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Mutations in one of at least eight different genes cause bone marrow failure, chromosome instability, and predisposition to cancer associated with the rare genetic syndrome Fanconi anemia (FA). The cloning of seven genes has provided the tools to study the molecular pathway disrupted in Fanconi anemia patients. The structure of the genes and their gene products provided few clues to their functional role. We report here the use of 3 FA proteins, FANCA, FANCC, and FANCG, as "baits" in the hunt for interactors to obtain clues for FA protein functions. Using five different human cDNA libraries we screened 36.5x10(6) clones with the technique of the yeast two-hybrid system. We identified 69 proteins which have not previously been linked to the FA pathway as direct interactors of FANCA, FANCC, or FANCG. Most of these proteins are associated with four functional classes including transcription regulation (21 proteins), signaling (13 proteins), oxidative metabolism (10 proteins), and intracellular transport (11 proteins). Interaction with 6 proteins, DAXX, Ran, IkappaBgamma, USP14, and the previously reported SNX5 and FAZF, was additionally confirmed by coimmunoprecipitation and/or colocalization studies. Taken together, our data strongly support the hypothesis that FA proteins are functionally involved in several complex cellular pathways including transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

Survivin is expressed in most cancers but is undetectable in differentiated adult cells, and plays an important role both in the suppression of apoptosis and mitotic spindle checkpoint; thus it has attracted great interest as a potential drug target. In this study, we investigated the antigene and antiproliferative effects of triplex-forming oligodeoxynucleotides (TFO) targeting survivin in human lung carcinoma A549 cells. Survivin-specific TFOs form stable triplexes under physiological conditions as tested by electrophoretic mobility shift assays. Treatment of A549 cells with survivin-specific but not control TFOs at a concentration of 400 nM in the presence of uptake-enhancing liposome significantly reduced survivin protein level, inhibited cell proliferation, and induced cell apoptosis as demonstrated by immunoblot, cell number counting, and Annexin V-staining. Moreover, we found that the triplex-forming potential of TFOs measured in vitro does not necessarily correlate with the ability of TFOs to affect expression of a targeted gene in vivo. Our results indicate that targeting survivin is a promising alternative strategy for the development of novel anticancer therapeutics.

Biological markers in nasal secretions provide valuable information on nasal pathophysiology. However, published data on biomarker concentrations in nasal fluids are remarkably inconsistent, and the bias due to different sampling techniques, has not yet been systematically evaluated. Concentrations of various protein were repeatedly determined in nasal secretions of 16 healthy volunteers. The proteins were detected by using: 1) alpha2-macroglobulin as a marker for plasma contamination; 2) lactoferrin as a marker for glandular secretion; 3) lactate dehydrogenase as a marker for tissue injury; and 4) interleukin (IL)-1beta, IL-8, tumour necrosis factor-alpha, and eosinophil cationic protein and tryptase as indicators for tissue inflammation. A total of four different sampling methods, including nasal lavage (NL) and a new polyurethane foam sampler technique (PFST) were employed. Analyte concentrations in NL were approximately 10-times lower than in specimens obtained by PFST. Due to the unpredictable dilution during NL, various analytes were below the detection limit of the high sensitivity assays employed. With PFST, concentrations below the detection limit rarely occurred. The specimens did not significantly differ regarding plasma contamination, glandular secretion or tissue injury. The considerable variability of reported analyte concentrations in nasal secretions mainly results from different sampling techniques. To collect nasal secretions, samplers are considered superior to nasal lavage techniques.

The aim of this prospective study was to determine the local concentrations of inflammatory mediators in various tissue types frequently affected by trauma to estimate the role of prestored cytokine release by mechanical tissue trauma in the induction of a systemic inflammatory response syndrome. The degree of tissue damage, evaluated by its systemic release of inflammatory mediators, represents an important factor concerning the outcome of trauma patients. Clinical trials indicate that the kind of traumatized tissue influences the cytokine pattern measured in patients blood afterwards. However, the tissue-specific mediator composition underlying this systemic mediator release is rarely elucidated. Upon approval of the local IRB/EC, skin, subcutaneous fat, muscle, cancellous bone, and lung tissue were obtained during standard surgical procedures. The protein-based concentrations of Interleukin (IL)-6, IL-8, IL-10, and IL-12 were determined in tissue homogenates by enzyme-linked immunoabsorbant assay (ELISA; n = 60 samples). Albumin was measured to evaluate the degree of blood contamination of tissue samples. IL-6 and IL-8 were consistently detectable in more than 95% of the tissue specimens. Lung and cancellous bone presented by far the highest concentrations of these cytokines, whereas skin, subcutaneous fat, and muscle showed significantly lower levels. IL-10 was not detectable in 88%; IL-12 could not be measured in 63% of the samples. Cytokine concentrations did not correlate with the amount of albumin measured in tissue specimens. Due to their consistent presence at the tissue level, high systemic concentrations of IL-6 and IL-8 in patients blood, seen after pulmonary trauma, long bone fractures, or soft tissue injury, may be interpreted as an overspill of local trauma mediators. This indicates their relevance in post-traumatic monitoring. Furthermore, albumin is a suitable and necessary indicator to evaluate influences of possible blood contamination in tissue samples.

Aim: The aim of this study was to enhance the delivery and thus anti-tumoral efficiency of antisense bcl-2 oligonucleotides (ODN's)., Methods: Bcl-2 overexpressing DoHH2 lymphoma and HeLa-cells were transfected with ODN's using a polycationic liposome preparation. Specific hybridization of antisense ODN's was demonstrated by gel-shift assays and in vitro transcription/translation studies. Cellular uptake of oligonucleotides was evaluated by fluorescence microscopy. Inhibition of bcl-2 translation was demonstrated by quantitative RT-PCR and Western Blot. TUNEL assay, ANNEXIN V-binding and Apo-2.7 expression were performed to evaluate induction of apoptosis., Results: Using polycationic liposomes, a ODN transfection rate of 95% in HeLa and 45% in DoHH2 cells were demonstrated by fluorescence microscopy. 24 hours after transfection quantitative RT-PCR detected a 56% decrease of bcl-2 mRNA in antisense and a 7% decrease in sense transfected DoHH2 cells (p < 0.05). In HeLa-cells, bcl-2 expression was almost completely inhibited 72 hours after antisense ODN transfection. Antisense treated cells also showed significant induction of apoptosis., Conclusions: Polycationic liposome-mediated transfection of bcl-2 antisense ODN's causes enhanced cellular uptake and efficient bcl-2 downregulation in bcl-2 overexpressing cell lines. This delivery strategy may explain why significant induction of apoptosis was achieved at low oligonucleotide concentrations (approximately 200 pmol/5 x 10(5) tumor cells).

The effect of long-term stagnation on copper corrosion by-product release and corrosion rates was studied in pipe-rigs according to the German standard DIN 50931, Part 1. The analysis of the water phase was supplemented by surface analysis of corrosion scales. Copper concentration during stagnation did not follow a solubility process. The characteristic curves obtained can be explained by subsequent copper release and copper refixation processes. Oxygen consumption can be described by the first-order kinetic rate law. The corrosion scales consisted of cuprite (Cu2O) and malachite (CuCO3 x Cu(OH)2). Malachite grew in well-defined crystals during stagnation, served as sink for dissolved copper and did not protect the pipe against corrosion attack. Copper concentrations measured after long-term stagnation (up to 122 h) correspond to the solubility of malachite in the testwater.

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre-tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III-dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5'-and 3'-end processing, nucleotide modification and splicing of intron-containing pre-tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre-tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron-containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2' phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2'-phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, phi27 and phi35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to phi requires an intron-containing substrate.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide exerting its effects predominantly by paracrine and autocrine mechanisms. ET-1 acts as a mitogen and co-mitogen on vascular smooth muscle cells, and accumulating evidence suggests that ET-1 is involved in the pathogenesis of atherosclerosis. Deposition of low density lipoproteins (LDL) in the vessel wall is known to play a crucial role in the development of atherosclerotic lesions. In the present study, we have investigated the effect of native LDL (nLDL) and oxidatively modified LDL (oxLDL) on ET-1 synthesis and endothelin receptor expression in cultured human coronary artery smooth muscle cells and human monocyte-derived macrophages. Native LDL and oxLDL induced a significant stimulation of ET-1 release and ET-1 mRNA expression in human coronary artery smooth muscle cells and monocyte-derived macrophages. Antibodies against the scavenger receptor CD36 significantly reduced the oxLDL-induced stimulation of ET-1 synthesis. The antioxidants trolox and probucol did not significantly inhibit the LDL-induced rise of ET-1 release. Endothelin B receptor expression was up-regulated in both cell types after incubation with nLDL and oxLDL. In coronary smooth muscle cells, endothelin A receptor expression was slightly increased by LDL, whereas endothelin A receptor was not detectable in monocyte-derived macrophages. Coronary artery smooth muscle cells secreted a more than 150-fold higher amount of immunoreactive ET-1 into the cell culture medium than monocyte-derived macrophages. In summary, the present data, demonstrating a LDL-induced up-regulation of the endothelin system in coronary smooth muscle cells and in monocyte-derived macrophages, provide further support for a pathophysiological role of endothelin in coronary atherosclerosis and suggest that ET-1 might be involved in mediating the atherogenic effects of LDL., (Copyright 2001 Academic Press.)

A high variability of RNase P RNA structures is seen among members of the Mycoplasma group. To gain further insight into the structure-function relations of this ribozyme, we have searched for the RNase P RNA gene from more distant relatives, the phytoplasmas. These mycoplasma-like organisms are the aetiological agents of many severe plant diseases. We report the sequence and catalytic properties of RNase P RNA from the phytoplasma causing apple proliferation disease. The primary and postulated secondary structure of this 443 nt long RNA are most similar to those of Acholeplasma, supporting the phylogenetic position of this pathogen. Remarkably, the extremely AT-rich (73.6%) phytoplasma RNA differs from the known bacterial consensus sequence by a single base pair, which is positioned close to the substrate cleavage site in current three-dimensional models. Phytoplasma RNase P RNA functions as an efficient ribozyme in vitro. Conversion of its sequence to the full consensus and kinetic analysis of the resulting mutant RNAs suggests that neither the sequence alone, nor the type of pairing at this position is crucial for substrate binding or catalysis by the RNase P ribozyme. These results refine the bacterial consensus structure close to the catalytic core and thus improve our understanding of RNase P RNA function.

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.

Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products. (Blood. 2000;95:719-720)

We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421-435). PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis. Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis. Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum. Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type I 1.38 +/- 0.09-fold of control and c-fibronectin 1.89 +/- 0.18-fold of control. Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 +/- 0.2-fold and 2.80 +/- 0.05-fold of control, respectively. Northern blot demonstrated an increased expression of the collagen-I-(alpha-1)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants. Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants. Unstimulated macrophages released 1.97 pg TGFbeta1/microgram of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFbeta1/microgram of DNA over 24 hours. These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFbeta as the responsible mediator. In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFbeta1 released by activated macrophages. We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis.

It has been reported recently that naturally occurring catalytic RNAs like hammerhead and hairpin ribozyme do not require metal ions for efficient catalysis. It seems that the folded tertiary structure of the RNA contributes more to the catalytic function than was initially recognized. We found that a highly specific self-cleavage reaction can occur within a small bulge loop of four nucleotides in a mini-substrate derived from Arabidopsis thaliana intron-containing pre-tRNA(Tyr) in the absence of metal ions. NH(4)(+) cations and non-ionic or zwitter-ionic detergents at or above their critical micelle concentration are sufficient to catalyze this reaction. The dependence on micelles for the reaction leads to the assumption that physical properties, i.e. the hydrophobic interior of a micelle, are essential for this self-cleavage reaction. We suggest that NH(4)(+)-ions play a crucial role for the entry of the negatively charged RNA into the hydrophobic interior of a detergent micelle. A change of the pattern of hydration or hydrogen bonds caused by the hydrophobic surrounding enhances the reaction by a factor of 100. These findings suggest that highly structured RNAs may shift pK(a) values towards neutrality via the local environment and thereby enhance their ability to perform general acid-base catalysis without the participation of metal ions.

Age-related changes in functional subsets of lymphocytes may influence the potential to build up immune responses. In particular, the capacity of tonsillar lymphocytes to counter infections may be altered during ageing. In order to address this question we investigated the proportional distribution of several subsets of tonsillar T and B cells with regard to ageing. Tonsils were derived from 119 patients between 2 and 65 years of age. Lymphocyte subsets were monitored by three-colour fluorescence of relevant CD markers in flow cytometry. As a general tendency the percentage of CD3+ T cells steadily increased whereas that of CD19+ B cells decreased at the same time. No significant differences were observed between lymphocytes of patients with and without inflammatory history of the tonsils. The percentage of CD8+ T cells declined whereas that of CD4+ T cells increased during the same time span. CD45RA+ T cells increased during the first two decades of life and gradually decreased thereafter. In contrast, CD45RO+ T cells showed an opposite trend. No differences were seen in the population of CD3-/CD56+ natural killer (NK) cells. The mature B cell marker CD40 showed no significant changes during ageing. However, CD38+ B cells, representing B cells of late maturation stages, dramatically declined up to the age of 65. In a similar manner the CD5+ subpopulation of B cells decreased during ageing. Substantial changes in major tonsillar T and B cell populations as shown in this study may have an impact on the ageing process of the immune system.

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5' and 3' ends, posttranscriptional addition of the 3' CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5' and 3' ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5' and 3' terminal ends, and the TpsiC loop are critical for exportin-t binding.

In mammals, most of the selenium contained in their body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is normally recognized as a translational stop signal, it is intriguing how cells recognize and distinguish the UGA Sec codon from the UGA stop codon. In eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated Sec insertion sequence (SECIS) located in the 3'-untranslated regions is required for recognition of UGA as a Sec codon. Although some proteins (SBPs) have been reported to bind to SECIS, it is not clear how the SECIS element can mediate Sec insertion at UGA. Eukaryotic Sec-tRNA(Sec) is not recognized by elongation factor EF-1alpha, but is recognized specifically by a Sec-tRNA(Sec) protecting factor, SePF, in bovine liver extracts. In this study, we provide evidence that SePF is distinct from SBP by chromatography. Upon UV irradiation, the SECIS RNA was cross-linked to a 47.5 kDa protein, a likely candidate of SBP, that is contained in the complex with a molecular mass of 150 kDa. These results suggest that SBP and SePF play different roles for the Sec incorporation. To our knowledge, this is the first demonstration that SBP is discriminated from the factor which directly recognizes Sec-tRNA(Sec), providing a novel clue to the mechanism of selenocysteine decoding in eukaryotes.

Various lines of evidence indicate that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease. We have investigated the effect of modified (oxidized) low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis in cultured human coronary artery smooth muscle cells (HCA-SMC). As shown by immunofluorescence microscopy and time-resolved fluorescence immunoassay, oxLDL dose-dependently stimulated collagen type I and fibronectin synthesis in cultured HCA-SMC. The effect on matrix synthesis was biphasic, with a maximum effect at concentrations between 1 and 10 microg/ml oxLDL. Higher oxLDL concentrations (>25 microg/ml) were cytotoxic. Beside oxLDL, malondialdehyde-modified LDL also stimulated extracellular matrix synthesis. In the presence of 100 microg/ml ascorbic acid, 25, 50 and 100 microg/ml oxLDL induced apoptosis within 6-8 hours (demonstrated by TUNEL-reaction, annexin-V binding and APO-2.7-expression). Apoptosis was not induced by normal (unmodified) LDL and malondialdehyde-modified LDL. The radical scavengers and antioxidants TROLOX and probucol and the hydrogen peroxide eliminator catalase significantly reduced oxLDL-induced apoptosis. Our results demonstrate that low concentrations of oxLDL are profibrogenic by stimulating extracellular matrix synthesis, whereas higher oxLDL concentrations induce oxidative stress and apoptosis in coronary artery smooth muscle cells. The profibrogenic effect might be relevant in the formation of atherosclerotic plaques, and the proapoptotic effect might contribute to an increased plaque vulnerability.

The carbohydrate-deficient glycoprotein syndromes (CDGS) are genetic, multisystemic diseases characterized by deficiencies in the glycosylation of many secretory glycoproteins, lysosomal enzymes, and possibly cell surface glycoproteins resulting in central nervous system abnormalities and frequent early death by infection. Here we examined whether membranous glycoconjugates of lymphocytes are affected by this disorder. For this, we analyzed cell surface-expressed sialoglycans of Epstein Barr virus (EBV)-transformed B cell lines derived from peripheral B lymphocytes of several patients with CDGS I A. These CDG-LCL (lymphoblastoid cell lines) expressed differentiation markers comparable to those of other EBV-transformed B cell lines. No apparent defects in the gross glycosylation process of defined complex glycosylated proteins such as the surface-expressed major histocompatibility complex class I glycoprotein or secreted immunoglobulin (IgM) were identified. However, using a novel flow cytometric enzyme assay to measure cell surface alpha2,6 sialylation on live cells we found that CDG-LCL express less alpha2,6 sialylated glycans in comparison to other EBV-transformed B cell lines. Also, CDG-LCL bound less of the B lymphocyte lectin CD22, specific for alpha2,6 sialylated lactosamines and known to modulate B cell receptor mediated signaling, as demonstrated by using a soluble CD22-immunoglobulin fusion protein in flow cytometry. CDG-LCL showed stronger surface staining with the monoclonal antibody 1B2 which detects a distinct group of surface-expressed lactosaminyl epitopes. After pretreatment with neuraminidase of Newcastle disease virus (NDVN) it became apparent that in CDG-LCL a significantly larger portion of the 1B2 epitopes was sialylated in alpha2,3 linkage as compared to other B cell lines. Intracellular alpha2,6 sialyltransferase activity as well as polymerase chain reaction products specific for four different sialyltransferases did not significantly differ in CDG-LCL as compared to other EBV-B cell lines. Differences in sialylation may be caused by the respective oligosaccharide core structures available for alpha2,6 or alpha2,3 sialylation in CDG-LCL. Therefore, lymphocytes derived from CDGS patients have distinct deviations in their surface-expressed lactosaminoglycan structures which may affect functions as exemplified by reduced interactions of CD22 with its ligands.

The siglecs, formerly called sialoadhesins, are a family of I-type lectins binding to sialic acids on the cell surface. Five members of this family have been identified: sialoadhesin, myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), CD22 and CD33. We have investigated the relevance of substituents at position C-9 and in the N-acetyl group of N-acetylneuraminic acid, using a series of synthetic sialic-acid analogues either on resialylated human erythrocytes or as free alpha-glycosides in hapten inhibition. All five siglecs require the hydroxy group at C-9 for binding, suggesting hydrogen bonding of this substituent with the binding site. Remarkable differences were found among the proteins in their specificity for modifications of the N-acetyl group. Whereas sialoadhesin, MAG and SMP do not tolerate a hydroxy group as in N-glycolylneuraminic acid, they bind to halogenated acetyl residues. In the case of MAG, N-fluoroacetylneuraminic acid is bound about 17-fold better than N-acetylneuraminic acid. In contrast, human and murine CD22 both show good affinity for N-glycolylneuraminic acid, but only human CD22 bound the halogenated compounds. In conclusion, our data indicate that interactions of the hydroxy group at position 9 and the N-acyl substituent contribute significantly to the binding strength.

Mature tRNAPhe from Escherichia coli and the transcript of its gene lacking modified nucleotides were compared by a variety of physical techniques. Melting experiments revealed that at a low Mg2+level the transcript was partially denatured, while the mature tRNA possessed intact tertiary interactions. Mg2+binding to both tRNAs was studied by CD and UV techniques as well as by using the Mg2+-sensitive fluorescence indicator, 8-hydroxyquinoline 5-sulfonic acid. Both tRNA forms exhibited a single strong Mg2+-binding site, its dissociation constant was 10-fold higher for the transcript. Conformational changes in response to Mg2+ addition measured by CD and UV spectrometry revealed no difference for the estimated binding cooperativity and strong differences for affinities of Mg2+-binding sites for the two tRNA forms. Conformational transitions in mature and in in vitro synthesized tRNA required the binding of two Mg2+ ions per molecule and therefore should be associated not only with a single strong binding site. The Mg2+ dependence of Stokes radii measured by gel-filtration revealed insignificant differences between the overall sizes of the two tRNA forms at physiological Mg2+ levels (>1 mM). Taken together, these results suggest that modified nucleotides stabilize tertiary interactions and increase the structure stability without affecting the mechanism of Mg2+binding and overall folding of the tRNA molecule. This conclusion is supported by the known biological activity of the E. coli tRNAPhe gene transcript.

The recognition process of tRNA(Ser) and tRNA(Sec) by human seryl-tRNA synthetase (SerRS) was studied using T7 transcripts representing defined regions of human tRNA(Ser) or tRNA(Sec) and the influence of the tRNA elements on serylation and tertiary structure was elucidated. The anticodon arms of both tRNAs showed no contribution to serylation in contrast to the acceptor stems and the long extra arms. D and T arms were only involved in formation of the L-shaped tRNA structure, not in the recognition process between tRNAs and SerRS. This is the first report of microhelices adapted from human tRNAs being aminoacylated by their homologous synthetase.

Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream. Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H. The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.

Background and Objectives: Pr1,2,3, PrM, Sa and Sia-l1, -b1, -lb1 are sialic acid (NeuNAc)-dependent antigens recognized by human cold agglutinins. Pr and Sa antigens are the O-glycans of glycophorins containing alpha 2,3NeuNAc (to galactose) and/or alpha 2,6NeuNAc (to galactosamine). The antigens of the Sia-l1, -b1, -lb1 complex are gangliosides that may carry alpha 2,3NeuNAc (to galactose) and/or alpha 2,8NeuNAc (to NeuNAc). We studied the NeuNAc groups involved in the antigens., Materials and Methods: From 74 sera with cold agglutinins against NeuNAc-dependent antigens, anti-T-free preparations were made and tested against human red cells, treated with an alpha 2,3-specific recombinant sialidase., Results: Most (51/62) Pr antigens use alpha 2,3NeuNAc, some (8/62) use alpha 2,6NeuNAc and a few (3/62) use both sialyl groups as immunodominant components on glycophorins. The immunodominant component of Sa and Sia-l1, -b1, -lb1 determinants was alpha 2,3NeuNAc in all cases., Conclusion: The red cell target structures for cold agglutinins against NeuNAc-dependent antigens have been identified. We propose a Pr nomenclature to reflect this. The binding of anti-Pr to gangliosides may be the basis for anti-Pr-induced peripheral neuropathy.

Gangliosides with a terminal 9-O-acetylated disialosyl group (CDw60 structures) show a restricted surface expression on human leukocytes. Hithereto, they have only been detected on subpopulations of human T lymphocytes. Using the defined CDw60 antibody UM4D4 and two new antibodies with preferential CDw60 activities, F6 and Z17, we demonstrate for the first time that CDw60 is an activation marker on human B lymphocytes. In vitro phorbol ester-stimulated human peripheral blood B lymphocytes as well as in vivo activated tonsillar B lymphocytes became CDw60 positive. CDw60 expression of these cells exceeds that of resting and activated T-lymphocytes.

Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (aaRS) is crucial to the maintenance of translational fidelity. The discriminator base A73 in human tRNALeuis critical for its specific recognition by the aaRS. Exchanging A73 for G abolishes leucine acceptance and converts it into a serine acceptor in vitro . Two RNA microhelices of 24 nt length that correspond to the tRNALeuacceptor stem and differ only in the discriminator base were synthesized: a wild-type tRNALeumicrohelix, where nt 21 corresponds to the discriminator base position 73, and an A21G mutant microhelix. To investigate whether different identities of both tRNAs are caused by conformational differences, NMR and UV melting experiments were performed on both microhelices. Two-dimentional NOESY spectra showed both microhelices to exhibit the same overall conformation at their 3'-CCA ends. Thermodynamic analysis and melting behaviour of the base-paired imino protons observed by NMR spectroscopy suggest that the A21G (A73G in tRNA) exchange results in a decrease of melting transition cooperativity and a destabilization of the terminal G1-C20 (G1-C72 in tRNA) base pair. Furthermore, the fact that this 3'-terminal imino proton is more solvent-exposed at physiological temperature might be another indication for the importance of the stability of the terminal base pair for specific tRNA recognition.

Oncogenic transformation is often accompanied by alterations of glycosylation on a tumor cell's surface, which may contribute to uncontrolled cell growth. The sialoglycans and degree of sialylation on the cell surface are of increasing interest because of their possible role in metastasis and tissue invasion. Since primary tumors and metastases may differ in the degree of sialylation, we examined the expression of sialic acid as a terminal constituent of lactosaminyl glycans on the cell surfaces of 30 cervical lymph-node metastases and 30 squamous-cell carcinomas of the oropharynx and oral cavity. Cell-surface sialylation was determined by a new histobiochemical assay on cryostat sections and was based on the enzymatic introduction of a fluorescence-labelled sialic acid into lactosaminyl type (Gal-beta 1-4 GlcNAc) oligosaccharide chains of cell-surface-expressed glycoproteins. To this end, tissues were incubated in the presence of 5-acetamido-9-deoxy-9-fluoresceinyl-thioureido neuraminic acid (CMP-9-fluoresceinyl-NeuAc) and alpha-2,6-sialyltransferase. In order to compare the degree of sialylation with the potential total amount of sialylation sites, pretreatment with sialidase for desialylation was required. We observed a significantly higher amount of lactosaminyl-type binding sites for sialic acid on metastases compared to the primary tumors (P = 0.001), indicating a lower degree of sialylation in metastases. In primary tumors no correlation was seen between the amount of binding sites and tumor localization, TNM stage or histologic grading. Pretreatment of specimens with sialidase demonstrated a significant degree of sialylation on both primary tumors and lymph-node metastases, but no difference between primary tumors and metastases. When tumor stroma of primary tumors and metastases was compared, tumor cells showed a higher degree of free binding sites for sialic acid, but a low degree of sialylation. Our results suggest that differences in the degree of sialylation of glycoconjugates on a tumor cell's surface may play an important role in the process of cell metastasis. Our histobiochemical method turned out to be very reliable, effective and readily performed.

We constructed a set of mammalian tRNA(Lys)3-pseudogenes, whose pre-tRNAs are processing-deficient in HeLa extract. The 3'-flanking region was designed partially or completely complementary to the PBS-flanking nucleotides of the U5-region of HIV-1 genomic RNA. We show that only the pre-tRNA with completely hybridized 3'-flanking region is efficiently extended by the HIV-1 reverse transcriptase, whereas the partially complementary pre-tRNA(Lys)3, the 3'-terminal 10 nucleotides of which are not hybridized to the viral template, is unable to prime the cDNA-synthesis.

We investigated ribonucleases from Saccharomyces cerevisiae which are active in pre-tRNA 3'-processing in vitro. Two pre-tRNA 3'-exonucleases with molecular masses of 33 and 60 kDa, two pre-tRNA 3'-endonucleases with molecular masses of 45 kDa/60 kDa and 55 kDa and 70-kDa 3'-pre-tRNase were purified from yeast whole cell extracts by several successive chromatographic purification steps. The purified exonucleases are non-processive 3'-exonucleases that catalyze the exonucleolytic processing of 3'-trailer sequences of pre-tRNAs to produce mature tRNAs. The 45-kDa/60-kDa 3'-endonuclease is tRNA-specific and catalyzes the processing of pre-tRNAs in a single endonucleolytic step. Two isoenzymes of this activity (p45 and p60) were identified by chromatography. The second endonuclease, p55, is dependent on monovalent ions and cleaves about three nucleotides downstream the mature 3'-end. All of the purified 3'-pre-tRNases accept homologous as well as heterologous pre-tRNA substrates. Pre-tRNAs carrying a 5'-leader are processed with almost the same efficiency as those lacking this 5'-leader. Mature tRNAs carrying the CCA 3'-sequence and tRNA pseudogene products carrying mutations in the mature domain are processed by the 3'-exonucleases, not by the 3'-endonucleases. The specific endonuclease p45/p60 discriminates between UUUOH as a 3'-flank, which is cleaved, and the CCA 3'-end of mature tRNAs, which is not cleaved. This study suggests that several 3'-pre-tRNases are active on tRNA precursors in vitro and might therefore in pre-tRNA 3'-processing in yeast, partly in a cooperative manner.

The mechanism of selenocysteine insertion into proteins is distinct from all other amino acids in all lines of descent in that it needs specific protein cofactors and a structurally unique tRNA(Sec). It is first aminoacylated with serine and further recognized among all other serylated serine isoacceptors by a selenocysteine synthase and is converted to selenocysteyl-tRNA(Sec). We present here the complete set of identity elements for selenylation of mammalian seryl-tRNA(Sec) and show that the transplantation of these elements into normal serine tRNA allows its selenylation. Four particular structural motifs differentiate eukaryotic tRNA(Sec) from normal tRNA(Ser): the orientation of the extra arm, the short 4 bp T psi C-stem, the extra long 9 bp acceptor-stem and the elongated 6 bp dihydrouridine-stem. Only the last two are essential and only together sufficient for selenocysteine synthesis, whereby the additional base-pairs of the acceptor-stem may be replaced by non-paired nucleotides. Each exchange of the first three structural motifs mentioned above between tRNA(Ser) and tRNA(Sec) resulted in a significant loss of serylation, indicating that the overall composition of particular structure elements is necessary to maintain normal functions of tRNA(Sec). Since we find that all seryl-tRNAs which are selenylated are also substrates for serine phosphorylation we propose that phosphoseryl-tRNA(Sec) is a storage form of seryl-tRNA(Sec).

Potassium peroxonitrite (ONOOK) and [Fe(EDTA)]2- were used to analyze the influence of chemically entirely different hydroxyl radical sources on tRNA cleavage profiles. [Fe(EDTA)]2- gives rise to hydroxyl radicals via a Fenton-like reaction during the oxidation of chelated Fe2+, while ONOOK generates hydroxyl radicals via its conjugate acid (ONOOH) when adding a stable alkaline solution of ONOOK in samples buffered at neutral pH. [Fe(EDTA)]2- is known to induce oxidative strand scission at sugar moieties thought to be solvent accessible, while those residues located in the 'inside' of structured RNAs are protected. Although ONOOH is neutral and significantly smaller than the metal complex, both reagents generate the same protection pattern on tRNAs, suggesting that access of the commonly formed hydroxyl radical, rather than access of its source, is the determining factor when probing the higher order structure of RNA. Strong difference in reactivity is only seen at the modified 2-thiouridine S34 of tRNA(Lys3) which shows hyperreactivity towards ONOOK treatment. This particular reaction may require interaction between the peroxonitrite anion and the thiocarbonyl group of the base, since hyperreactivity is not observed when probing the dethiolated tRNA(Lys3).

The intervening sequences of nuclear tRNA precursors are known to be excised by tRNA splicing endonuclease. We show here that a T7 transcript corresponding to a pre-tRNA(Tyr) from Arabidopsis thaliana has a highly specific activity for autolytic intron excision. Self-cleavage occurs precisely at the authentic 3'-splice site and at the phosphodiester bond one nucleotide downstream of the authentic 5'-splice site. The reaction results in fragments with 2',3'-cyclic phosphate and 5'-OH termini. It is resistant to proteinase K and/or SDS treatment and is not inhibited by added tRNA. The self-cleavage depends on Mg2+ and is stimulated by spermine and Triton X-100. A set of sequence variants at the cleavage sites has been analysed for autolytic intron excision and, in parallel, for enzymatic in vitro splicing in wheat germ S23 extract. Single-stranded loops are a prerequisite for both reactions. Self-cleavage not only occurs at pyrimidine-A but also at U-U bonds. Since intron self-excision is only about five times slower than the enzymatic intron excision in a wheat germ S23 extract, we propose that the splicing endonuclease may function by improving the preciseness and efficiency of an inherent pre-tRNA self-cleavage activity.

The recognition of human tRNA(Leu) or tRNA(Ser) by cognate aminoacyl- tRNA synthetases has distinct requirements. Only one base change (A73-->G) in tRNA(Leu) is required to generate an efficient serine acceptor in vitro, whereas several changes in three structural domains (the acceptor stem, DHU loop and long extra arm) of tRNA(Ser) are necessary in order to produce a leucine acceptor. Hence, the molecular basis for the discrimination between human tRNA(Ser) and tRNA(Leu) by the seryl-tRNA synthetase depends almost exclusively on a highly specific recognition of the discriminator base G73. In order to elucidate the specific role of the functional groups of this base in discrimination, tRNA(Ser) constructs were made which contain the artificial base analogues 2-aminopurine riboside or inosine at the discriminator position 73. Aminoacylation of these constructs by a HeLa S100 extract showed that molecules with 2-aminopurine riboside, but not with inosine, in position 73 could be serylated at low efficiency. However, the 2-aminopurine riboside and the inosine derivatives of tRNA(Ser) were equally efficient competitive inhibitors of serylation, whereas tRNAs(Ser) with any other natural base at position 73 did not competitively inhibit serylation of tRNA(Ser). This was in contrast to leucylation of tRNA(Leu), where tRNA(Leu) transcripts with any other nucleotide in the discriminator position acted as strong competitive inhibitors. These results suggest that the discriminator bases in human tRNA(Ser) and tRNA(Leu) play completely different roles in recognition of the tRNAs by their cognate aminoacyl-tRNA synthetases.

This is the first report which gives a general survey about viroid variant composition in a vineyard and within single plants. A German vineyard with 20-year-old grapevines (Vitis vinifera) of the cultivars 'Bacchus' and 'Kerner' was analysed for viroid infections. Only grapevine yellow speckle viroid 1 (GYSVd1) and the grapevine isolate of hop stunt viroid (HSVdg) were detected. Both viroids occur in several sequence variations. Eighteen novel GYSVd1 variants and two previously published HSVdg main variants with six new minor variants were found. They were randomly spread in the vineyard. The distribution of GYSVd1 and HSVdg main variants and their accompanying subvariants differed even in neighbouring plants. We conclude that these individual viroid variant profiles are the result of 20 years of independent evolution, i.e. mutation and selection, in each single plant. Four of the nine GYSVd1 main variants were mutated in the inverted repeats bordering the central conserved region. These base substitutions decreased the thermodynamic stability of a metastable structure called hairpin I.

We present kinetic studies on the enzymatic transfer of several synthetic sialic acid analogues, modified at C-5, to distinct glycoprotein glycans by sialyltransferases differing in acceptor- and linkage-specificity. Biochemical properties of sialic acids were modified by introducing formyl-, trifluoroacetyl-, benzyloxy-carbonyl-, and aminoacetyl-groups to the amino group at C-5 of neuraminic acid. The latter substitution renders the corresponding alpha-glycoside resistant towards sialidases. The respective CMP-sialic acid analogues were prepared by CMP-sialic acid synthase with a yield of 13-55%. The kinetic parameters of several sialyltransferases for the 5-substituted CMP-glycosides differed significantly. Relative to parent CMP-NeuAc, reaction rates of human- and rat liver Gal beta 1, 4GlcNAc alpha 2,6-sialyltransferases ranged from 50 to 170%, of GalNAc alpha 2,6-sialyltransferases from 40-140%, and of Gal beta 1,3Gal-NAc alpha 2,3-sialyltransferase from 20-50%. Resialylation of asialo-alpha 1-acid glycoprotein by 5-N-formyl- and 5-N-aminoacetyl-neuraminic acid employing rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase proceeded to about 80% of galactose sites which is identical to the extent achieved with parent NeuAc. According to our data, neosialoglycoconjugates which carry sialic acids modified at the N-acetyl group can be prepared for structure-function analysis, as this position seems crucial for recognition of adhesion proteins and influenza viruses.

A synthetic sialic acid analog, N-acetyl-9-acetamido-9-deoxy-neuraminic acid, can be used by influenza C virus as a receptor determinant for attachment to cells. In contrast to the natural determinant, N-acetyl-9-O-acetylneuraminic acid, the synthetic sialic acid is resistant to the action of the receptor-destroying acetylesterase of this virus. The sialic acid analog was enzymatically transferred to the surface of Madin-Darby canine kidney cells that are resistant to infection because of a lack of receptors. Influenza C virus was able to infect the modified cells through requiring a 10-fold larger amount of the sialic acid analogue on the cell surface compared to the natural receptor determinant. The quantitative difference is accounted for mainly by a less efficient binding of influenza C virus to the analog. Thus, in our system, inactivation of the receptor by the viral esterase is not required for the initiation of an influenza C virus infection.

Cell surface expressed lactosaminyl glycans were determined on live cells by flow cytometry using a sialyltransferase mediated labeling procedure. Fluorescent CMP-sialic acid and Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase were applied to probe expression of acceptor glycans on untreated or sialidase pretreated erythrocytes. After enzymatic fluorescence labeling, erythrocytes were treated with endo-beta-galactosidase or trypsin to distinguish polylactosaminyl- and complex-type glycans. The expression of lactosaminyl sequences on cord- was 20% lower than on adult cells. After sialidase treatment fluorescence incorporation on both cell types increased twofold compared to untreated cells indicating a low sialylation extent. A recombinant alpha 2,3-sialyltransferase was preferentially labeling polylactosaminyl glycans. Taking advantage of the different fine specificity as determined here, alpha 2,6- and alpha 2,3-sialyltransferase can be applied to distinguish certain types of lactosaminyl glycans.

Anti-Pr agglutinins (CAs) with the subspecificities anti-Pr1h, -Pr1d, -Pr2, -Pr3h, -Pr3d, -PrM and anti-Sa CAs recognize immunodominant N-acetylneuraminic acid (NeuN Ac) groups of tetra and/or trisaccharides (O-glycans) of glycophorin. These O-glycans are sialylated in alpha 2,3- and/or alpha 2,6-linkages. Sa and most Pr antigens have been inactivated by alpha 2,3-specific sialidases. Antigenicity was reconstituted on desialylated glycophorin by alpha 2,3-specific Gal beta 1,3GalN Ac-sialyltransferase indicating that alpha 2,3-linked NeuN Ac groups are the immunodominant components of Sa and most Pr antigens. Some Pr antigens were resistant to alpha 2,3-specific sialidase and were not reconstituted by alpha 2,3-specific Gal beta 1,3GalN Ac-sialyltransferase, which indicates that alpha 2,6-linked NeuN Ac group represents an immunodominant component of some Pr antigens.

We have previously shown that the exchange of the discriminator base A73 of human tRNA(Leu) for G is alone sufficient to achieve complete loss of leucine acceptance and to create an efficient serine acceptor. The reverse identity switch, however, which was studied using T7 RNA polymerase transcripts of in vitro mutagenized tRNA genes, reveals a far more complex pattern of identity elements for tRNA(Leu). Introduction of the following tRNA(Leu)-specific structures is necessary to transform human tRNA(Ser) into an efficient leucine acceptor: the discriminator base A73, the base pairs C3:G70, A4:U69 and G5:C68 of the acceptor stem, C20a of the DHU loop and the long extra arm. In contrast to tRNA(Ser), human tRNA(Leu) identity requires both the sequence and the correct orientation of the long extra arm, whereas only its orientation is essential for serine identity.

Six of 13 cloned members of the human tRNA(Val) gene family code for tRNA(Val) pseudogenes, of which all but one are transcribed efficiently in HeLa cell extracts. Due to single or multiple mismatches in stem regions, the corresponding pre-tRNAs are resistant against the action of human 5'- and 3'-processing enzymes and are thus prevented from being converted to mature tRNAs. Surprisingly, all of them are accurately and efficiently processed to mature-sized tRNA in yeast nuclear extract. This is in agreement with corresponding studies of plant pre-tRNAs which are not processed in wheat germ extract but are rapidly processed in yeast extract. These observations imply that the yeast pre-tRNA 5'- and 3'-maturases do not monitor the three-dimensional structure of their substrates as stringently as mammalian and plant enzymes, possibly because tRNA pseudogenes do not occur in yeast.

Two rapid microscale procedures for isolation of total RNA from grapevine leaves are described. Homogenized leaf tissue is phenol extracted and polyphenols and polysaccharides are removed by either an aqueous two-phase system followed by DEAE-cellulose chromatography or by differential precipitation with the solvent 2-butoxyethanol. The resulting RNA, about 5-15 micrograms from 0.25 g grapevine leaves, is of high quality and can be employed for reverse transcription and polymerase chain reaction (PCR) amplification or Northern blot analysis to detect grapevine viroids or other RNAs. The two procedures are optimized for small-scale examination of many samples in a short time. Thus RNA from 12-24 leaf samples can be analyzed per day, including reverse transcription and PCR amplification. The application of these procedures led to the first detection of grapevine yellow speckle viroid 1 in German grapevines.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.

A flow cytometric assay was developed to detect rare cancer cells in blood and bone marrow. Multiple markers; each identified by a separate color of immunofluorescence (yellow and two shades of red), are used to reliably identify the cancer cells. Blood or bone marrow cells, which are not of interest but interfere in detecting the cancer cells, are identified by a panel of immunofluorescence markers, each of which has the same color (green). Thus, the rare cancer cells of interest are yellow and two different shades of red but not green. The requirement that the rare cancer cell be simultaneously positive for three separate colors (the specific markers) and negative for a fourth color (the exclusion color) allowed detection of as few as one cancer cell in 10(7) nucleated blood cells (a frequency of 10(-7). To test this rare-event assay prior to clinical studies, a model study was performed in which the clinical sample was simulated by mixing small numbers of cells from the breast carcinoma line BT-20 with peripheral blood mononuclear cells. We detected statistically significant numbers of BT-20 cells at mixing frequencies of 10(-5), 10(-6), and 10(-7). In control samples, no target events (BT-20) were observed when more than 10(8) cells were analyzed. For additional confirmation that the BT-20 cells in the model study were correctly identified and counted, the BT-20 cells (and only BT-20 cells) were covalently stained with a fifth fluorescence dye, 7-amino-4-chloromethylcoumarin (CMAC). CMAC fluorescence data were not used in the assay for detecting BT-20 cells. Only after the analysis using data from the specific and exclusion colors had been completed were the events identified as BT-20 cells checked for CMAC fluorescence. The putative BT-20 events were always found to be positive for CMAC fluorescence, which further increases confidence in the assay. Manual data analysis and an automated computer program were compared. Results were comparable with the manual and automated methods, but the automated "genetic algorithm" always found more BT-20 events. Cell sorting of BT-20 cells from samples that contained BT-20 at frequencies of 10(-5), 10(-6), and 10(-7) provided further evidence that these rare cells could be reliably detected. The good performance of the assay with the model system will encourage further studies on clinical samples.

An increasing number of mammalian cell adhesion molecules, including sialoadhesion, CD22 and the family of selectins, have been found to bind cell surface glycoconjugates containing sialic acids. Here we describe how the structural diversity of this sugar influences cell adhesion mediated by the related molecules sialoadhesin and CD22 in murine macrophages and B-cells respectively. We show that the 9-O-acetyl group of Neu5,9Ac2 and the N-glycoloyl residue of Neu5Gc interfere with sialoadhesin binding. In contrast, CD22 binds more strongly to Neu5Gc compared to Neu5Ac. Of two synthetic sialic acids tested, only CD22 bound the N-formyl derivative, whereas a N-trifluoroacetyl residue was accepted by sialoadhesin. The potential significance for the regulation of sialic acid dependent cell adhesion phenomena is discussed.

Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.

Although the tRNA species directing selenocysteine insertion in prokaryotes differ greatly in their primary structure from that of their eukaryotic homologues they share very similar three-dimensional structures. To analyse whether this conservation of the overall shape of the molecules reflects a conservation of their functional interactions it was tested whether the selenocysteine inserting tRNA species from Homo sapiens supports selenoprotein synthesis in E. coli. It was found that the expression of the human tRNA(Sec) gene in E.coli can complement a lesion in the tRNA(Sec) gene of this organism. Transcripts of the Homo sapiens and Xenopus laevis tRNA(Sec) genes synthesised in vitro were amino-acylated by the E.coli seryl-tRNA ligase although at a very low rate and the resulting seryl-tRNA(Sec) was bound to and converted into selenocysteyl-tRNA(Sec) by the selenocysteine synthase of this organism. Selenocysteyl-tRNA(Sec) from both eukaryotes was able to form a complex with translation factor SELB from E.coli. Although the mechanism of selenocysteine incorporation into seleno-proteins appears to be rather different in E.coli and in vertebrates, we observe here a surprising conservation of functions over an enormous evolutionary distance.

Selenocysteine tRNA [tRNA(Ser)Sec] has been shown to be serylated by tRNA(Ser) synthetase. The serine moiety of seryl-tRNA(Ser)Sec in vertebrates is further phosphorylated by a kinase, in addition to being converted into selenocysteine. Using site-directed mutagenesis we have introduced a number of mutations into T7 RNA polymerase transcripts of human tRNA(Ser)Sec. Our results show that most of the unique structural features of tRNA(Ser)(Sec), like the 5'-triphosphate, the 9 bp long acceptor stem and the anticodon, are not identity elements for phosphorylation of human seryl-tRNA(Ser)Sec. However, the length and secondary structure of the D-stem (6 bp in contrast with 4 bp in the canonical serine tRNA) of human tRNA(Ser)Sec, but not its sequence, are the major identity determinants which discriminate this tRNA from common tRNA(Ser) and identify it as the substrate for phosphorylation by seryl-tRNA(Ser)Sec kinase. This notion is confirmed by the fact that normal seryl-tRNA(Ser), which is not a substrate for serine phosphorylation, becomes a substrate if two additional base pairs are introduced into its D-stem.

Selenocysteine tRNA [tRNA((Ser)Sec)] is charged with serine by the same seryl-tRNA synthetase (SerRS) as the canonical serine tRNAs. Using site-directed mutagenesis, we have introduced a series of mutations into human tRNA((Ser)Sec) and tRNA(Ser) in order to study the identity elements of tRNA((Ser)Sec) for serylation and the effect of the orientation of the extra arm. Our results show that the long extra arm is one of the major identity elements for both tRNA(Ser) and tRNA((Ser)Sec) and gel retardation assays reveal that it appears to be a prerequisite for binding to the cognate synthetase. The long extra arm functions in an orientation-dependent, but not in a sequence-specific manner. The discriminator base G73 is another important identity element of tRNA((Ser)Sec), whereas the T- and D-arms play a minor role for the serylation efficiency.

It has been reported that selenocysteine tRNA from bovine liver is completely edited to two isoacceptor species, called tRNA([Ser]SecNCA) and tRNA([Ser]SecCmCa), which differ from the gene sequence. We used direct tRNA sequencing, mobility shift analyses, primer extension, restriction enzyme digestion and single strand conformational polymorphism (SSCP) analyses of products from reverse transcription coupled with polymerase chain reaction (RT/PCR), sequencing of RT/PCR products and HPLC-coupled mass spectrometry to reproduce this result and show here that editing of these tRNAs does not occur.