Patients with IgE antibodies against the carbohydrate epitope galactose-α-1,3-galactose (α-Gal) have reported severe allergic reactions after consumption of red meat. Investigations have revealed associations between IgE to α-Gal and tick bites. We provide the first direct evidence that α-Gal is present within ticks thus potentially explaining the relationship between tick exposure and sensitization to α-Gal, with development of red meat allergy as a secondary phenomena. Serum from Swedish patients with delayed severe reactions to red meat was included in the study. A dose-dependent inhibition of IgE responses to α-Gal by the tick Ixodes ricinus is demonstrated. Furthermore, using cryostat-cut sections of I. ricinus, we show that both a monoclonal and a polyclonal antibody against α-Gal stains the gastrointestinal tract of the tick. The same pattern is seen when staining with patient sera IgE positive to α-Gal. These results confirm that the α-Gal epitope is present in I. ricinus and imply host exposure to α-Gal during a tick bite. This provides further evidence that tick bites are associated with IgE responses to α-Gal and red meat allergy.

The binding of L-selectin to the HEV-derived ligand GlyCAM-1 bears a strict requirement for oligosaccharide sulfation. In the companion study [Hemmerich, S., Bertozzi, C.R., Leffler, H., & Rosen, S. D. (1994) Biochemistry 33, 4820-4829], we identified the major sulfated mono- and disaccharides of GlyCAM-1 as Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Gal beta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. Sialic acid and fucose are also critical to the recognition determinants on GlyCAM-1. However, the hydrolysis conditions employed in the previous study resulted in cleavage of these moieties, precluding their positional assignment. Here, we employ lectins of defined specificity in conjunction with specific exoglycosidases to identify a major GlyCAM-1 capping structure that includes all three critical elements. The complementary reactivity of Maackia amurensis agglutinin with fully sialylated, undersulfated GlyCAM-1 and Sambucus nigra agglutinin/Trichosanthes japonica agglutinin with desialylated but normally sulfated GlyCAM-1 indicates the presence of terminal 6'-sulfated sialyllactosamine. alpha (1-->3/4)Fucosidase removes fucose almost quantitatively from asialo-GlyCAM-1 while substantially enhancing its binding to Lycopersican esculentum agglutinin (specific for beta 1-->4-linked GlcNAc), indicating the presence of Fuc in an alpha 1-->3 linkage to GlcNAc. The strict requirement for desialylation to achieve defucosylation indicates a proximal location of Fuc with respect to terminal sialic acid. The nature of the capping group was further defined by studying the effects of sulfation, sialylation, and fucosylation on the ability of exo-beta(1-->4)galactosidase to release [3H]Gal from GlyCAM-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzyme deficiency leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal alpha-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human alpha-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, alpha-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20-35% of that of the normal mice. The transduced animals continued to show higher alpha-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or alpha-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Previously we showed that a large number of endothelial cells in vein grafts undergo apoptosis or necrosis during the first few days followed by endothelial regeneration. In the present study, we investigated endothelial cell death and regeneration in vein grafts using transgenic mice carrying LacZ genes driven by an endothelial TIE2 promoter. When a vein fragment from TIE2-LacZ was isografted into the carotid artery of wild-type mice, the number of beta-gal+ cells were reduced at 3 days and disappeared completely by 4 weeks after grafting. Conversely, beta-gal+ cells were observed on the surface of vein segments donated by wild-type mice isografted into TIE2-LacZ mice at 1 week and reached confluence by 4 weeks, suggesting recipient origins of endothelial cells. Interestingly, beta-gal+ cells were evenly distributed on the surface of the whole vein segment grafted into TIE2-LacZ mice, indicating a contribution of circulating progenitor cells. When wild-type veins were grafted into a chimeric mouse carrying TIE2-LacZ genes in bone marrow cells, a proportion of cells displayed a beta-gal+ staining. Furthermore, the number of CD34+ and Flk+ progenitor cells in blood of apoE-deficient mice were significantly lower than those of wild-type controls, which coincided with diminished beta-gal+ endothelial cells on the surface of vein grafts in TIE2-LacZ/apoE-/- mice. Thus, we provide the first evidence that endothelial cells of vein grafts are derived from circulating progenitor cells, of which one-third are derived from bone marrow progenitor cells. Hyperlipidemia due to apoE deficiency results in a lower number of endothelial progenitors in blood and correlated with enhanced atherosclerosis. The full text of this article is available online at http://www.circresaha.org.

The UDP-Glc:glycoprotein glucosyltransferase was purified to homogeneity from the fission yeast Schizosaccharomyces pombe. The enzyme has been recently suggested to be involved in the mechanism by which unfolded, partially folded, or misfolded glycoproteins are retained in the endoplasmic reticulum. The pure yeast glucosyltransferase formed protein-linked Glc1-Man9GlcNAc2,Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 when incubated with UDP-Glc and denatured thyroglobulin. The same compounds were formed upon glucosylation of endogenous acceptors by crude microsomes. The enzyme was a soluble microsomal protein that required Ca2+ for activity, used UDP-Glc and not TDP-Glc, ADP-Glc, or UDP-Gal as sugar donor, had an almost neutral optimum pH value, and as the glucosyl-transferase obtained from rat liver, glucosylated denatured but not native glycoproteins or glycopeptides. A similar enzymatic activity could not be detected in Saccharomyces cerevisiae microsomes and transient glucosylation of glycoproteins (addition of a single glucose unit to glucose-free oligosaccharides by the glucosyltransferase followed by its removal by glucosidase II) could not be detected in intact S. cerevisiae cells. These are the only eukaryotic cells described so far in which these processing reactions of the endoplasmic reticulum do not occur. Availability of the pure S. pombe enzyme will eventually allow testing the possible involvement of the glucosyltransferase in sensing glycoprotein tertiary structures in the endoplasmic reticulum.

Transgenic pigs are promising donor organisms for xenotransplantation as they share many anatomical and physiological characteristics with humans. The most profound barrier to pig-to-primate xenotransplantation is the rejection of the grafted organ by a cascade of immune mechanisms commonly referred to as hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), immune cell-mediated rejection, and chronic rejection. Various strategies for the genetic modification of pigs facilitate tailoring them to be donors for organ transplantation. Genetically modified pigs lacking alpha-1,3-Gal epitopes, the major xenoantigens triggering HAR of pig-to-primate xenografts, are considered to be the basis for further genetic modifications that can address other rejection mechanisms and incompatibilities between the porcine and primate blood coagulation systems. These modifications include expression of human complement regulatory proteins, CD39, endothelial protein C receptor, heme oxygenase 1, thrombomodulin, tissue factor pathway inhibitor as well as modulators of the cellular immune system such as human TNF alpha-related apoptosis inducing ligand, HLA-E/beta-2-microglobulin, and CTLA-4Ig. In addition, transgenic strategies have been developed to reduce the potential risk of infections by endogenous porcine retroviruses. The protective efficacy of all these strategies is strictly dependent on a sufficiently high expression level of the respective factors with the required spatial distribution. This review provides an overview of the transgenic approaches that have been used to generate donor pigs for xenotransplantation, as well as their biological effects in in vitro tests and in preclinical transplantation studies. A future challenge will be to combine the most important and efficient genetic modifications in multi-transgenic pigs for clinical xenotransplantation.

It is now well recognized that there are several groups of cholinergic neurons in the basal forebrain with direct projections to various cortical regions. Immunohistochemical investigations of the distribution of the neuropeptide galanin (GAL) have shown that two of these brain areas, the medial septum and diagonal band, contained large numbers of GAL-immunoreactive neurons. In the present study, double staining techniques using antibodies raised against choline acetyltransferase (ChAT) revealed that GAL- and ChAT-like immunoreactivities are colocalized within a subpopulation of the cholinergic neurons within the medial septum and diagonal band. This colocalization of GAL- and ChAT-immunoreactivities was not seen to occur within other groups of forebrain cholinergic neurons. Immunohistochemistry carried out subsequent to injections of fluorescent retrograde tracers into the hippocampal formation revealed that both ChAT/GAL- and ChAT-containing neurons project to the hippocampal formation. The question of GAL as a modulator of cholinergic transmission in this projection is discussed.

Galactosyl beta-1,3-N-acetyl galactosamine (Gal beta-1,3-GalNAc) (Thomsen Friedenreich antigen), the Class I core sequence in O-linked oligosaccharide chains, behaves as an oncofetal antigen showing increased expression in many epithelial malignancies. Previous work has shown that peanut agglutinin (PNA), a lectin that binds Gal beta-1,3-GalNAc, stimulates proliferation in HT-29 (human colon cancer) cells and normal human colonic epithelium and this implies that cell surface glycoproteins which express Gal beta-1,3-GalNAc may play an important role in the regulation of epithelial cell proliferation. We have now studied the effect on epithelial cells of another dietary Gal beta-1,3-GalNAc-binding lectin, the edible mushroom Agaricus bisporus lectin (ABL). This differs from PNA in its ability to bind also to sialylated Gal beta-1,3-GalNAc. In contrast to PNA, ABL (25 micrograms/ml) inhibited incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87% (95% confidence limit, 85-89%), Caco-2 colon cancer cells by 16% (95% confidence limit, 12-20%), MCF-7 breast cancer cells by 50% (95% confidence limit, 47-52%), and Rama-27 rat mammary fibroblasts by 55% (95% confidence limit, 51-60%) when these cells were grown for 24 h in serum-free medium. When assessed by cell count, similar inhibition of proliferation of HT29 cells by ABL was found. In the presence of 2% fetal calf serum (which contains the ABL-binding glycoprotein fetuin), the inhibitory effect of ABL on cell proliferation was still demonstrable but at increased ABL concentration (60 micrograms/ml for 49% inhibition). Ten micrograms/ml ABL completely abolished the stimulatory effect on [3H]thymidine incorporation of epidermal growth factor (100 pg/ml) and PNA (25 micrograms/ml) and markedly inhibited the stimulatory effect of insulin (50 ng/ml). ABL (0.2 mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50 micrograms/ml ABL was reversible after removal of the lectin. Binding studies with 125I-labeled ABL suggested a single class of binding site with an apparent Kd value of (4.12 +/- 0.29) x 10(-7) M with (3.6 +/- 0.3) x 10(7) binding sites/cell. A. bisporus lectin is a reversible noncytotoxic inhibitor of epithelial cell proliferation which deserves study as a potential agent for cancer therapy.

This paper describes a hypothesis that attempts to account for how changes in noradrenergic systems in the brain can affect depression-related behaviors and symptoms. It is hypothesized that increased activity of the locus coeruleus (LC) neurons, the principal norepinephrine (NE)-containing cells in the brain, causes release of galanin (GAL) in the ventral tegmentum (VTA) from LC axon terminals in which GAL is colocalized with NE. It is proposed that GAL release in VTA inhibits the activity of dopaminergic cell bodies in this region whose axons project to forebrain, thereby resulting in two of the principal symptoms seen in depression, decreased motor activation and decreased appreciation of pleasurable stimuli (anhedonia). The genesis of this hypothesis, which derives from studies using an animal model of depression, is described as well as recent data consistent with the hypothesis. The formulation proposed suggests that GAL antagonists may be of therapeutic benefit in the treatment of depression.

Adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus, is gaining attention as a vector for its potential use in human gene therapy. However, few studies have examined the safety and the efficacy of this vector system in vivo. We report here that recombinant AAV vectors, when directly injected intravenously in mice, accumulated predominantly in liver cells, suggesting that AAV may possess in vivo organ-tropism for liver. The transduced lacZ reporter gene was expressed in hepatocytes in the liver and, at the level examined, did not appear to induce any detectable cytotoxic T lymphocyte response against beta Gal. AAV-mediated transduction of murine hematopoietic progenitor cells ex vivo followed by transplantation into lethally irradiated syngeneic mice also revealed high-efficiency gene transfer into progeny cells without any observable cytotoxicity or deleterious effect. The transduced reporter gene sequences were also expressed in mice in vivo. The AAV-based vectors may thus prove useful as a potentially safe alternative to the more commonly used retrovirus- and adenovirus-based vector systems.

Three model systems were used to demonstrate the immunogenicity of highly attenuated and replication-defective recombinant MVA. (1) Intramuscular inoculation of MVA-IN-Fha/np induced humoral and cell-mediated immune responses in mice and protectively immunized them against a lethal respiratory challenge with influenza virus. Intranasal vaccination was also protective, although higher doses were needed. (2) In rhesus macaques, an immunization scheme involving intramuscular injections of MVA-SIVenv/gag/pol greatly reduced the severity of disease caused by an SIV challenge. (3) In a murine cancer model, immunization with MVA-beta gal prevented the establishment of tumor metastases and even prolonged life in animals with established tumors. These results, together with previous data on the safety of MVA in humans, suggest the potential usefulness of recombinant MVA for prophylactic vaccination and therapeutic treatment of infectious diseases and cancer.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. beta-Galactosidase (beta-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E 9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5-15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

Double strand breaks (DSBs) can cause damage to the genomic integrity of a cell as well as initiate genetic recombination processes. The HO and I-SceI endonucleases from budding yeast have provided a way to study these events by inducing a unique DSB in vivo under the control of a galactose-inducible promoter. The GAL::HO construct has been used extensively to study processes such as nonhomologous end joining, intra- and interchromosomal gene conversion, single strand annealing and break-induced recombination. Synchronously induced DSBs have also been important in the study of the DNA damage checkpoint, adaptation, and recovery pathways of yeast. This chapter describes methods of using GAL::HO to physically monitor the progression of events following a DSB, specifically the events leading to the switching of mating type by gene conversion of MAT using the silent donors at HML and HMR. Southern blot analysis can be used to follow the overall events in this process such as the formation of the DSB and product. Denaturing alkaline gels and slot blot techniques can be employed to follow the 5' to 3' resection of DNA starting at the DSB. After resection, the 3' tail initiates a homology search and then strand invades its homologous sequence at the donor cassette. Polymerase chain reaction is an important means to assay strand invasion and the priming of new DNA synthesis as well as the completion of gene conversion. Methods such as chromatin immunoprecipitation have provided a means to study many proteins that associate with a DSB, including not only recombination proteins, but also proteins involved in nonhomologous end joining, cell cycle arrest, chromatin remodeling, cohesin function, and mismatch repair.

We report here the discovery of 137 previously unappreciated genes in yeast through a widely applicable and highly scalable approach integrating methods of gene-trapping, microarray-based expression analysis, and genome-wide homology searching. Our approach is a multistep process in which expressed sequences are first trapped using a modified transposon that produces protein fusions to beta-galactosidase (beta-gal); non-annotated open reading frames (ORFs) translated as beta-gal chimeras are selected as a candidate pool of potential genes. To verify expression of these sequences, labeled RNA is hybridized against a microarray of oligonucleotides designed to detect gene transcripts in a strand-specific manner. In complement to this experimental method, novel genes are also identified in silico by homology to previously annotated proteins. As these methods are capable of identifying both short ORFs and antisense ORFs, our approach provides an effective supplement to current gene-finding schemes. In total, the genes discovered using this approach constitute 2% of the yeast genome and represent a wealth of overlooked biology.

Replacement of the CRP-binding site of the gal control region by curved sequences can lead to the restoration of promoter strength in vivo. One curved sequence called 5A6A, however, failed to do so. The gene hns exerts a strong negative control on the resulting 5A6A gal promoter as well as on the distant bla promoter, specifically in a 5A6A gal context. The product of this gene, H-NS, displays a better affinity for this particular insert compared to other curved sequences. Mechanisms by which H-NS may repress promoters both at short and long distances from a favoured binding site are discussed.

The cell wall of Mycobacterium tuberculosis and related genera is unique among prokaryotes, consisting of a covalently bound complex of mycolic acids, D-arabinan and D-galactan, which is linked to peptidoglycan via a special linkage unit consisting of Rhap-(1-->3)-GlcNAc-P. Information concerning the biosynthesis of this entire polymer is now emerging with the promise of new drug targets against tuberculosis. Accordingly, we have developed a galactosyltransferase assay that utilizes the disaccharide neoglycolipid acceptors beta-d-Galf-(1-->5)-beta-D-Galf-O-C(10:1) and beta-D-Galf-(1-->6)-beta-D-Galf-O-C(10:1), with UDP-Gal in conjunction with isolated membranes. Chemical analysis of the subsequent reaction products established that the enzymatically synthesized products contained both beta-D-Galf linkages ((1-->5) and (1-->6)) found within the mycobacterial cell, as well as in an alternating (1-->5) and (1-->6) fashion consistent with the established structure of the cell wall. Furthermore, through a detailed examination of the M. tuberculosis genome, we have shown that the gene product of Rv3808c, now termed glfT, is a novel UDP-galactofuranosyltransferase. This enzyme possesses dual functionality in performing both (1-->5) and (1-->6) galactofuranosyltransferase reactions with the above neoglycolipid acceptors, using membranes isolated from the heterologous host Escherichia coli expressing Rv3808c. Thus, at a biochemical and genetic level, the polymerization of the galactan region of the mycolyl-arabinogalactan complex has been defined, allowing the possibility of further studies toward substrate recognition and catalysis and assay development. Ultimately, this may also lead to a more rational approach to drug design to be explored in the context of mycobacterial infections.

The mechanism whereby lovastatin can counteract steroid-induced osteonecrosis and osteoporosis is poorly understood. We assessed the effect of lovastatin on a multipotential cell line, D1, which is capable of differentiating into either the osteoblast or the adipocyte lineage. The expression of bone cell and fat cell transcription factors Cbfa1/Runx2 and PPARgamma2, respectively, were determined. 422aP2 gene expression was analyzed. Osteocalcin promoter activity was measured by cotransfecting the cells with the phOC-luc and pSV beta-Gal plasmids. Lovastatin enhanced osteoblast differentiation as assessed by a 1.8x increase in expression of Cbfa1/Runx2 and by a 5x increase in osteocalcin promoter activity. Expression of PPARgamma2 was decreased by 60%. By enhancing osteoblast gene expression and by inhibiting adipogenesis, lovastatin may shunt uncommitted osteoprogenitor cells in marrow from the adipocytic to the osteoblastic differentiation pathway. Future evaluation of lovastatin and other lipid-lowering drugs will help determine their potential as therapeutic agents for osteonecrosis and osteoporosis.

Recent studies have suggested that the signal peptide-less cytokine, interleukin (IL)-1 alpha, may play a role as an intracellular regulator of human endothelial cell proliferation in vitro (Garfinkel, S., Haines, D. S., Brown, S., Wessendorf, J., Gillespie, D. H., and Maciag, T. (1992) J. Biol. Chem. 267, 24375-24378). In order to determine the intracellular locale of the IL-1 alpha precursor, we fused the open reading frame of the IL-1 alpha precursor to the reporter gene beta-galactosidase (Gal) and studied the cellular distribution of the chimera in NIH 3T3 cells after transfection. Immunological and enzymatic analysis demonstrated that the IL-1 alpha:beta-Gal fusion protein was associated with the nucleus. To further define the region responsible for this activity, we ligated the mature form of IL-1 alpha (IL-1 alpha 113-271) and the IL-1 alpha precursor domain (IL-1 alpha 1-112) to beta-Gal. Analysis of the intracellular distribution of these chimeric polypeptides following transfection demonstrated a differential distribution of IL-1 alpha 1-112:beta-Gal in the nucleus and IL-1 alpha 113-271:beta-Gal in the cytosol. Because the IL-1 alpha precursor domain contains a sequence that resembles a nuclear translocation signal (KVLKKRRL, residues 79-86), we prepared an IL-1 alpha precursor point mutant in which Lys82 was replaced by Glu. Transfection of NIH 3T3 cells with the IL-1 alpha precursor point mutant (IL-1 alpha 1-271 Glu82:beta-Gal) resulted in a significant reduction in the ability of the IL-1 alpha precursor to associate with the nucleus and similar data were obtained as a result of Lys82 mutagenesis in the IL-1 alpha precursor domain (IL-1 alpha 1-112 Glu82:beta-Gal). These data suggest that the IL-1 alpha precursor contains a functional nuclear localization sequence within the structure of the precursor domain and Lys82 is critical for its function.

The study of gene regulation in many organisms has been facilitated by the development of reporter genes. The authors report the use of lacZ from Streptococcus thermophilus, a gene encoding a beta-galactosidase, as a reporter for the fungal pathogen Candida albicans. As test cases, Strep. thermophilus lacZ was placed under control of three different C. albicans promoters: MAL2 (maltase), inducible by maltose; HWP1 (hyphal cell wall protein), induced by conditions that promote filamentous growth; and ACT1 (actin). These constructs were each integrated into the C. albicans genome and beta-galactosidase activity was readily detected from these strains, but only under the appropriate growth conditions. Beta-galactosidase activity could be detected by several methods: quantitative liquid assays using permeabilized cells, colorimetric assays of colonies replicated to paper filters, and in situ coloration of colonies growing on medium containing the indicator X-Gal. These results show the usefulness of STREP: thermophilus lacZ as a monitor of gene regulation in this medically important yeast.

Yah1p, an [Fe 2S 2]-containing ferredoxin located in the matrix of Saccharomyces cerevisiae mitochondria, functions in the synthesis of Fe/S clusters and heme a prosthetic groups. EPR, Mossbauer spectroscopy, and electron microscopy were used to characterize the Fe that accumulates in Yah1p-depleted isolated intact mitochondria. Gal- YAH1 cells were grown in standard rich media (YPD and YPGal) under O 2 or argon atmospheres. Mitochondria were isolated anaerobically, then prepared in the as-isolated redox state, the dithionite-treated state, and the O 2-treated state. The absence of strong EPR signals from Fe/S clusters when Yah1p was depleted confirms that Yah1p is required in Fe/S cluster assembly. Yah1p-depleted mitochondria, grown with O 2 bubbling through the media, accumulated excess Fe (up to 10 mM) that was present as 2-4 nm diameter ferric nanoparticles, similar to those observed in mitochondria from yfh1Delta cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe (3+) ions of the nanoparticles to become reduced and largely exported from the mitochondria. Fe did not accumulate in mitochondria isolated from cells grown under Ar; a significant portion of the Fe in these organelles was in the high-spin Fe (2+) state. This suggests that the O 2 used during growth of Gal- YAH1 cells is responsible, either directly or indirectly, for Fe accumulation and for oxidizing Fe (2+) ->> Fe (3+) prior to aggregation. Models are proposed in which the accumulation of ferric nanoparticles is caused either by the absence of a ligand that prevents such precipitation in wild-type mitochondria or by a more oxidizing environment within the mitochondria of Yah1p-depleted cells exposed to O 2. The efficacy of reducing accumulated Fe along with chelating it should be considered as a strategy for its removal in diseases involving such accumulations.

Anti-Gal is a human natural antibody which interacts specifically with the mammalian carbohydrate structure Gal alpha 1-3Gal beta 1-4GlcNAc-R, termed, the alpha-galactosyl epitope. This antibody constitutes approximately 1% of circulating IgG in human serum and is produced, upon stimulation, by 1% of circulating B lymphocytes. Anti-Gal is also present as IgA antibodies in body secretions such as saliva, milk and colostrum. The antigenic source for the constant production of anti-Gal seems to be the alpha-galactosyl-like epitopes found on many bacteria of the gastrointestinal flora. Whereas anti-Gal is abundant in humans, apes and Old World monkeys, it is absent from New World monkeys, prosimians and nonprimate mammals. The latter group of species produces, however, large amounts of alpha-galactosyl epitopes >> 10(6) epitopes per cell). It is estimated that anti-Gal appeared in ancestral Old World primates less than 28 million years ago, possibly as a result of an evolutionary event which exerted a selective pressure for the suppression of alpha-galactosyl epitopes expression by inactivation of the gene for the enzyme alpha 1,3 galactosyltransferase. This also resulted in the loss of immune tolerance to the alpha-galactosyl epitope and the production of anti-Gal. The physiologic role of this antibody is not clear as yet. It may participate in the protection against gastrointestinal bacteria. In addition it seems to contribute to the removal of normal and pathologically senescent red cells by interacting with the few hundred cryptic alpha-galactosyl epitopes which are exposed de novo in the course of red cell aging, thereby opsonizing these cells for phagocytosis by reticuloendothelial macrophages. The alpha-galactosyl epitope has been found to be aberrantly expressed on human cells and the interaction of anti-Gal with such epitopes may result in autoimmune disease. Preliminary data suggest such a mechanism in Graves' disease. Anti-Gal has been found to interact with therapeutic recombinant proteins expressing alpha-galactosyl epitopes, but so far there is no indication that it affects the half-life in the circulation and the biologic activity. Detection of anti-Gal in the seminal fluid and in the cerebrospinal fluid may serve as a simple means for assessment of damage to the blood-genital tract barrier or the blood-brain barrier. Studies on the interaction of anti-Gal with aberrantly expressed alpha-galactosyl epitopes on human cells may elucidate the possible role of anti-Gal in human autoimmune diseases.

The binding of Artocarpus integrifolia lectin to N-dansylgalactosamine (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) leads to a 100% increase in dansyl fluorescence with a concomitant blue shift in the emission maximum by 10 nm. This binding is carbohydrate-specific and has an association constant of 1.74 X 10(4) M-1 at 20 degrees C. The lectin has two binding sites for N-dansylgalactosamine. The values of -delta H and -delta S for the binding of N-dansylgalactosamine are in the range of values reported for several lectin-monosaccharide interactions, indicating an absence of nonpolar interaction of the dansyl moiety of the sugar with the combining region of the protein. Dissociation of the bound N-dansylgalactosamine from its complex with the lectin and the consequent change in its fluorescence on addition of nonfluorescent sugars allowed evaluation of the association constant for competing ligands. The thermodynamic parameters for the binding of monosaccharides suggest that the OH groups at C-2, C-3, C-4, and C-6 in the D-galactose configuration are important loci for interaction with the lectin. The acetamido group at C-2 of 2-acetamido-2-deoxygalactopyranose and a methoxyl group at C-1 of methyl-alpha-D-galactopyranoside are presumably also involved in binding through nonpolar and van der Waals' interactions. The T-antigenic disaccharide Gal beta 1----3GalNAc binds very strongly to the lectin when compared with methyl-beta-D-galactopyranoside, the beta(1----3)-linked disaccharides such as Gal beta 1----3GlcNAc, and the beta(1----4)-linked disaccharides, N-acetyllactosamine and lactose. The major stabilizing force for the avid binding of T-antigenic disaccharide appears to be a favorable enthalpic contribution. The combining site of the lectin is, therefore, extended. These data taken together suggest that the Artocarpus lectin is specific toward the Thomsen-Friedenreich (T) antigen. There are subtle differences in the overall topography of its combining site when compared with that of peanut (Arachis hypogaea) agglutinin. The results of stopped flow spectrometry for the binding of N-dansylgalactosamine tot he Artocarpus lectin are consistent with a simple single-step bimolecular association and unimolecular dissociation rate processes. The value of K+1 and K-1 at 21 degrees C are 8.1 X 10(5) M-1 s-1 and 50 s-1, respectively. The activation parameters indicate an enthalpy-controlled association process.

Two monoclonal antibodies directed to the Type II carbohydrate chain of glycosphingolipids have been prepared by the murine hybridoma technique. Their reactivity was determined by liposome lysis, plate-binding assay, complement fixation, and hemagglutination inhibition. One antibody was specific for glycolipids having the nonreducing terminal N-acetyllactosamine (Gal beta 1 leads to 4GlcNAc beta 1 leads to R) structure and did not react with glycolipids having either a Type I chain (Gal beta 1 leads to 3GlcNAc beta 1 leads to R), a ganglio-series structure (Gal beta 1 leads to 3GalNAc beta 1 leads to R), or a sialosyl or fucosyl substitution at the N-acetyllactosamine residue. This antibody readily agglutinated adult human erythrocytes of all ABO types but did not agglutinate umbilical cord cells. The other antibody reacted with the Type II chain H structure (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to R) but reacted only weakly with the Type I chain H (Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc beta 1 leads to R) and ganglio-series H (Fuc alpha 1 leads to 2Gal beta 1 leads to 3GalNAc beta 1 leads to R) structures. At 37 degrees C this antibody agglutinated human erythrocytes of types O and A2 but was unable to cause detectable agglutination of types B and A1. Because of their narrow, well defined specificity, these monoclonal anticarbohydrate reagents will be useful in the study of the distribution, quantity, and function of specific carbohydrates on the cell surface.

Previous studies showed that nonsense mutations in either of two genes (capR or capS) or an undefined mutation in a third gene (capT) led to pleiotropic effects: (i) increased capsular polysaccharide synthesis (mucoid phenotype); (ii) increased synthesis of enzymes specified by at least four spatially separated operons involved in synthesis of capsular polysaccharide including the product of the galE gene, UDP-galactose-4-epimerase (EC 5.1.3.2) in capR mutants. The present study demonstrated that the entire galactose (gal) operon (galE, galT, and galK) is derepressed by mutations in either the capR or the capT genes, but not by mutation in capS. Double mutants (capR9 capT) were no more derepressed than the capR9 mutant, indicating that capR9 and capT regulate the gal operon via a common pathway. Isogenic double mutants containing either galR(+), galR(-), galR(s), or galO(c) in combination with either capR(+) or capR9 were prepared and analyzed for enzymes of the gal operon. The results demonstrated that capR9 caused derepression as compared to capR(+) in all of the combinations. Strains with a galR(s) mutation are not induced, for the gal operon, by any galactose compound including d-fucose, and this was confirmed in the present study using d-fucose. Nevertheless, the derepression of galR(s) capR9 compared to galR(s) capR(+) was four- to sixfold. The same derepression was observed when galR(+)capR9 was compared to galR(+)capR(+). The data eliminate the explanation that internal induction of the gal operon by a galactose derivative was causing increased gal operon enzyme synthesis in capR or capT mutants. Furthermore, the same data suggest that the galR and capR genes are acting independently to derepress the gal operon. A modified model for the structure of the gal operon is proposed to explain these results. The new feature of the model is that two operator sites are suggested, one to combine with the galR repressor and one to combine with the capR repressor.