Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. I. Histochemical distribution of lectin binding sites in normal alimentary tract as well as in benign and malignant gastric neoplasms

Labeled lectins with binding specificity to the hexose components of mucus glycoproteins (HPA, RCA I, PNA, Con A, WGA, and UEA I) were used to demonstrate structural differences in the glycoprotein composition of various cell types of the normal, benign and malignant gastrointestinal mucosa. While in the RCA I, UEA I, and WGA binding of normal mucus secreting cell types only quantitative differences were observed, the mucus in the surface epithelial cells of gastric mucosa and in the colonic goblet cells was characterized by the absence of PNA, Con A, and PNA, HPA binding sites, respectively. These lectins, however, showed a strong binding to the supranuclear, Golgi-region in the undifferentiated or activated forms of these cells. Even the staining intensity of the luminal membrane surfaces of the non mucinous parietal and chief cells was often stronger by PNA, HPA, and RCA I lectins than that of the mucus secretions in the highly differentiated mucus cells. These results indicate the existence of either heterogeneous glycoprotein components or mucus molecules with variations in the degree of glycosylation of their oligosaccharide chains in the different cells. The latter seems more likely since in benign and malignant alterations lectin binding sites appear in great density, which were found to be characteristic of the undifferentiated mucus cells or for the non mucinous cells of the normal gastric mucosa. Similarly in some gastric cancers which do not stain with the periodic acid-Schiff reaction at all, large amount of free or neuraminic acid substituted PNA binding sites can be detected.     

Vitelline coat of Unio elongatulus: III. Glycan chain analysis of the 220- and 180-kD components by means of lectins

Lectins of different binding specificity were used to analyze the oligosaccharide chains of the 220- and 180-kD proteins of the Unio elongatulus egg vitelline coat (vc). The lectins ConA and RCA₁ reacted with both glycoproteins, and four other lectins reacted with one or other vc components. The lectin from Galanthus nivalis, which recognizes terminal mannose residues of N-linked high mannose type oligosaccharide chains, bound specifically to the 180-kD protein. Binding sites for this lectin were found throughout the vc of the differentiating oocyte and the mature egg. Lectins specific for the O-linked oligosaccharide chains, such as AIA and PNA, reacted only with the 220-kD protein species. Binding sites for these lectins were found only in the crater region. The presence of fucosyl residues on the glycan chains was investigated with lectins from Lotus tetragonolobus and Aleuria aurantia. The latter was positive on both glycoproteins, whereas LTA was only positive to the 220-kD species. The binding sites of both these lectins were in the same areas as those of PNA and AIA. These results suggest that while the 180-kD protein is part of the entire vc structure, the 220-kD protein is prevalently accumulated in the crater region. Since this is where sperm recognition and interaction take place, it has been suggested the 220-kD protein acts as a ligand molecule in the sperm-egg interaction. © 1995 Wiley-Liss, Inc.     

Isolation and characterization of human and canine gastric mucosal glycoproteins and their degradation by proteases and acid hydrolases

High molecular weight glycoproteins were isolated and purified from canine antral and fundic mucosal tissue by means of non-degrading techniques. The results disclosed the advantage of urea extraction technique over the culture method in isolating the native glycoproteins. The glycoproteins were susceptible to degradation by protease, thus yielding low molecular weight glycopeptides. Chemical analysis of these glycopeptides and their parent macromolecules revealed that the oligosaccharide residues are attached to threonine, serine and proline residues of the protein chains. Similarly, high molecular weight glycoproteins isolated from human gastric gel mucin showed the same characteristics of canine gastric glycoproteins. Canine fundic glycoprotein or glycopeptide released their prosthetic carbohydrate groups under the lytic effect of fundic acid hydrolases.     

Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.     

Structures of sulfated oligosaccharides in human trachea mucin glycoproteins

The structures of high molecular weight sulfated oligosaccharide chains in mucins purified from the sputum of a patient with cystic fibrosis and blood group H determinant were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by ion exchange chromatography on DEAE-Agarose and a fraction containing multisulfated chains was further purified by lectin affinity chromatography to completely remove small amounts of sialylated chains. A major sulfated oligosaccharide fraction containing chains with an average of 160 to 200 sugar residues was isolated by gel filtration on BioGel P-10 columns and individual subfractions were characterized by methylation analysis, periodate oxidation and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GldNAc in a ratio of 1:2:2.1 and only one galactosaminitol residue for every 160-to 200 sugar residues. The average molecular weight of oligosaccharide chains in these fractions was between 27,000 and 40,000 daltons. Structural analysis showed that these high molecular weight chains contained varying amounts of the repeating unit shown in the following oligosaccharide. Only one in about every 10 repeating units contained sulfate esters.Several shorter chains which contain 2 to 3 sulfate esters were also isolated from this multisulfated oligosaccharide fraction. The structures proposed for these oligosaccharides indicate that they are lower molecular weight chains with the same general structure as those found in the high molecular weight sulfated oligosaccharides. Taken collectively, the results of these studies show that a major sulfated oligosaccharide fraction in resporatory mucin purified from the mucus of patients with cystic fibrosis contains high molecular weight branched chains that consist of a repeating oligosaccharide sequence with sulfate linked to the 6 positions of galactose and possibly GlcNAc residues in the side chains.     

Prolactin isoforms secreted by human prolactinomas.

Prolactin (hPRL) secreted by human prolactinoma cells in culture was purified by gel filtration, lectin affinity chromatography and gel electrophoresis in order to identify the different isoforms of the hormone and to test their respective immunoreactivities and bioactivities. The nonglycosylated hPRL (NG-hPRL), unbound to lectins, was the major form and was a species (NG1-hPRL), of 23,000 (M(r)) apparent molecular weight. The lectin-bound glycosylated hPRL (G-hPRL) consisted of three forms, G1-, G2- and G3-hPRL, of identical molecular weights (25,000 M(r)). Endoglycosidase treatment indicated that these three forms differed by the heterogeneity of their carbohydrate chains. These G-PRLs proved to be 68% less immunoreactive and 50% less bioactive than NG-hPRL. It is concluded from these data that, in prolactinomas, the main variant of the hormone is the nonglycosylated form of PRL.     

Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells

The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by siman virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines.The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of ³⁵SO₄ were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons.These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.     

Two types of rat gastric mucus glycoprotein subunits

Gastric mucus glycoproteins were extracted with 2% Triton X-100 from rat gastric corpus and antrum and purified by CsCl equilibrium centrifugation. Corpus mucus glycoproteins were degraded into what appeared to be two "subunits" (Mw 4.4 x 10(5) and 6 x 10(6)) by the reduction of disulfide bonds. Papain digestion of the latter produced glycopeptides with a molecular weight of approximately 4.4 x 10(5). This type of subunit had carbohydrate chains with about 9 sugars attached to every 2 amino acid residues. Papain digestion of the former type of subunit revealed no change in the elution profile on Bio-Gel A-15m. This type of subunit had carbohydrate chains with 17-19 sugars attached to every 3 amino acid residues. The subunit of antral mucus glycoproteins was essentially the same as the former type of corpus subunits in molecular weight (Mw 4.4 x 10(5)) and average oligosaccharide chain length. These results suggest that there are two distinct types of mucus glycoprotein subunits in rat stomach.     

Synthesis and secretion of colonic glycoproteins: Evidence for shedding in vivo of low molecular weight membrane components

In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [³H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M α-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen.     

LAMP-1 in CHO cells is a primary carrier of poly-N-acetyllactosamine chains and is bound preferentially by a mammalian S-type lectin

Recent studies indicate that some mammalian S-type lectins bind preferentially to oligosaccharides containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1]n or poly-N-acetyllactosamine (PL) sequence. We report here our investigation on the distribution of these sequences in glycoproteins in Chinese hamster ovary (CHO) cells and the interaction of glycoproteins containing PL chains with an immobilized S-type lectin (L14) from calf heart tissue. Our results demonstrate that PL chains are carried by a few high molecular weight glycoproteins which are bound by tomato-lectin Sepharose and one of these was precipitated by antibody to LAMP-1 (a lysosomal-associated membrane glycoprotein). More importantly, these high molecular weight glycoproteins, including LAMP-1, were bound with high affinity by L14. These results indicate that mammalian S-type lectins are highly specific in their interactions with glycoproteins and that LAMPs carry important recognition sequences for these lectins.     

Study of membrane glycoproteins of human erythrocytes using lectins

A method to study the glycoprotein composition of cell membranes, in particular of human red blood cells, has been developed. It includes the separation of membrane components by the SDS-polyacrylamide gradient slab gel electrophoresis, electroblotting of the phoretograms onto the nitrocellulose sheets and detection of glycoprotein fractions with FITC and peroxidase labeled lectins. PNA detected asialoglycoproteins with O-linked oligosaccharide chains, corresponding to all the PAS-positive bands of the phoretogram. SBA interacted more selectively and revealed only certain PAS-positive bands. Glycoproteins with N-linked carbohydrate chains were PAS-negative and can be identified only by the interaction with WGA, LCL, RCA. Group-specific agglutinins have shown that the ABO antigenic determinants are located in N-linked carbohydrate chains of membrane glycoproteins.     

Ultrastructural localization of glycoconjugates in human bronchial glands: the subcellular organization of N- and O-linked oligosaccharide chains.

We investigated the glycoconjugates of the human bronchial glands at light and electron microscopic level by means of lectin histochemistry in combination with neuraminidase digestion and beta-elimination reaction. Both direct and indirect techniques using lectin-peroxidase, lectin-gold, and glycoprotein-gold complexes were applied. The binding pattern of the six lectins (ConA, HPA, DSA, WGA, LEA, and PNA) used in the present study suggests that mucous and serous cells of human bronchial glands contain both N- and O-glycosylated proteins in the secretory granules. Asparagine-linked oligosaccharides containing Gal(beta-1,4) GlcNAc and Man residues were abundant in serous cells. The demonstration of both the terminal Neu 5Ac (alpha-2,3, or 6) Gal (beta-1,4) GlcNAc sequence in the N-linked oligosaccharides of mucous cells and the terminal disaccharide Gal (beta-1,4) GlcNAc in the N-linked oligosaccharide chains of serous cells suggests the existence of complex type sugar chains N-glycosidically linked to the peptide region of the glycoproteins. The binding pattern of the DSA and the neuraminidase-DSA sequence provides evidence for the existence of sialyltransferase activity in the forming mucous granules of mucous bronchial cells.     

Unusual lectin-binding properties of a herpes simplex virus type 1-specific glycoprotein

Lysates from herpes simplex virus type 1-infected cells were subjected to affinity chromatography on soybean and Helix pomatia lectins. One of the virus-specified glycoproteins, probably the herpes simplex virus type 1-specific gC glycoprotein, bound to the lectins and was eluted with N-acetylgalactosamine. The affinity chromatography permitted a high degree of purification of the type-specific glycoprotein with respect to both host cell components and other viral glycoproteins. The lectin affinity pattern of this glycoprotein indicates the presence of a terminal alpha-N-acetylgalactosamine in an oligosaccharide, a finding not reported previously for glycoproteins of enveloped viruses.     

Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV

Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

Phosphonoglycoprotein from Metridium senile--heterogeneity of glycoproteins containing aminoethylphosphonic acid.

1. After separation by SDS gel-chromatography, analysis of AEP-containing glycoproteins from M. senile, indicated 66% amino acids with 220 AEP res./1000 res. and 30% carbohydrate for high mol. wt (greater than 10(7) forms and 80% amino acids with 25-50 AEP res./1000 res. and 10% carbohydrate for low mol. wt (2-4 x 10(4) forms. 2. Uronic acids, sulfate, lipid, and sialic acids were absent. 3. Mild base digestion released AEP-hexosamine containing oligosaccharides and destroyed ser-thr residues in the high mol. wt components. 4. Phosphonoglycoproteins appear to be acidic connective tissue components with AEP linked to hexosamine containing oligosaccharide side chains.     

Histochemical characterization of glycoproteins present in jejunal and colonic goblet cells of pigs on different diets. A biopsy study using chemical methods and peroxidase-labelled lectins

We examined the glycoprotein composition of intestinal goblet cells in jejunal and colonic biopsies obtained from pigs on different diets. Paraffin sections were stained both chemically and with the following horseradish-peroxidase conjugated lectins: Canavalia ensiformis (Con-A), Limulus polyphemus (LPA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA1), Glycine max (SBA) and Triticum vulgaris (WGA). Using chemical staining procedures, only small quantitative differences were noted between the two organs. With respect to lectin staining, the mucus of the jejunum was characterized by the absence of Con-A binding sites, and colonic mucus consistently exhibited an absence of SBA affinity. After dietary modifications, O-acetyl sialic acid reactivity was lowered in the jejunum but was enhanced in the colon. In the jejunum, the glycoproteins became neuraminidase susceptible, whereas the colon became characterized by the absence of neutral mucins. The affinity for the tested lectins after the different diets was variable, but the most striking effects were observed after the fibreless diet (milk alone). Our data suggest the existence of marked regional variations in goblet-cell mucus and indicate significant differences between the glycoprotein components of the jejunal and colonic mucosa. Furthermore, the biosynthesis of mucins in both regions was altered by even only short-term feeding modifications.     

Specific Lectin Binding to Beta1 Integrin and Fibronectin on the Apical Membrane of Madin-Darby Canine Kidney Cells

Although lectins have previously been used to identify specific cell types in the kidney and various other tissues, the proteins labeled were not identified. We hypothesized that fluorescently labeled lectins could provide a useful tool for direct labeling of membrane-associated glycoproteins. Protein fractions from Madin-Darby canine kidney (MDCK) cells were exposed to a panel of 16 fluorescently labeled lectins to identify suitable lectin-protein pairs. Peanut agglutinin (PNA) selectively bound a 220-240 kDa O-linked glycoprotein with a slightly acidic isoelectric point, while Sambucus nigra agglutinin (SNA) labeled a 130 kDa glycoprotein with a highly acidic isoelectric point. Both proteins were readily labeled by lectins applied to the apical surface of living confluent cells. The proteins were isolated by lectin affinity columns and identified by mass spectrometry. Peptides from the PNA-binding protein shared molecular weight and amino acid composition with fibronectin. Fragments of the SNA-binding protein showed amino-acid identity with peptides from beta1 integrin. The identities of these proteins were validated by Western blotting. Binding of PNA to a 220 kDa protein was inhibited by an anti-fibronectin antibody, and binding of a 130 kDa protein by SNA was diminished by an anti-beta1 integrin antibody. We conclude that PNA and SNA can be used as specific markers for fibronectin and beta1 integrin, respectively, in MDCK cells.     

Glycoproteins and glycolipids of oxyntic cell microsomes I. Glycoproteins: Carbohydrate composition,analytical and preparative fractionation

Isotopically-labeled sugars were incorporated into glycoproteins of isolated bullfrog gastric mucosa. The majority of the label was found in gastric microsomal fractions which were shown to contain membranes derived from the oxyntic cell tubular membrane system and were not significantly contaminated with mucus. The tubular membranes contained exceptionally large quantities of carbohydrate (approx. 260 μg/mg protein). Most of the sugar (73%) was associated with protein in the following molar ratios: hexose, 1.0: fucose, 0.42; hexosamine, 0.62; sialic acid, <0.02. The remaining sugar, predominantly hexose, could be extracted into lipid solvents and was presumably glycolipid.Gastric microsomes were dissolves in sodium dodecyl sulfate and subjected to acrylamide gel electrophoresis and Sephadex G-200 fractionation. The latter preparative procedure yielded several molecular weight classes, each of which contained different sets of proteins and/or glycoproteins; however, the molar ratios of the sugars found in the two carbohydrate containing classes were quite similar.Significant quantities of carbohydrate were also found in gastric microsomal fractions from other species, e.g. pig and rabbit. Furthermore, characteristic proteins and glycoproteins were not present in tadpole gastric microsomes until the later stages of metamorphosis when HCl secretory capability had been established. The above findings suggest that glycoproteins may play an important role in oxyntic cell functions; the possibility of a membrane protective role is discussed.     

Adhesion of Lactobacillus fermentum 104-S to Porcine Stomach Mucus

The adhesion to whole and fractionated porcine gastric mucus of both Lactobacillus fermentum 104-S cells and a saccharide extracted from this strain was investigated. It has been shown previously that this saccharide had affinity for nonsecreting gastric epithelium. The mucus component(s) with affinity the bacterial cells was partly characterized by gel filtration and treatment with protease or metaperiodate. L. fermentum 104-S extracts containing the saccharide were radioactively labeled, fractionated by gel filtration, and tested for affinity for the gastric mucus component showing receptor activity for the whole cells of strain 104-S. The mucus material with affinity for the bacterial cells had a relative molecular weight of 30–70 K. From the results of treatment with protease or metaperiodate, it is proposed that the mucus components(s) that adhered to the whole bacterial cells contained glycoprotein groups. The radioactively labeled saccharide extracted from L. fermentum 104-S cells did not bind to the mucus fraction that had affinity for the whole cells. Conclusively, we suggest that the mechanism by which cells of L. fermentum 104-S adhere to the gastric mucus is different from the mechanism mediating the adhesion of this strain to the nonsecreting gastric epithelium. Cells of L. fermentum 104-S adhere to a glycoproteinaceous mucus component with a relative molecular weight of 30–70 K. Received: 29 August 1995 / Accepted: 26 December 1995