Abstract Changes in the expression of peanut lectin (PNA) were examined in keratinocytes of oral keratosis showing a mixture of hyperortho- and hyperparakeratinized epithelium. In the hyperorthokeratinized epithelium, which was reacted with anti-filaggrin antibody in both granular and cornified cells, PNA bound to the surface of keratinocytes from the spinous layer to the granular layer. Neither anti-filaggrin nor PNA reactions were detected in keratinocytes of the hyperparakeratinized epithelium. After neuraminidase pretreatment, however, PNA staining appeared in all cells, except cornified cells, of both hyperortho- and hyperparakeratinized epithelia. These findings suggest that PNA-binding epitopes in keratinocytes were modified by sialic acid during the hyperparakeratotic process of oral keratosis.

Although musculoskeletal pain is one of the most common causes of chronic pain and physical disability in both developing and developed countries, relatively little is known about the nerve fibers and mechanisms that drive skeletal pain. Small diameter sensory nerve fibers, most of which are C-fiber nociceptors, can be separated into two broad populations: the peptide-rich and peptide-poor nerve fibers. Peptide-rich nerve fibers express substance P (SP) and calcitonin gene related peptide (CGRP). In contrast, the peptide-poor nerve fibers bind to isolectin B4 (IB(4)) and express the purinergic receptor P(2)X(3) and Mas-related G protein-coupled receptor member d (Mrgprd). In the present report, we used mice in which the Mrgprd(+) nerve fibers express genetically encoded axonal tracers to determine the peptide-rich and peptide-poor sensory nerve fibers that innervate the glabrous skin of the hindpaw as compared to the bone marrow, mineralized bone and periosteum of the femur. Whereas the skin is richly innervated by CGRP(+), SP(+), P(2)X(3)(+) and Mrgprd(+) sensory nerve fibers, the bone marrow, mineralized bone and periosteum receive a significant innervation by SP(+) and CGRP(+), but not Mrgprd(+)and P(2)X(3)(+) nerve fibers. This lack of redundancy in the populations of C-fibers that innervate the bone may present a unique therapeutic opportunity for targeting skeletal pain as the peptide-rich and peptide-poor sensory nerve fibers generally express a different repertoire of receptors and channels to detect noxious stimuli. Thus, therapies that target the specific types of C-nerve fibers that innervate the bone may be uniquely effective in attenuating skeletal pain as compared to skin pain.

After traumatic spinal cord injury (SCI), disruption and plasticity of the microvasculature within injured spinal tissue contribute to the pathological cascades associated with the evolution of both primary and secondary injury. Conversely, preserved vascular function most likely results in tissue sparing and subsequent functional recovery. It has been difficult to identify subclasses of damaged or regenerating blood vessels at the cellular level. Here, adult mice received a single intravenous injection of the Griffonia simplicifolia isolectin B4 (IB4) at 1-28 days following a moderate thoracic (T9) contusion. Vascular binding of IB4 was maximally observed 7 days following injury, a time associated with multiple pathologic aspects of the intrinsic adaptive angiogenesis, with numbers of IB4 vascular profiles decreasing by 21 days postinjury. Quantitative assessment of IB4 binding shows that it occurs within the evolving lesion epicenter, with affected vessels expressing a temporally specific dysfunctional tight junctional phenotype as assessed by occludin, claudin-5, and ZO-1 immunoreactivities. Taken together, these results demonstrate that intravascular lectin delivery following SCI is a useful approach not only for observing the functional status of neovascular formation but also for definitively identifying specific subpopulations of reactive spinal microvascular elements. J. Comp. Neurol. 507:1031-1052, 2008.(c) 2007 Wiley-Liss, Inc.

Expression of sugar residues and the nature of oligosaccharide linkage during keratinocyte maturation in the epidermis of the Breton dog were studied with the use of lectin histochemistry. Thirteen lectins were used. Labelling was not observed with GSA I-B4, GSA II, UEA-I, and LTA. The cytoplasm of keratinocytes reacted with PNA, HPA, Con A, and WGA from the basal layer to the granular layer. PNA and Con A showed highest reactivity in the granular cell layer. The cell surface showed increased reactivity with PNA, HPA, and WGA with maturation of keratinocytes. KOH-neuraminidase treatment (KOH-Neu) increased PNA and RCA120 staining during keratinocyte differentiation thus indicating an increase in oligosaccharides terminating with sialic acid-Galbeta(1,3)GalNAc and sialic acid-Galbeta(1,4)GlcNAc, respectively. Labelling of the glycocalyx of basal and spinous keratinocytes with SNA and MAA revealed terminal Neu5acalpha(2,6)Gal/GalNAc and Neu5acalpha(2,3)Galbeta(1,4)GlcNAc. KOH-Neu-DBA showed oligosaccharides terminating with sialic acid-GalNAcalpha(1,3)GalNAc in the spinous and granular layers. A selective glycocalyx labelling of granular keratinocytes was observed with DBA and SBA. Reactions with MAA, PNA, DBA, RCA120, SBA, HPA, and WGA disappeared after the beta-elimination reaction. Our findings indicate that Breton dog epidermis contains more O-linked than N-linked oligosaccharides and confirm that different subpopulations of keratinocytes can be distinguished by lectin histochemistry.

BACKGROUND: In order to extend previous observations on the heterogeneity in labeling of rat vascular endothelium by the lectins GS I and LEA [1,2], we have conducted a survey of several organs with a lectin panel with greater variety of carbohydrate specificity.MATERIAL AND METHODS: Submandibular gland, duodenum, distal colon, liver, kidney, heart, skeletal muscle, adrenal gland, ovary, cerebral and cerebellar cortex of a rat have been examined by light and electron microscopy using lectin-gold probes on Lowicryl K4M embedded tissue.RESULTS: Five lectins (LCA, con A, LEA, RCA, WGA) labeled vascular endothelial cells in the connective tissue of all the organs studied, while PNA, EEA, TPA, LABA, UEA I expressed no detectable affinity towards endothelial cells. HPA and GS I reacted with most endothelial cells, except those in the kidney glomeruli, liver sinusoids and zona fasciculata of adrenal gland. In cerebral and cerebellar cortex GS I reacted witn pericytes of large vessels, but not with endothelial cells of the capillary bed. SDS-PAGE extracts of heart, skeletal muscle and cerebral cortex reveal that differences in GS I labeling depend, at least in part, on 40 and 200 kD glycoproteins, present in heart and skeletal muscle, but not cerebral cortex.CONCLUSIONS: Data indicate, that GS I, widely used for selective histochemical labeling of rat endothelial cells is not uniform endothelial marker in all organs. More precise investigation of these lectin reactive determinants in rat vascular endothelium, including developmental changes, isolation and enzyme-FACE sequencing of their carbohydrate moieties, generation of antibodies and their possible organ specific binding affinities could give new insight into the physiological role of GS I and HPA binding glycoproteins in rat endothelium.

Quantification of changes in gastrointestinal morphology and mucus gel has been difficult to study. In the present study, we investigated changes in rat intestine under total parenteral nutrition (TPN) using fluoresceinated lectin staining and image analysis. Wistar rats (n = 34) were divided into two groups: one group received TPN for 2 weeks, and a control group received standard rat chow and water ad libitum for the same period. A 1-cm segment of distal ileum was removed and cut into cross sections. Sections were stained with hematoxylin and eosin, and to stain the mucus, periodic acid-Schiff (PAS), alcian blue (AB), and fluoresceinated lectin, that is, FITC-labeled Ulex europaeus agglutinin I (FITC-UEA-I), were used. Light microscope images were stored in a personal computer and analyzed using image analysis. We measured perimeter length, mucosal thickness, villus area, villus surface area index, mucus stain-positive area, mucosal area ratio, and mucosal surface area ratio. Perimeter length, mucosal thickness, villus area, and villus surface area index in the TPN group were significantly less than those in the control group (P < 0.001 for each parameter). In all mucus stainings, the stain-positive area in the TPN group was significantly less than that in the control group. However, there were no significant differences in mucosal area or mucosal surface area ratios between the two groups. The FITC-UEA-I-positive area was significantly greater than the PAS- or and AB-positive area. There were significant positive correlations between the FITC-UEA-I-positive area and both the PAS-positive and AB-positive areas. TPN for 2 weeks promoted intestinal atrophy and decreased absolute quantity of mucus gel. We successfully introduced the FITC-UEA-I staining method to evaluate changes in mucus gel.

The activity of seven different types of biotinylated lectin were examined in normal and tumorous lacrimal gland tissue. In the normal lacrimal gland tissue, the glandular cells and the tubular epithelium were labeled by maclura pomifera agglutinin (MPA), soybean agglutinin (SBA), and bauhinia purpurea agglutinin (BPA). The myoepithelial cells were stained by griffonia simplicifolia agglutinin 1 (GS1). In the primary pleomorphic adenoma tissue, the epithelial components were labeled by MPA, ulex europaeus agglutinin, SBA, peanut agglutinin, and BPA. The mesenchymal components showed negative labeling. In contrast, the recurrent pleomorphic adenoma tissue showed a positive reaction in the mesenchymal components when GS1 and BPA were used. These results revealed differences in the glycoconjugate composition among normal and tumorous lacrimal gland tissues from patients with primary and recurrent pleomorphic adenomas.

The acidic glycoconjugates of mouse ileum Paneth cells were examined with the aid of light and electron microscopy, using cationic colloidal gold (CCG) as a probe. Specimens of mouse ilea were fixed in half-strength Karnovsky's fixative and embedded in Lowicryl K4M resin. Semithin and ultrathin sections were cut of examination with light and electron microscopy, respectively. Examination of the sections using light microscopy revealed the positive staining of CCG at pH 1.0 and pH 2.5, which was detected at the rim of secretory granules and at the supranuclear regions of the Paneth cells. At pH 4.0, in addition to staining of the secretory granule rim, weak staining was observed in the granule core. At pH 7.2, the cytoplasm other than secretory granules exhibited positive CCG staining. Examination of the sections using electron microscopy, at pH 1.0, the trans lamellae of the Golgi apparatus, the rim of the secretory granules, and lysosomes were labeled selectively by CCG. At pH 2.5, labeling was also discernible over the same structures in the cells. However, at this pH, the labeling intensity was stronger than that at pH 1.0, due to the dual labeling of sulfated and sialylated glycoconjugates in these structures. At pH 4.0, the Golgi apparatus, rims and cores of secretory granules and ribosomes were labeled. Lysosomes and nuclei were also positively stained. At pH 7.2, the rims of secretory granules were not stained. The present results indicate that the CCG method gives good resolution and contrast when applied to staining, and therefore is useful for the specific staining of glycoconjugates such as sulfated, sialylated and phosphated glycoconjugates for light and electron microscopy.

Lectins are polyvalent proteins of non-immune origin with exquisite carbohydrate binding specificity making them ideal for investigation of cell surface glycoprotein and glycolipid antigens. We examined the cell surface lectin binding phenotypes of 20 UM-SCC squamous cell carcinoma cell lines established from 17 patients with head and neck cancers using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of binding. Conconavalin A (Con A) from Canavalia ensiformis and the peanut agglutinin (PNA) from Arachis hypogaea bound all SCC cell lines tested and wheat germ agglutinin (WGA) from Triticum vulgaris bound to 12 of 13 tumor cell lines. The blood group O specific lectin UEA 1 from Ulex europeus also bound to all cell lines regardless of the donor blood type. Lectins of Dolichos biflorus (DBA) and Griffonia simplicifolia (GS I-B4 or BSA I-B4) with binding specificity for glycoproteins associated with blood group A and B respectively, had reactivity that did not directly correlate with blood group antigen expression. In contrast to the other lectins in our panel which exhibited broad reactivity with SCC antigens, the BSA-II lectin from Griffonia simplicfolia,(GS II or BSA II) which has sugar binding specificity for terminal non-reducing GlcNAc, did not bind to any of the screened cell lines. Our results demonstrate a common pattern of lectin-defined carbohydrate expression on the cell surface of squamous cell carcinomas of head and neck that appears promising in defining the malignant cellular phenotype. Lectin binding profile may be useful in differentiating benign from malignant histopathology.

Hydrolysis of glucosylceramides (GlcCer) by beta-glucocerebrosidase generates ceramides, critical components of the epidermal permeability barrier. Ceramides also are involved in the regulation of cellular proliferation and differentiation in a variety of cell types. Whereas most studies have focused on ceramides and their sphingoid base metabolites as growth inhibitors, GlcCer apparently acts oppositely (i.e., as a mitogen). To determine whether enhancement of GlcCer content stimulates epidermal mitogenesis, we examined the response of hairless mouse epidermis to alterations in endogenous and/or exogenous GlcCer. Topical applications of conduritol B epoxide, a specific irreversible inhibitor of beta-glucocerebrosidase, increased epidermal GlcCer levels twofold, an alteration localized largely to the basal, proliferative cell layer (fourfold increase); and stimulated epidermal proliferation (2.3-fold elevation in [3H]thymidine incorporation; P < or = 0.001), localized autoradiographically again to the basal layer, and resulting in epidermal hyperplasia. Intracutaneous administration of GlcCer (2.0 mg) also stimulated epidermal DNA synthesis, while simultaneous treatment with conduritol B epoxide plus GlcCer resulted in an additive increase in DNA synthesis. These increases in epidermal proliferation could not be attributed either to altered epidermal permeability barrier function, or to nonspecific irritant effects, as determined by four separate criteria. These results strongly suggest that GlcCer directly stimulates epidermal mitogenesis.

Regulation in epidermal differentiation can best be studied if molecular mechanism can be associated with structural and functional changes. Such recognized associations include the cessation of mitosis through inhibition of DNA replication by a G1-inhibitor present in the suprabasal cells, the biosynthesis of a tonofilament-protein as an early event in keratinization, the biosynthesis of HRP0 (histidine-rich protein) and its polymerization to HRPI during the formation of keratohyalin, the conversion of HRPI to HRPII coincident with the loss of the nucleus from the granular cell, and the aggregation of the stratum corneum basic protein and keratin filaments to form fibers in the cornified cell. To this list can now be added changes in specificity for lectin-binding to the cell surface as the keratinocyte progresses toward the cutaneous surface. This report presents data on a) the conversion of HRPI to HRPII and b) the differential lectin-binding in the epidermis of the newborn rat. HRPI (Mol. Wgt. greater than or equal to 10(6)) and HRPII (Mol. Wgt. 6 X 10(4)) have similar unique amino acid compositions and exhibit extensive-but not complete-homology in primary structures as determined by peptide mapping after exposure to trypsin. When labeled by exposure in vivo to radioactivity histidine, about 75 of the labeled histidine from both HRPI and HRPII appeared in one peptide fraction in the map, HRPI appears to have on histidine-containing fragment which is not present in HRPII. This peptide appears to contain phosphate and to account for the organically-bound phosphate which was found in HRPI but not defected in HRPII. Changes which occur in the lectin-binding specificity of the cell during differentiation may result from either movement or chemical change in carbohydrates at the cell surface. Immunofluorescent studies have shown that an isolectin from Bandieraea simplicifolia with specificity for alpha-D-galactose binds to the surface of basal and lower spinous cells, a lectin from Ulex europaeus with specificity for alpha-L-focus labels spinous cells, and a second lectin from B. simplicifolia with specificity for N-acetyl-D-glucosamine labels cornified cells. The relationship fo these alterations in the carbohydrates of the cell surface in intracellular structural and/or functional changes in unknown.

Lectins are polyvalent proteins of non-immune origin with exquisite carbohydrate binding specificity making them ideal for investigation of cell surface glycoprotein and glycolipid antigens. We examined the cell surface lectin binding phenotypes of 20 UM-SCC squamous cell carcinoma cell lines established from 17 patients with head and neck cancers using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of binding. Conconavalin A (Con A) from Canavalia ensiformis and the peanut agglutinin (PNA) from Arachis hypogaea bound all SCC cell lines tested and wheat germ agglutinin (WGA) from Triticum vulgaris bound to 12 of 13 tumor cell lines. The blood group O specific lectin UEA 1 from Ulex europeus also bound to all cell lines regardless of the donor blood type. Lectins of Dolichos biflorus (DBA) and Griffonia simplicifolia (GS I-B4 or BSA I-B4) with binding specificity for glycoproteins associated with blood group A and B respectively, had reactivity that did not directly correlate with blood group antigen expression. In contrast to the other lectins in our panel which exhibited broad reactivity with SCC antigens, the BSA-II lectin from Griffonia simplicfolia,(GS II or BSA II) which has sugar binding specificity for terminal non-reducing GlcNAc, did not bind to any of the screened cell lines. Our results demonstrate a common pattern of lectin-defined carbohydrate expression on the cell surface of squamous cell carcinomas of head and neck that appears promising in defining the malignant cellular phenotype. Lectin binding profile may be useful in differentiating benign from malignant histopathology.

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10(-5) M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of cells with a microfilament-disrupting drug, cytochalasin B (10 micrograms/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5--15 min. A morphological abnormality, microtubule paracrystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.

The objective of this work was to investigate the existence of new polymorphs and pseudopolymorphs of atorvastatin and the transformation of crystal forms. Three crystal forms of atorvastatin have been isolated by recrystallization and characterized by powder X-ray diffractometry (PXRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TG). The PXRD and DSC patterns of the three crystal forms were different respectively and it was confirmed that Form 3 is a new crystal form. After storage of 2 years at room temperature, this new crystal form was not transformed and it was shown to have a good physical stability.

Some sugar binding active peptides from the lectins of Bauhinia purpurea, lentil and Ulex europaeus have been synthesized by the solid synthesis method. The sugar binding activity of these peptides with neoglycoproteins and di/trisaccharides was observed by capillary electrophoresis, showing relative specificity.

Lectins have been detected in the nuclear matrix of nerve tissue cells, and an extraction procedure for the protein fraction with lectin activity has been developed. The lectins are characterized by hemagglutinating activity that is inhibited by D-GlcNAc, D-Gal, Lac, and D-Glc. The existence of lectins with similar molecular masses (from 7 to 20 kD) in the nuclear matrix of calf and rat brain has been shown.

Using experimental transcription model in vitro, and method of electron-topological calculations the elements of pharmacological and structural community have been found in the groups of physiologically active compounds (PAC), blocking and activating external cellular receptors. This finding confirms the necessity of subdivision special groups of bio-substrates blockators and activators for hierarchical classification, of PAC.

Several viruses have been shown to code for proteins that specifically bind to double-stranded RNA or RNA with large amounts of secondary structure. These proteins have been implicated in providing interferon resistance to viruses and in inhibiting induction of apoptosis by viruses, and have been suggested to be involved in regulation of viral and cellular protein synthesis in infected cells. This article describes methods for detecting and analyzing viral double-stranded RNA-binding proteins.

The correlation between the characteristics of K(+)-transport across the mitochondrial and bilayer lipid membranes formed form mitochondrial lipids has been demonstrated. It has been shown that different modes of K(+)-efflux activation in mitochondria result in the appearance of K(+)-transporting phospholipid forms. The experiments described in this work suggest that current fluctuations similar to those observed for biological channels can be registered in unmodified bilayer lipid membranes containing no protein components.

Physico-chemical and microbiological properties of three different forms (crystalline and two amorphous ones) of the 1-acetoxyethyl ester of cefuroxime and bioavailability after oral administration to rats have been investigated. Relation between physico-chemical properties of these forms and their bioavailability is observed. It is shown, that for oral administration the most appropriate is an amorphous form obtained by rapid evaporation of a solvent from solution of the ester.

Several analogues of the standard M13 sequencing primer that contain up to five 3'-deoxypsicothymidines, or one or two such units labeled with fluorescein at the 1'-position, have been prepared. All these oligonucleotides have been shown to prime the DNA-polymerase-catalyzed synthesis of DNA.

Studies have been made on the activity of various digestive enzymes and their distribution between the mucosal, submucosal and muscle-serosal layers of the small intestine in Wistar and Sprague-Dawley rats. Three groups of the enzymes were found, their activities being maximal either in the mucosal, or in muscular layers, or being evenly distributed between three intestinal layers.

Many members of the herpesvirus family have been shown to bind heparan sulfate on the surface of target cells. A previous report on bovine herpesvirus-1 (BHV-1) glycoprotein interactions indicated that only gC bound to heparin-agarose columns. In this report, we demonstrated that both gC and gB of wild-type BHV-1 bound to heparin-agarose columns and that gC-minus virus infectivity was inhibited by heparin.