate (inhibition constant K _i=0.080 μmol · l⁻¹), a competitive inhibitor of lysozyme binding to its receptor. PACAP-38 is a more potent and specific agonist for the lysozyme receptor than either intact lysozyme or CB2, a 24-amino acid fragment of lysozyme. Accepted: 11 October 1999     

c-Fms Signaling Mediates Neurofibromatosis Type-1 Osteoclast Gain-In-Functions

Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1) patients. NF1 (Nf1) haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1^+/− mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21^Ras in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF). Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1^{+/ −} osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1^+/− osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1 ^+/− osteoporotic mice induced by ovariectomy (OVX) and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1^+/−-OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.     

Prostaglandin-E2 is produced by adult human epidermal melanocytes in response to UVB in a melanogenesis-independent manner

Excessive ultraviolet radiation (UVR) exposure induces erythema, mediated in part by prostaglandin-E₂ (PGE₂). While keratinocytes are a major PGE₂ source, epidermal melanocytes (EM) also express PGE₂-production machinery. It is unclear whether EM-produced PGE₂ contributes to UVR-induced skin inflammation, and whether this is correlated with melanogenesis. Epidermal melanocytes were cultured from skin phototype-1 and -4 donors, followed by assessment of PGE₂ production and melanogenesis. Epidermal melanocytes expressed cytoplasmic phospholipase-A₂, cyclooxygenase-1, cytoplasmic prostaglandin-E synthase and microsomal prostaglandin-E synthase-1, -2. Epidermal melanocytes produced PGE₂ under basal conditions, which increased further after arachidonic acid stimulation. Epidermal melanocytes expressed cyclooxygenase-2 (COX-2) mRNA and a selective COX-2 inhibitor (NS-398) reduced PGE₂ production. Ultraviolet B-induced PGE₂ production was positively correlated with skin phototype-1, despite variability between individual EM donors. By contrast, there was no correlation between PGE₂ production by EM and their melanogenic status. Thus, EM may contribute to UVR-induced erythema, with role of donor skin phototype more important than their melanogenic status.     

Development of polymorphic microsatellite loci for North American river otters (Lontra canadensis) and amplification in related Mustelids

In order to complement ecological research on native and reintroduced populations of the North American river otter (Lontra canadensis), we developed 10 polymorphic microsatellite loci that would permit us to evaluate population genetic structure and conduct fecal DNA analyses. The number of alleles per locus ranged from 3 to 14 and observed heterozygosity ranged from 0.23 to 0.68. Preliminary screening revealed that primers were polymorphic in a subset of other Mustelids.     

Population Genetics of the Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis) across Multiple Spatial Scales

Conservation genetics is a powerful tool to assess the population structure of species and provides a framework for informing management of freshwater ecosystems. As lotic habitats become fragmented, the need to assess gene flow for species of conservation management becomes a priority. The eastern hellbender (Cryptobranchus alleganiensis alleganiensis) is a large, fully aquatic paedamorphic salamander. Many populations are experiencing declines throughout their geographic range, yet the genetic ramifications of these declines are currently unknown. To this end, we examined levels of genetic variation and genetic structure at both range-wide and drainage (hierarchical) scales. We collected 1,203 individuals from 77 rivers throughout nine states from June 2007 to August 2011. Levels of genetic diversity were relatively high among all sampling locations. We detected significant genetic structure across populations (F_st values ranged from 0.001 between rivers within a single watershed to 0.218 between states). We identified two genetically differentiated groups at the range-wide scale: 1) the Ohio River drainage and 2) the Tennessee River drainage. An analysis of molecular variance (AMOVA) based on landscape-scale sampling of basins within the Tennessee River drainage revealed the majority of genetic variation (∼94–98%) occurs within rivers. Eastern hellbenders show a strong pattern of isolation by stream distance (IBSD) at the drainage level. Understanding levels of genetic variation and differentiation at multiple spatial and biological scales will enable natural resource managers to make more informed decisions and plan effective conservation strategies for cryptic, lotic species.     

Ultrastructure of complex carbohydrates of rodent and monkey ependymal glycocalyx and meninges

The surfaces of the brain offer metabolic and mechanical support to the underlying parenchyma. Mouse, rat, and monkey brains were fixed by immersion in a glutaraldehyde fixative or glutaraldehyde with cetylpyridinium chloride, followed by block staining for complex carbohydrates using alcian blue with OsO4 postfixation, or OsO4 postfixative solution containing ruthenium red, or alcian blue and then ruthenium red-OsO4 treatment. The ependyma in these species had a glycocalyx extending into the ventricular fluid as a finely filamentous network when stained with alcian blue or with alcian blue followed by ruthenium red-OsO4. Mice in the middle age range had stained material in this glycocalyx resembling the hyaluronic acid reported in the ocular vitreous body. Similar material was seen in the arachnoidal space of these mice and in the inner connective tissue matrix of the dura mater. Both the mouse and monkey had a cell-free zone, termed the inner dural matrix zone, between the thick fibrous dura and its innermost cellular layer. This zone contained filamentous and globular alcian blue-stained material. The complex carbohydrates of the mouse ependymal glycocalyx and inner dural matrix zone underwent changes developmentally. Aged rats were injected intraventricularly with latex beads, which, along with extravasated erythrocytes, were seen to adhere to the ependymal glycocalyx. A similar adhesion of erythrocytes was seen in the mouse and monkey ependymal glycocalyx and in the filamentous network of the mouse and monkey inner dural matrix zone. The ependymal glycocalyx, formed in part of complex carbohydrates, is much thicker than previously demonstrated. Some activities related to the ependymal lining of the ventricles, including the movement of cells or particles, the penetration of metabolites or serum-protein fractions (e.g., immunoglobulins), and cell-surface hydration, probably depend in part on complex carbohydrates that provide a sticky, electrically negative, hydrophilic environment. The complex carbohydrates in the inner dural matrix zone might provide mechanical buffering. Complex carbohydrates in the arachnoidal space may help to maintain a loose tissue that needs not only to be hydrated, but also to be open enough to provide cerebrospinal fluid circulation.     

Factors affecting the outcome of territorial contests between hatchery and naturally reared coho salmon parr in the laboratory

In aquarium experiments using coho salmon as a model species, prior residents dominated intruders of the same size but intruders with a 6% length advantage were equally matched against prior residents. Prior winning experience (distinct from individual recognition) also strongly influenced competitive success and overcame a prior residence effect. Coho salmon reared in a hatchery dominated size-matched fish from the same parental population reared in a stream. Hatchery-reared salmon also dominated naturally spawned salmon, even when the wild salmon were prior residents. Thus the combined effects of greater size and rearing experience of hatchery-produced salmon were sufficient to overcome a wild salmon's advantage of prior residence. Efforts to rehabilitate salmonid populations must consider such behavioural interactions if displacement of wild fish is to be prevented.     

The binding characteristics and utilization of Aleuria aurantia,Lens culinaris and few other lectins in the elucidation of fucosyltransferase activities resembling cloned FT VI and apparently unique to colon cancer cells

Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.     

Insertional inactivation of the methionine s-methyltransferase gene eliminates the s-methylmethionine cycle and increases the methylation ratio

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met and S-adenosyl-Met (Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met to S-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lower S-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%-60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.