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The mistaken idea that biologically important molecules belong to mutually exclusive four categories has been inhibiting glycoconjugate research for many years. We are aware now that the great majority of secrtory and membrane bound proteins are glycosylated. So are many mambrane bound lipids. Recent advances in glycobiology have demonstrated that glycosylation is not limited to surface and extracellular proteins only, but is widespread also in the nucleus and the cytoplasm of the cell. Different sizes, chemical, physical and antigenic properties of glycans determine the variety of biological functions which are now slowly being unravelled. The overwhelming body of evidence indicates that the carbohydrates attached to glycoconjugates at the surface of cells play pivotal roles in the early stages of infection by pathogenic invaders, such as bacteria and viruses. They are important for defence and inflammation, for fertilization and development, for memory consolidation and rejection of xenografts. They explained many storage deseases and are of primary importance in the progression of cancer and metastasis thus providing a gauge of malignancy. Immunologists and physicians have begun to realise that the absence of the normal saccharide portions from recombinant therapeutics and adjuvants provokes an immune response from some patients during trials. Thus sugars are laying claim to important physiological and pharmaceutical properties, and can no longer be ignored.

Nearly all proteins are glycosylated. In more than a half of all proteins carbohydrates are a significant structural component. Regulatory modification of Ser and Thr by covalent addition of a single GlcNAc is as abundant or even more abundant than phosphorylation, but this type of glycosylation is catalysed by a single enzyme, contrary to phosphorylation that involves thousands of kinases. The glycocalyx is the predominant surface structure on cells. Cell-cell interaction are mediated by lectins of one cell recognising glycan targets on an other cell surface. Glycan - lectin interactions are essential in inflammation, and in fertilization. Even pathogens start their communication with the host cell through carbohydrate - lectin recognitions. As with many other life sciences, glycobiology has been revolutionised by the development of the fast and powerful techniques, both physical and those of molecular biology. As a result, structural glycobiology is rapidly advacing. Radiant achievements of the rapidly developing and branching glycosciences are reviewed. New information on glycoproteins, glycolipids and their receptors, their structure, biosynthesis and function, as well as on their potential to support medicine and to provide new therapeutics has been briefly discussed.

Background Schizophrenia is the collective term for an exclusively clinically diagnosed, heterogeneous group of mental disorders with still obscure biological roots. Based on the assumption that valuable information about relevant genetic and environmental disease mechanisms can be obtained by association studies on patient cohorts of ≥ 1000 patients, if performed on detailed clinical datasets and quantifiable biological readouts, we generated a new schizophrenia data base, the GRAS (Göttingen Research Association for Schizophrenia) data collection. GRAS is the necessary ground to study genetic causes of the schizophrenic phenotype in a 'phenotype-based genetic association study' (PGAS). This approach is different from and complementary to the genome-wide association studies (GWAS) on schizophrenia. Methods For this purpose, 1085 patients were recruited between 2005 and 2010 by an invariable team of traveling investigators in a cross-sectional field study that comprised 23 German psychiatric hospitals. Additionally, chart records and discharge letters of all patients were collected. Results The corresponding dataset extracted and presented in form of an overview here, comprises biographic information, disease history, medication including side effects, and results of comprehensive cross-sectional psychopathological, neuropsychological, and neurological examinations. With >3000 data points per schizophrenic subject, this data base of living patients, who are also accessible for follow-up studies, provides a wide-ranging and standardized phenotype characterization of as yet unprecedented detail. Conclusions The GRAS data base will serve as prerequisite for PGAS, a novel approach to better understanding 'the schizophrenias' through exploring the contribution of genetic variation to the schizophrenic phenotypes.

Povijest otkrića dvostruke uzvojnice i značenje za razvoj humane genetike

Molekuläre Biowissenschaften decken das Lebensgesetz auf. In der lebenden Welt funktioniert jeder Organismus aufgrund des hierarhischen Organisation, jeder Prozess, jede Struktur und jede Energie haben ihren Zweck. Gewirtschaftet wird wirksam und sparsam. Stoffe werden wiederverwendet und zugelassen sind nur vorsehbar nützliche Vorräte. Die Nutzung und Lenkung von Informationen, Kommunikation und Aufteilung von Funktionen &#8211 ; ; alles koordiniert und unter Aufsicht, und Signalmoleküle alarmieren wenn etwas nicht in Ordnung ist. Molekuläre Beschützer schützen xenofobisch die Integrität des eigenen Organismus und kämpfen gegen fremde Eindringlinge. Die Zweckmässigkeit und Ordnung sind wichtige Elemente des Systems der lebenden Materie, bzw. des Lebensgesetzes, die alles der Erhaltung der harmonischen Ganzheit unterordnet. Die Gesellschaft ist nur ein Teil der Biosphäre.

E. coli is by far the most common cause of urinary tract infections. Standard methods for identification of uropathogenic E. coli (UPEC) in urine are semi-quantitative and usually take 2-3 days, so development of a more rapid, molecular approach to detect UPEC may lead to earlier diagnosis and improve management of urinary tract infections. Attachment of UPEC to host cell receptors is mediated by interaction of the bacterial lectin (adhesin) localized at the top of fimbriae and specific carbohydrate moiety of the host membrane glycoconjugates. The most common type of UPEC causing pyelonephritis expresses type P adhesin (UPEC-P) that specifically recognizes P1 antigen (Gal alpha(1-4)Gal moiety - galabiose). Pigeon glycoproteins are rich in galabiose sequences at the terminal positions of N-glycans that makes them potent inhibitors of UPEC-P adhesion. Oligosaccharides were purified from pigeon ovalbumin, reductively aminated with hydrazine and labelled with digoxin that was previously activated with CNBr. Microtitar plates were coated with purified pigeon ovalbumin and incubated with solution containing bacteria or a model lectin. Pigeon ovalbumin glycans conjugated with digoxin were added and detected with antibodies against digoxigenin (that also recognize digoxin) conjugated with alkaline phosphatase Linear response was observed in a wide range of concentrations, indicating that this model might be useful for quantifying bacteria. As far as we know this is the first case of labelling glycans with digoxin as well as their application. Our preliminary study indicates that specific glycoproteins and glycans labeled with digoxin can be used for development of a rapid, species-specific molecular tool for detection of uropathogenic E. coli. Similar approach may be further utilized for detection of other bacterial species, measurement of bacterial virulence and sensitivity of antibiotic.

Pregledno iznesene središnje teme suvremene glikobiologije predstavljene na naprednom tečaju Federacije europskih društava za biokemiju i molekularnu biologiju pod naslovom "Glycoconjugates -Intriguing Structures and Versatile Functions"

Consequences of depleted uranium interferre with those of the exposure to the stress of war. Pathological changes in soldiers experienecing the 1991 war in Croatia are discussed.

Molekularne bioznanosti otkrivaju zakone života i opstanka koji se temelje na hijerarhijskom ustroju žive tvari, kooperativnosti, komplementarnosti, uključenju i usmjeravanju svih procesa na dobrobit cjeline. Genetički determinizam predviđa slobodu ponašanja radi prilagodbe uvjetima života, obrane i opstanka pojedinca i vrste. Slobodu sve češće ugrožavaju društveni pritisci koji ne mare za biološke zakone života, a iz njih izvire temeljna etika življenja i ponašanja.

Prikaz značenja Simonićeve knjige "Znastost najveća avantura čovječanstva" u vrijeme globalizacije tržišta: raspravlja o triumfima ljudskih spoznaja, o znanju koje se ne pretvara u mudrost, koje se podjednako zloupotrebljava koliko donosi dobrobiti čovječanstvu.

In order to study the effects of stress on copper (Cu), zinc (Zn), Zn- related enzymes, and metallothionein (MT) in liver, we used the immobilization procedure as an experimental model of stress. Acute immobilization stress caused changes of Zn, Cu, and MT concentrations in rat liver. The Zn level in total liver homogenates increased for 39% (p < 0.00011). This increase originated from the Zn concentration in nuclear fraction, suggesting a higher content of Zn-dependent enzymes and large molecular weight proteins. The stress induced increase of alkaline phosphatase (ALP), which is Zn- related metalloenzyme, closely followed the changes in the Zn concentration in liver. However, the activity of another Zn-dependent enzyme, 5'-nucleotidase (5'-NT), was lower after acute stress. The total Cu concentration was about twice higher in the stressed group in comparison with the control group, but no redistribution of Cu within hepatocytes was found. MT concentration was measured in the mitochondrial-lysosomal fraction of liver homogenates. It was found that acute immobilization stress caused about a 100% increase in the MT concentration in liver.

Serum glycoproteins from individuals under chronic stress (prisoners of war from detention camps, n - 30) were analyzed by the Western-blot method applying a set of five different lectins (DSA, GNA, MAA, PNA and SNA). Glycoprotein patterns of samples from the stressed group revealed significant changes if compared to the control sera (from apparently non-stressed volunteers): both galactose-specific DSA lectin and sialic acid-specific SNA lectin recognized a novel nonconsitutional glycoprotein with an apparent size of 57 kDa in the stress group, which was completely absent in the control group. In addition, concentration of a 45 kDa SNA reactive glycoprotein increased more than 500 fold. The high incidence of disturbed patterns of glycoproteins in the sera of stressed subjects (95%) points to their important role in stress response and qualifies them for widespread testing as potential diagnostic markers of the stress syndrome.

In recent years it has become clear that carbohydrate portions of glycoconjugates are performing numerous vital physiological functions in higher organisms. However, since glycobiology is a relatively new science, and carbohydrate structures are highly complex, the continuous development of novel analytical techniques is necessary to support the process of understanding the intricate nature of glycoconjugate structure and function. The introduction of digoxin as a novel tag for labelling of lectins that are being used to analyse glycoconjugates in immunoassay-like techniques is described. Lectins labelled with digoxin have significant advantages over biotin- or digoxigenin-labelled lectins and will hopefully prove to be a useful addition to the repertoire of glycobiological tools.

Mutations in the LDL-receptor gene cause a marked elevation of plasma cholesterol, in particular of the LDL subfraction, and the clinical phenotype of familial hypercholesterolemia. Mutation analysis was performed in 127 hypercholesterolemic patients from Zagreb. The promoter region and all 18 exons of the LDL-receptor gene were screened using single-strand conformation polymorphism method. The molecular defects detected were then characterized by direct sequencing. Nucleotide changes were detected in 9.4% patients with hypercholesterolemia. About five different mutations in the low-density lipoprotein receptor gene were identified in 12 patients. Four missense mutations (C6W, C127R, D200G and S265T) were located in ligand binding domain and one (G457R) was located in the epidermal growth factor precursor homology domain. One of these mutations, C127R (FH Zagreb), has not been described before in any other population. Five genetic polymorphisms (C6C, A370T, R450R, N570N and V632V) were detected in other. Knowledge of genetic basis of hypercholesterolemia may contribute to designing a specific diagnostic analysis scheme. Therefore, for molecular diagnosis of familial hypercholesterolemia in Croatia, the whole low density lipoprotein receptor gene of each patient must be screened for new and recurrent mutations.

Glycans are the most abundant and most diverse biopolymers in nature. Because of their highly specific interactions with physiological receptors, they participate in many crucial biological processes. All these processes are potential targets for therapeutic intervention, and carbohydrate-based drugs are rapidly being taken up by the modern biotechnology and pharmaceutical industry. Recent developments in the field of glycobiology have overcome the problem of glycan analysis and synthesis ; and many compounds based on carbohydrates are now in various stages of clinical trials. This article presents glycoproteins in a new light, as an important biopharmaceutical targets, giving an overview of their potential use as therapeutic glycoproteins and proteoglycans, inflammation blockers, cancer therapeutics and vaccines, inhibitors of pathogenic microbes, viral inhibitors and potential aids in the treatment of lysosomal diseases, neurological diseases and transplantation rejection.

A new method is described which is suitable for reliably analysing apoptotic fragmentation in small amounts of DNA. After isolation, DNA was labelled with biotin-4-dUTP using Klenow polymerase. Then DNA was size-separated by agarose gel electrophoresis, blot transferred and subsequently visualized by the streptavidin alkaline phosphatase-BCIP/NBT procedure. This non-radioactive method was used to detect apoptotic DNA in rat pheochromocytoma PC12 cells, treated with tributyltin (1 nM). While only 30 ng of DNA is required for analysis of apoptotic DNA using the new blot technique, 100-fold more material is needed to identify the fragmentation of DNA after separation by agarose gel electrophoresis and direct staining with ethidium bromide. In a further set of experiments, rat cortical cells were incubated with human immunodeficiency virus type 1 viral glycoprotein of M(r) of 120 kDa (gp120) to induce apoptosis. More than 0.3 ng of gp 120/ml are required to detect apoptotic DNA by the direct procedure; only 0.1 ng gp120/ml or less were sufficient to document clear DNA fragmentation using the non-radioactive blotting technique described here. These results demonstrate that the new procedure can be used to analyse very small amounts of apoptotic DNA and shows that gp120-induced apoptosis can be measured at low concentrations of the viral protein.

Lectins are defined as carbohydrate binding proteins other than enzymes and antibodies. Until recently lectins were considered to be exclusively plant proteins and as such drew only limited attention of biochemists. As soon as they have been detected in animal cells and after their involvement in vital processes such as development, differentiation and neoplastic growth was recognized, widespread studies have focused on their specificity, structure, localization and specific biological functions. Very recently several ?-galactose specific lectins were organized into a new lectin family - galectins. Members of this family are present in nearly all animals from lover invertebrates such as sponges and nematodes to mammals, including humans. Though not completely understood, the awareness of their physiological importance is growing as their properties are unraveled, and the interest for them is ascending as multiple implications of their presence are emerging.

Bacterial resistance to macrolide-lincosamide-streptogramin B (MLS) antibiotics is based on activity of methyltransferases of Erm family. Erm methyltransferases mono- and dimethylate specific adenine residue within 23S rRNA, thus preventing antibiotic binding to the ribosome. Here we describe the optimum procedure for the expression, isolation and purification of ErmC', which is biochemically more stabile than most members of Erm family. ermC' gene was isolated and amplified from plasmid pIM13 from Bacillus subtilis BD1167 via polymerase chain reaction. Simultaneously, new restriction sites were introduced at both ends of the gene to enable its cloning to an expression vector pET-25b(+). Expression host strain BL21(DE3)pLysS was then transformed with recombinant vector pET-25b(+)ermC'. Protein expression was induced with IPTG and optimized for obtaining soluble protein at a greater rate. Optimum procedure for purification of protein from bacterial protein extract was designed after trying of several purification methods. The procedure involves two subsequent steps of cation exchange, with use of stepwise salt gradient in the first step, and a linear salt gradient in a second one. Highly purified ErmC' has been obtained in large quantities to be used in future studies of ErmC' activity, as a contribution to the intensive worldwide efforts to find potent and specific inhibitors for overcoming the MLS resistance.

Cortisol is one of the main hormonal regulators of the stress response. In saliva cortisol is traditionally analyzed by radioimmunoassays (RIA). The use of radioisotopes, hazardous solvents and expensive scintillation counters is sometimes not desirable, thus there was a need to develop equally sensitive, yet low-cost, high throughput and safe method. Here we describe an enzyme-linked immunosorbent assay (ELISA) for indirect assay of cortisol in saliva. In the assay, an immobilized anti-cortisol antibody is used to capture cortisol in the sample. Using competition with biotin-labeled cortisol (tracer) and streptavidin/alkaline-phosphatase detection system we were able to accurately measure cortisol at levels normally found in saliva (1- 30 nmol/l) with acceptable inter- and intra-assay coefficients of variation (6,96% and 2,67%). Using this method we have analyzed circadian variations of cortisol in patients diagnosed with post-traumatic stress disorder (PTSD), active professional soldiers and control individuals. Interestingly, though PTSD is considered to be a single disorder, we found that PTSD patients separate into two well characterized groups, one with significantly smaller and one with significantly increased circadian variations of cortisol.

Galectin-3 is a 32 kDa lectin that specifically binds to β-galactose on the non-reducing termini of oligosaccharide structures. It was shown to be involved in many different biological processes, including growth regulation, cell differentiation, adhesion, neoplastic transformation and tumor metastasis. Using M3/38 monoclonal antibody we analyzed effects of UV radiation on the expression of galectin-3 in A1235 glioblastoma cells. We found that exposure to UV-C light (λ=254 nm, 80 J/m2) nearly doubles the expression of galectin-3 within the 1 h after the exposure, and that galectin-3 stays on the approximately same level for the next 24 h. Based on the published structure of the promotor region of human galectin-3 gene (LGALS3) and roles of AP-1 and NF-kB in the stress response, we speculated that these two transcription factors might also have a role in the regulation of galectin-3 expression after exposure to UV-C irradiation. To test this hypothesis we have used antisense-jun oligonucleotides and zL3-vs, a specific proteasome inhibitor that prevents degradation of IkB, and blocks transcriptional activation through NF-kB. Treatment of cells with antisense-jun oligonucleotides caused slow, but statistically significant, and continuous decrease in galectin-3. After 10 h of cultivation in the presence of antisense-jun, the level of galectin-3 was about 60% of the initial value. In the same time, in cells that were treated with nonsense-jun oligonucleotides, levels of galectin-3 did not change, suggesting that Jun is involved in the regulation of the basal level of galectin-3 expression. However, even when the expression of Jun was significantly attenuated by antisense-jun oligonucleotides, UV-C irradiation still caused some increase in galectin-3 and moreover this increase started within minutes after the exposure, when it is to early to see effects of newly-synthesized Jun. To see whether NF-kB is involved in the induction of galectin-3 by UV-C light, we have used zL3-vs to block transcriptional activation through NF-kB. Treatment of cells with zL3-vs did not have any effect on the basal level of galectin-3 in the first 4 h of incubation. However, when cells pre-treated with zL3-vs were exposed to UV-C radiation, the increase of galectin-3 that was visible in the control cells, was nearly completely absent. This suggested that the early phase of induction of galectin-3 following the exposure to UV-C radiation is mediated by NF-kB.

Excessive stress is one of the major problems in a modern society. A number of epidemiological and experimental studies clearly demonstrated a link between psychological stress and the development and course of many diseases, from simple virus infections and gastric ulcers, to cardiovascular diseases and cancer. Metabolic adaptation to adverse conditions is highly important for survival in ever-changing environment, but excessive activation of the stress response appears to be associated with various detrimental effects. However, exact molecular mechanisms that link psychological stress and the development of disease are still mostly elusive. Previously we have shown specific changes in protein glycosylation and expression of lectins that occur during the stress response. Here we report that stress also affects activity of sialyltransferases (STs). Using asialofetuin as an acceptor and 14C-CMP-NeuAc as a donor of sialic acid we have measured total ST activity in liver, kidney, heart, skeletal muscle, adrenal gland, medulla spinalis, hippocampus, cerebellum and cortex of rats exposed to immobilization stress, either acutely (one 2 h episode), or chronically (multiple immobilization, swim, and foot-shock stresses for eight days). Interestingly, despite the fact that all analyzed tissues were exposed to same hormonal signals, the impact of these signals on ST activity varied significantly. In liver and spleen acute stress caused approximately 40% increase in ST activity. However, when the exposure to stressors was prolonged, their effects in liver and spleen were opposite. While ST activity in spleen nearly doubled (193ą81% of control), in liver it decreased to 82ą12% of the activity in control rats. Beside in liver and spleen, ST activity in other analyzed non-neural tissues did not appear to be significantly influenced by stress. In neural tissues there was generally a reduction of ST activity as a consequence of stress. The only exception was the medulla spinalis where we did not observe any changes. Interestingly, despite significantly changed ST activity in some tissues, in the same tissues we did not observe any changes in sialoglycoprotein patterns.

Streptomyces species and related genera synthesize a large number of secondary metabolites many of which are biologically active. Among them polyketides are the largest class. Polyketides are a structurally diverse family of natural products with a broad range of biological activities and pharmacological properties. Polyketide antibiotics, antifungals, antiparasitics, animal growth promotants, natural insecticides, cytostatics, immunosuppressants and anticholesterolemics are in com-mercial use. The evidences accumulated up till now have indicated a substantial analogy between the formation of long chain fatty acids and biosynthesis of polyketides carried out by Type I and Type II polyketide synthases (Hopwood, Chem. Rev., 97, 2465, 1997). During the last decade many polyketide gene-clusters have been cloned and sequenced. DNA sequencing showed considerable DNA homology hence they originated from the same ancestor. This allowed the use of combinatorial biology in creation of novel chemical entities. Combinatorial biology aims to create biodiversity by engineering the way the natural molecules are formed to generate "natural" substances that Nature never imagined. In principal, two approaches have been used: targeted manipulation (e.g. disruption and replacement of certain genes involved in the biosynthetic pathway ; McDaniel et al., Proc. Natl. Acad. Sci. USA, 96, 1846, 1999) and random approach (e.g. gene shuffling technology ; Del Cardayre et al., WO 00/4190). In PLIVA, the targeted approach that generated several novel scaffolds by the manipu-lation of the S. rimosus oxytetreacycline gene-cluster has been used (Petković et al., J. Biol. Chem., 274, 32829, 1999 ; Perić et al., P-990153A). In this talk the latest data from this field will be presented.

Gangliosides are predominant sialogycoconjugates in vertebrate nervous system. The most abundant structures in adult and fetal brain are GM1, GD1a, GD1b and GT1a. Their share same core structure consisting of ceramide lipid and neutral tetrasaccharide core (Gal b3 GalNAc b4 Gal b4 Glc) and veried in number and position of sialic acid. Neurobiological role for these major structures has not been defined. Recently developed knockout mice line of b1,4 GalNAc-T gene lacking all complex ganglio-series gangliosides showed decreased central myelination, axonal degeneration in central and peripheral nervous system, and demyelination in peripheral nerves. These results are in agreement with observation that mice lacking myelin-associated glycoprotein (MAG), known as in vitro receptor for GD1a and GT1b, show similar pathological features suggesting role of this structures in myelination. More elaborate study of distribution and function of major gangliosides in developing and adult brain was obstructed by lack of high-affinity IgG antibodies due to conservation of this molecules across mammalian and avian species. Knockout mice for b1,4 GalNAc-T gene are immunologically naďve to complex gangliosides and give robust respond upon immunization. We were able to develop high–affinity IgG antibodies against all four major brain gangliosides: GM1, GD1a, Gd1b and GT1a. Study of mice brain shows developmentally regulated pattern of expression different for each tested antibody giving insight in different metabolic regulation of glycosphingolipid synthesis for different brain structures.

Immunoglobulin G (IgG) is a 150 kD glycoprotein and the most abundant type among human antibodies. The molecule exhibits very complex tertiary structure with variable domains, hypervariable regions and conserved N-glycosylation whose changes are shown to be connected with several autoimmune diseases. Recent analysis of the IgG glycosylation in Juvenile Chronic Arthritis (JCA, an autoimmune rheumatic disease) and related autoimmune diseases indicated a high increase of O-linked fucosylation which is potentially significant in early diagnostics of these diseases (patent pending). Determination of the targeted fucosylation sites could have profound importance in elucidation of their molecular pathogeneses, but the variability of the molecule, hence the heterogeneity of IgG samples purified directly from human sera, hampers any straightforward analysis with standard biochemical and MS methods. The aim of this work was to challenge a method for rapid structural analysis of fucosylated peptides with ESI Q-TOF MS. Using this recently developed strategy, mapping of the possible fucosylation sites on IgG molecules isolated directly from sera of JCA patients was performed. Large portions of the IgG constant regions were found to be excluded as targets for O-fucosylation and at least one O-fucosylation site on the Igg1 chain was determined.

More than a half of all known proteins contain covalently attached oligosaccharides that represent a significant part of their molecular structure. It is estimated that over 500 specific enzymes (glycosidases and glycosyltransferases) are involved in the synthesis of carbohydrate structures on glycoconjugates, and the whole process is very complex and energetically expensive for the cell. Due to very high structural variability and the lack of adequate methods to analyze it, carbohydrate parts of glycoconjugates were ignored by most of the scientific community until only few years ago. However, thanks to the development of novel methods, in the last years there is an exponential increase in the knowledge about the physiological roles of carbohydrate structures. As well as many other diseases, rheumatoid arthritis is associated with specific changes in protein glycosylation. Majority of studies focused on galactosylation of IgG, but recently we demonstrated that the most prominent glycosylation change in both rheumatoid arthritis and juvenile rheumatoid arthritis is the increase in fucosylation. Using a novel method based on recently developed BioprobeŽ technology we were able to show that juvenile rheumatoid arthritis is also associated with changes in the activity of serum lectins. Although the functional significance of these changes are still not known, they could prove to be a valuable diagnostic marker for rheumatoid diseases.

GP62 is a member of the stress glycoprotein family that was proposed to have a chaperone-like function in the heat-shock response. Using lectin blotting we have analyzed glycosylation of GP62 in A1235 cells exposed to heat-shock (2 h at 42ºC), subculturing and UV-radiation. As expected, heat-shock resulted in the appearance of significant amounts of GP62, which was undetectable in the control cells. SNA, DSA and PHA-E lectins recognized GP62, while BSA I, Con A, RCA I, SJA, UEA-I, VVA and WGA did not. Based on this information, it is possible to identify some segments of carbohydrate structures attached to GP62. GP62 appears to contain a bianntenary complex-type oligosaccharide with at least one a(2,6)-linked sialic acid, N-acetyllactosamine and bissecting b(1,4)-linked N-acetylglucosamine. While performing these experiments we noticed that heat-shock is not necessary for the induction of GP62, and that even simple subculturing of cells was a sufficient stimulus. However, the dynamics of the induction was somewhat different. When GP62 was induced by a heat-shock, it appeared within 4 h after the end of heat treatment and remained on a relatively high level for at least next 24 h. After subculturing, GP62 appeared after only 2 h, stayed on a high level during the following 2 h, and virtually disappeared after 24 h. Interestingly, glycosylation of GP62 induced by subculturing was found to be different from glycosylation of GP62 induced by heat-shock. In addition to SNA, DSA and PHA-E lectins, which recognized GP62 induced by heat-shock, GP62 induced by subculturing was also recognized by RCA I and WGA lectins. Thus, in addition to carbohydrates present on GP62 induced by heat-shock, GP62 induced by subculturing contained terminal galactose and N-acetylglucosamine residues. Contrary to heat-shock and subculturing, when cells were exposed to UV-C radiation, GP62 did not appear, suggesting that it is associated with some, but not all, cellular stress-response pathway.

Over a half of all known proteins contain covalently linked oligosaccharide structures that presumably perform some specific physiological functions. Due to the lack of adequate methods, until recently only little was known about the role of oligosaccharide structures of glycoconjugates. However, in the recent years it was convincingly demonstrated that at least some of these structures are of utmost importance. One of the principal ways how oligosaccharides perform their functions is through interactions with their specific receptors named lectins. Recently we have developed glycoprobes, a novel set of compounds that enable direct measurement of lectin activity in complex biological samples. The glycoprobe consists of three vital parts: (i) glycan; (ii) digoxin tag; and (iii) photoreactive crosslinker. When incubated in dark, oligosaccharide part of the glycoprobe forms a complex with lectin. After illumination, covalent link between the probe and the lectin is formed resulting in a digoxin-tagged lectin. Using antibodies against digoxin, this complex can easily be identified by Western blots. Glycoprobes containing Man9 oligosaccharide and YEE(ahGalNAc)3 glycopeptide were prepared and used to analyze lectin activity in sera of 20 patients with juvenile rheumatoid arthritis and 20 control sera. Human serum (75 μg of total protein) was incubated in the presence of 0.8 mM mannose- or GalNAc-glycoprobe in dark for 60 min. After crosslinking by UV-illumination, proteins were separated by 8% SDS PAGE, transferred onto PVDF membranes and analyzed with anti-digoxin antibodies. As expected, we were able to detect several lectins that specifically bound glycoprobes. The variability of lectin content and activity was surprisingly high in both patients with juvenile rheumatoid arthritis and controls. Some of the changes were apparently the consequence of natural variability, but for several specific lectins we were able to demonstrate significantly different activities between the control group and patients suffering from juvenile rheumatoid arthritis.

Metabolic response to psychological stress is a very complex and demanding physiological process. It involves many organs and diversely affects individual organ systems. Though highly important for survival in ever changing environment, when excessively activated, it becomes associated with various detrimental effects. A number of epidemiological and experimental studies conducted during the past few years have clearly demonstrated a link between stress and the development and course of many diseases from simple virus infections and gastric ulcers to cardiovascular diseases and cancer1. However, main biochemical pathways linking stress to the development of diseases are still not understood. We have shown that stress causes tissue- and stressor-specific changes of glycoprotein structure and glycosyltransferase activity2. We have also noticed that the expression of carbohydrate-binding proteins vary depending on the type of stress experience. When studying the effects of stress on the expression of galactoside-specific galectin-3, we have found that the extent and direction of changes of galectin-3 expression are strongly dependent on the origin of disturbance. While immobilization, heat-shock, and subculturing decreased cellular levels of galectin-3, UV-C irradiation and alkylating agents induced its expression. Transcription factors NFкB and Jun were shown to be involved in at least some of these stress related processes. 1. M.Flögel and G.Lauc, in Encyclopedia of Stress, G. Fink ed., Academic Press, 2000, p. 678-673. 2. G.Lauc and M.Flögel, in Encyclopedia of Stress, G. Fink ed., Academic Press, 2000, p 276-282.