Interplay between the genetic clades of Micromonas and their viruses in the Western English Channel.

The genus Micromonas comprises distinct genetic clades that commonly dominate eukaryotic phytoplankton community from polar to tropical waters. This phytoplankter is also recurrently infected by abundant and genetically diverse prasinoviruses. Here we report on the interplay between prasinoviruses and Micromonas with regard to the genetic diversity of this host. For 1 year, we monitored the abundance of three clades of Micromonas and their viruses in the Western English Channel, both in the environment using clade-specific probes and flow cytometry, and in the laboratory using clonal strains of Micromonas clades to assay for their viruses by plaque-forming units. We showed that the seasonal fluctuations of Micromonas clades were closely mirrored by the abundance of their corresponding viruses, indicating that the members of Micromonas genus are susceptible to viral infection, regardless of their genetic affiliation. The characterization of 45 viral isolates revealed that Micromonas clades are attacked by specific virus populations, which exhibit distinctive clade specificity, life strategies and genetic diversity. However, some viruses can also cross-infect different host clades, suggesting a mechanism of horizontal gene transfer within the Micromonas genus. This study provides novel insights into the impact of viral infection for the ecology and evolution of the prominent phytoplankter Micromonas.

Allergic enterocolitis presenting as recurrent necrotizing enterocolitis in preterm neonates.

An uncommon clinical entity mimicking necrotizing enterocolitis (NEC) is allergic enterocolitis secondary to cow's milk protein allergy. Although milk protein allergy is the most common food allergy among infants and young children, the incidence and prevalence of this disease entity presenting as enterocolitis in neonates is not well documented. We report this case of milk protein-associated allergic enterocolitis to highlight the unusual recurrent presentation as NEC, (with recurrent pneumatosis, bloody stools) managed successfully with modification of milk formula.

Rapid quantification of the toxic alga Prymnesium parvum in natural samples by use of a specific monoclonal antibody and solid-phase cytometry.

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.

The first green lineage cdc25 dual-specificity phosphatase.

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G(2)/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G(2)/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been coevolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages.

Role of nuclear WW domains and proline-rich proteins in dinoflagellate transcription.

Dinoflagellates are unique among eukaryotes in their lack of histones and nucleosomes, and permanently condensed chromosomes. These unusual features raise questions as how chromatin condensation and gene expression are achieved. In this study, we investigated nuclear proteins potentially implicated in the regulation of the transcription. Dinap1 is a dinoflagellate nuclear protein that has a WW domain and is synthesized mainly in G1 and S phases of the cell cycle. In this study, we found that Dip1, a proline-rich potential ligand of Dinap1, and DapC, a Dip1 potential ligand, were both present in the nucleus of Crypthecodinium cohnii during the G1 phase. Dip1 contained a PPXY motif, and its domain organization was similar to that of the splicing factor FBP21 in that it possessed one zinc finger and two WW domains. Although DapC has no known homolog, 22 repeats of a PPXPXGX heptapeptide were identified at the N-terminus, and this structure is similar to that of the C-terminal part of the mouse splicing factor SAP62. Dinap1 was co-precipitated with Dip1 and DapC in vitro and in vivo, but despite their nuclear location, these three proteins did not bind directly to DNA. Dinap1 activated up to 40% of the basal transcription activity of C. cohnii in an in vitro assay, whereas DapC inhibited it by 40% and Dip1 had no effect. These dinoflagellate proteins appear to be the subunits of a nuclear complex that may be involved in regulating transcription.

Polyethylene glycol-derivatized cysteine-substitution variants of recombinant staphylokinase for single-bolus treatment of acute myocardial infarction.

BACKGROUND: Thrombolytic therapy of acute myocardial infarction (AMI) is evolving toward bolus administration. Derivatization of proteins with polyethylene glycol (PEG) may reduce their clearance. METHODS AND RESULTS: A staphylokinase (SakSTAR) variant with 12 amino acid substitutions to reduce its antigenicity, SakSTAR (K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R), and with Ser in position 3 mutated into Cys (code SY161), was derivatized with maleimide-PEG with M:(r) of 5,000 (P5), 10,000 (P10), or 20,000 (P20). The PEGylated variants recognized only one third of the antibodies elicited with wild-type SakSTAR in AMI patients. In experimental animals, plasma clearances were reduced 2. 5- to 5-fold with P5, 5- to 20-fold with P10, and 20-fold with P20, and bolus injection induced pulmonary plasma clot lysis at doses inversely related to their clearance. Intravenous bolus injection of 5 mg of the P5, P10, or P20 variants in AMI patients was associated with plasma half-lives (t(1/2alpha)) of 13, 30, and 120 minutes and clearances of 75, 43, and 8 mL/min, respectively, compared with 3 minutes and 360 mL/min for SakSTAR. Injection of 5 mg P5 variant restored TIMI-3 flow within 60 minutes in 14 of 18 AMI patients (78%, 95% CI 55% to 91%) and of 2.5 mg in 7 of 11 patients (63%, 95% CI 35% to 85%), both in the absence of fibrinogen degradation. The immunogenicity of the variants was significantly (P:<0.002) reduced. CONCLUSIONS: The staphylokinase variant SY161-P5, derivatized with one linear polyethylene glycol molecule of M:(r) 5000, is a promising fibrin-selective agent for single-bolus coronary thrombolysis.

Morphology and behaviour of dinoflagellate chromosomes during the cell cycle and mitosis.

The morphology and behaviour of the chromosomes of dinoflagellates during the cell cycle appear to be unique among eukaryotes. We used synchronized and aphidicolin-blocked cultures of the dinoflagellate Crypthecodinium cohnii to describe the successive morphological changes that chromosomes undergo during the cell cycle. The chromosomes in early G(1) phase appeared to be loosely condensed with numerous structures protruding toward the nucleoplasm. They condensed in late G(1), before unwinding in S phase. The chromosomes in cells in G(2) phase were tightly condensed and had a double number of arches, as visualised by electron microscopy. During prophase, chromosomes elongated and split longitudinally, into characteristic V or Y shapes. We also used confocal microscopy to show a metaphase-like alignment of the chromosomes, which has never been described in dinoflagellates. The metaphase-like nucleus appeared flattened and enlarged, and continued to do so into anaphase. Chromosome segregation occurred via binding to the nuclear envelope surrounding the cytoplasmic channels and microtubule bundles. Our findings are summarized in a model of chromosome behaviour during the cell cycle.

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.

Cyclic expression of a nuclear protein in a dinoflagellate.

Nuclei of the dinoflagellate Crypthecodinium cohnii strain Whd were isolated and nuclear proteins were extracted in three fractions, corresponding to the increasing affinity of these proteins to genomic DNA. One fraction contained two major bands (48- and 46-kDa) and antibodies specific to this fraction revealed two major bands by Western blot on nuclear extracts, corresponding to the 46- and 48-kDa bands. The 48-kDa protein was detected in G1 phase but not in M phase cells. An expression cDNA library of C. cohnii was screened with these antibodies, and two different open reading frames were isolated. Dinoflagellate nuclear associated protein (Dinap1), one of these coding sequences, was produced in E. coli and appeared to correspond to the 48-kDa nuclear protein. No homologue of this sequence was found in the data bases, but two regions were identified, one including two putative zinc finger repeats, and one coding for two potential W/W domains. The second coding sequence showed a low similarity to non-specific sterol carrier proteins. Immunocytolocalization with specific polyclonal antibodies to recombinant Dinap1 showed that the nucleus was immunoreactive only during the G1 phase: the nucleoplasm was immunostained, while chromosome cores and nuclear envelopes were negative.

beta-D-glucosyl-hydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres.

The unusual DNA base beta-D-glucosyl-hydroxymethyluracil, called "J, " replaces approximately 0.5-1% of Thy in DNA of African trypanosomes but has not been found in other organisms thus far. In Trypanosoma brucei, J is located predominantly in repetitive DNA, and its presence correlates with the silencing of telomeric genes. Using antibodies specific for J, we have developed sensitive assays to screen for J in a range of organisms and have found that J is not limited to trypanosomes that undergo antigenic variation but is conserved among Kinetoplastida. In all kinetoplastids tested, including the human pathogens Leishmania donovani and Trypanosoma cruzi, J was found to be abundantly present in the (GGGTTA)n telomere repeats. Outside Kinetoplastida, J was found only in Diplonema, a small phagotrophic marine flagellate, in which we also identified 5-MeCyt. Fractionation of Diplonema DNA showed that the two modifications are present in a common genome compartment, which suggests that they may have a similar function. Dinoflagellates appear to contain small amounts of modified bases that may be analogs of J. The evolutionary conservation of J in kinetoplastid protozoans suggests that it has a general function, repression of transcription or recombination, or a combination of both. T. brucei may have recruited J for the control of genes involved in antigenic variation.

Cloning, characterization and chromosomal localization of a repeated sequence in Crypthecodinium cohnii, a marine dinoflagellate.

Genomic DNA of Crypthecodinium cohnii has been extracted in the presence of cetylmethylammonium bromide and hydrolysed by 13 restriction enzymes. No typical ladder-like pattern or isolated band of satellite sequences were found with any of these enzymes. A "mini" genomic DNA library had been made and screened by reverse hybridization to isolate highly repeated sequences. Seven such DNA fragments were sequenced. The copy number of one of them (Cc18), 226 bp long, was estimated at around 25,000, representing 0.06% of the total genome. Cc18 was found to be included in a higher fragment of 3.0 kb by Southern blot analysis after cleavage by PstI. This higher molecular weight fragment could be composed either of tandemly repeated Cc18 sequences, or by only one or a very low copy number of Cc18. In this latter case, these fragments, also repeated 25,000 times would represent 1 to 2% of the total genome. Genomic localization of Cc18 by in situ hybridization on squashed C. cohnii cells showed that it was widely distributed on the different chromosomes. All the chromosomes observed displayed Cc18 labeling, which appeared homogeneously distributed. The ability of Cc18 to be a specific molecular marker to distinguish sibling C. cohnii species is discussed.

Procoagulant properties of intravenous staphylokinase versus tissue-type plasminogen activator.

The fibrin-specificity and procoagulant effects of recombinant staphylokinase (Sak42D) were compared with those of recombinant tissue-type plasminogen activator (rt-PA) in patients with acute myocardial infarction. Plasma samples were obtained at baseline and at 25 and 90 min, from 24 patients who were randomly assigned to a double bolus (15 mg each, 30 min apart) administration of Sak42D or to accelerated weight-adjusted rt-PA (maximum of 100 mg over 90 min). Baseline levels of fibrinopeptide A (FPA), prothrombin fragment 1 + 2 and thrombin-antithrombin III complex (TAT) were comparable in the Sak42D and rt-PA groups (p > or = 0.6). In patients treated with Sak42D, plasma levels of FPA, prothrombin fragment 1 + 2 and TAT did not markedly increase during treatment (p = 0.06, p = 0.4 and p = 0.03, respectively). In contrast, during administration of rt-PA the levels of FPA, prothrombin fragment 1 + 2 and TAT increased significantly over baseline (p = 0.003, p < 0.0001 and p = 0.001, respectively). As a result, the levels of all three procoagulant parameters were significantly lower during treatment with Sak42D as compared to rt-PA. Thus, FPA levels in the Sak42D group (median values) were 40 ng/ml at 25 min and 11 ng/ml at 90 min, as compared to 88 ng/ml and 50 ng/ml in the rt-PA group (p = 0.0007 and p = 0.009, respectively). Prothrombin fragment 1 + 2 levels in the Sak42D group were 1.3 nM at 25 min and 1.2 nM at 20 min, as compared to 11 nM and 5.3 nM in the rt-PA group (both p < 0.0001). TAT levels were 4.7 ng/ml at 25 min and 6.2 ng/ml at 90 min in the Sak42D group, with corresponding values of 16 ng/ml and 9.6 ng/ml in the rt-PA group (p = 0.02 and p = 0.03, respectively). In the patients treated with Sak42D, no significant systemic fibrinolytic activation was observed, as revealed by unaltered levels of clottable fibrinogen, plasminogen and alpha 2-antiplasmin up to 90 min after the start of therapy. In contrast, the corresponding residual levels at 90 min in patients treated with rt-PA decreased to (mean +/- SEM; n = 12) 62 +/- 6%, 45 +/- 5% and 52 +/- 10%, respectively (all p < or = 0.01 versus the Sak42D group). These data confirm the high degree of fibrin-specificity of Sak42D and demonstrate that this is associated with significantly less generation of procoagulant activity in plasma after intravenous administration in patients with acute myocardial infarction.

Recombinant staphylokinase variants with altered immunoreactivity. II: Thrombolytic properties and antibody induction.

BACKGROUND: The substitution variants K35A,E38A,K74A, E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A, D82A (SakSTAR.M89) of recombinant staphylokinase (SakSTAR) with reduced antibody reactivity were assayed for thrombolytic potency and antibody induction in animal models and in patients. METHODS AND RESULTS: In a 125I-fibrin-labeled pulmonary embolism model in the hamster, the doses giving 50% clot lysis in 90 minutes were 25 micrograms/kg for SakSTAR, 85 micrograms/kg for SakSTAR.M38, and 90 micrograms/kg for SakSTAR.M89. In rabbits with 125I-fibrin-labeled plasma clots incorporated into extracorporeal arteriovenous loops, lysis within 2 hours was 76 +/- 18% (mean +/- SD, n = 28) with 400 micrograms/kg SakSTAR, 53 +/- 13% (n = 8) with 1000 micrograms/kg SakSTAR.M38, and 39 +/- 13% (n = 6) with 800 micrograms/kg SakSTAR.M89. When groups of eight rabbits were immunized by intravenous administration of 0.2 to 1.0 micrograms/kg compound followed by subcutaneous injection of 0.4 mg in Freund's adjuvant at 2, 3, and 5 weeks, SakSTAR.M38 and SakSTAR.M89 elicited markedly less circulating neutralizing activity, compared with SakSTAR, when determined at 6 weeks (neutralizing 6.1 +/- 3.0 and 4.9 +/- 1.3 micrograms compound/mL plasma, respectively, versus 20 +/- 17 micrograms/mL; P = .02 and P = .01, respectively) and induced significantly less resistance to thrombolysis (residual thrombolytic potency producing 59 +/- 25% and 39 +/- 12% lysis, respectively, versus 8.5 +/- 5.7%; P = .008 and P = .006, respectively). In patients with peripheral arterial occlusion, intra-arterial administration of SakSTAR.M38 (n = 4) or SakSTAR.M89 (n = 4) induced significantly fewer circulating neutralizing antibodies (P = .03) and specific IgG (P = .01) at 2 to 3 weeks than SakSTAR (n = 8). CONCLUSIONS: SakSTAR.M38 and SakSTAR.M89 induce less antibody formation and might constitute preferred agents for thrombolytic therapy in humans.

mCD24 expression in the developing mouse brain and in zones of secondary neurogenesis in the adult.

Interactions mediated by cell surface glycoproteins are considered to be crucial during the formation of the nervous system. Using a monoclonal antibody directed to mCD24, a glycosylphos-phatidylinositol-anchored membrane glycoprotein, we have mapped its distribution throughout the mouse cerebral cortex during development and in young adult. Before birth, mCD24 immunoreactivity was observed in the intermediate zone, the cortical plate and the marginal zone, whereas the ventricular zones were immunonegative. After birth, mCD24 expression declined rapidly in the cortex, except in the corpus callosum (and other commissures in the brain) where immunoreactivity was still found until P20. Furthermore, mCD24 expression was maintained in young adults (until P60, at least) in zones of secondary neurogenesis, such as the granule cells of the dentate gyrus, the subventricular zone lining the anterior part of the lateral ventricles and a zone of cells extending between the striatum and the corpus callosum to the centre of the olfactory bulb. In this area mCD24 and polysialic acid neural cell adhesion molecule stainings were superimposed, and this corresponded to the pathway of migration of the olfactory immature neurons (subependymal layer). A layer of ciliated ependymal cells, lining all the ventricular walls, was also immunoreactive for mCD24. Thus, except for these epithelial-like cells, mCD24 was essentially found associated with differentiating postmitotic neurons. Its spatiotemporal expression, both during development and in the adult, is compatible with a role for this glycoprotein in cell surface recognition and in signalling events occurring during neuronal migration and axonal growth.

mCD24, a glycoprotein transiently expressed by neurons, is an inhibitor of neurite outgrowth.

In the immune system, mCD24, the mouse homolog of the human glycosyl phosphatidylinositol-anchored glycoprotein CD24, may play a role in cell adhesion. In the nervous system, the function of mCD24 has not been determined, but its transient expression by neurons suggests that it may be involved in axon growth in development. Here we show that retinal ganglion cells (RGCs) and dorsal root ganglion (DRG) neurons express mCD24 in the developing but not adult mouse in vivo and in DRG neurons of the injured adult peripheral nervous system (PNS). In vitro, mCD24 was expressed by immature neurons and by a subpopulation of adult DRG neurons. To analyze the possible function of mCD24 in the nervous system, we prepared rat C6 glioma cells stably transfected or retrovirally infected with mCD24 cDNA. The cells did not exhibit changes in their adhesive properties or cell division rate after transfection or infection. When mCD24-expressing cells were used as monolayer substrates for culturing RGCs and DRG neurons, neurite outgrowth was inhibited, depending on neuronal age and on the relative levels of mCD24 in the monolayer. This inhibition, however, was not dependent on the expression of mCD24 by the neurons themselves, because DRG neurons of a mouse deleted of the mCD24 gene showed the same response. These results show that mCD24 interacts in a heterophilic manner with a developmentally regulated molecule expressed by neurons, and they suggest that in vivo, mCD24 may inhibit the further extension or collateral branching of axons in late embryonic development.

Prevalence and induction of circulating antibodies against recombinant staphylokinase.

Streptokinase (SK) is a routinely used thrombolytic agent but it is immunogenic and allergenic; staphylokinase (STA) is a potential alternative agent which is under early clinical evaluation. The comparative prevalence of antibodies against recombinant STA (STAR) and against SK was studied in healthy subjects and their induction with intravenous administration in small groups of patients. Enzyme-linked immunosorbent assays, using microtiter plates coated with STAR or SK and calibration with affinospecific human antibodies, revealed 2.1 to 65 micrograms/ml (median 11 micrograms/ml) anti-STAR antibodies and 0.9 to 370 micrograms/ml (median 18 micrograms/ml) anti-SK antibodies (p < 0.001 vs anti-STAR antibodies) in plasma from 100 blood donors, with corresponding values of 0.6 to 100 micrograms/ml (median 7.1 micrograms/ml) and 0.4 to 120 micrograms/ml (median 7.3 micrograms/ml), respectively, in 104 patients with angina pectoris. Three out of 17 patients with Staphylococcus aureus bacteremia had significantly increased anti-STAR antibody levels (150, 75 and 75 micrograms/ml), and STAR neutralizing activities (2.2, 3.6 and 4.1 micrograms STAR neutralized per ml plasma, respectively). In 6 patients with acute myocardial infarction, given 10 mg STAR intravenously over 30 min, median anti-STAR antibody levels were 3.5 micrograms/ml at baseline, 2.9 micrograms/ml at 6 to 8 days and 1.2 mg/ml at 2 to 9 weeks, with median corresponding titers of STAR neutralizing activity at 2 to 9 weeks of 42 micrograms/ml plasma. Conversely, in 5 patients treated with 1,500,000 units SK over 60 min, median anti-SK antibodies increased from 2.9 micrograms/ml at baseline to 360 micrograms/ml at 5 to 10 days, with corresponding median SK neutralizing activities of 13 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Multicenter evaluation of commercially available methods for the immunological determination of plasminogen activator inhibitor-1 (PAI-1).

In order to evaluate the comparability of data obtained with various available kits for the immunological determination of PAI-1 antigen in plasma and in order to investigate the underlying cause of observed differences, e.g. problems of specificity or of proper calibration of the provided standard, a multicenter study was organised in the framework of the Subcommittee of Fibrinolysis of the Scientific and Standardization Committee. Eight different plasma samples were distributed among 16 laboratories: a pooled normal plasma, NIBSC 87/512, PAI-1 antigen depleted plasma, PAI-1 depleted plasma supplemented with 59 ng/ml active PAI-1 and four different individual plasma samples. A considerable variation in absolute values is observed between the various kits, e.g. in pooled normal plasma a value is found ranging between 7.4 and 28 ng/ml. Harmonization of all data relative to the PAI-1-depleted plasma supplemented with an exact amount of active PAI-1 (59 ng/ml), followed by a statistical analysis using a two way analysis of variance, revealed that 6 out of 7 kits yielded values that were not significantly different with coefficients of variation around 30%. Correlations between the values obtained with these kits yielded slopes between 0.75 and 1.44 with correlation coefficients between 0.973 and 0.999. Values obtained with one kit appeared to be significantly different (even after harmonization) from the other kits (p < 0.001 to p < 0.05). Comparison of PAI-1 antigen with the PAI activity values in the analysed samples suggests that one kit may deal with a problem of a difference in reactivity between active and latent PAI-1.(ABSTRACT TRUNCATED AT 250 WORDS)

CD24, a signal-transducing molecule expressed on human B lymphocytes, is a marker for human regenerating muscle.

The expression of the CD24 molecule, a glycoprotein expressed at the surface of most B lymphocytes and differentiating neuroblasts, was studied in developing nerve and muscle (after 16 weeks of gestation), normal adult and various diseased human muscles using immunohistochemistry and Western blot analysis. Immunohistochemical studies demonstrated that: (1) in developing muscles, fibers did not express CD24, whereas only some mesenchymal areas, also expressing neural cell adhesion molecule (N.CAM) and vimentin, and developing nerves were positive; (2) in normal adult muscles, CD24 immunoreactivity was observed only in some unmyelinated nerve fibers--intra and extra fusal muscle fibers, satellite cells and neuromuscular junctions were negative; and (3) in all diseased muscles studied here, CD24 expression was always associated with a subpopulation of regenerative fibers. These fibers also expressed vimentin, desmin, developmental myosin heavy chain, N.CAM and its polysialylated isoforms (PSA-N.CAM). The number of CD24-positive fibers was always lower than that of PSA-N.CAM-positive fibers. Denervated fibers and vacuolated muscle fibers never expressed CD24. Western blot analysis indicated that the apparent molecular mass of CD24 antigen was different between muscle and developing nervous tissues, suggesting that CD24 glycosylation is tissue specific. Since the molecule was not expressed in developing human muscle fibers, it strongly suggests that regenerative and fetal myotubes are different with respect to the CD24 molecule expression.

Galactosyl ceramide (or a closely related molecule) is the receptor for human immunodeficiency virus type 1 on human colon epithelial HT29 cells.

The gastrointestinal tract is considered to be a major route of infection for human immunodeficiency virus (HIV). Infection of human colon epithelial cells by HIV is not blocked by anti-CD4 antibodies known to block infection of lymphoid cells (J. Fantini, N. Yahi, and J. C. Chermann, Proc. Natl. Acad. Sci. USA 88:9297-9301, 1991), suggesting the presence of an alternate receptor for HIV on these cells. In this report, we show that (i) a monoclonal antibody specifically directed against galactosyl ceramide inhibited the infection of HT29 cells by two markedly different strains of HIV-1, as assessed by polymerase chain reaction amplification and reverse transcriptase assay; (ii) this antibody strongly labeled the surface of HT29 cells by immunofluorescence and electron microscopic immunolocalization; (iii) the labeling was preferentially but not totally restricted to the basolateral membrane domain of differentiated colonic cells, in agreement with the ability of HIV to infect both the apical and basolateral surfaces of these epithelial cells; and (iv) in thin-layer chromatography-immunostaining experiments with neutral glycolipids prepared from HT29 cells, the antibody specifically reacted with a ceramide monoglycoside fraction corresponding to galactosyl ceramide. We did not detect this glycolipid in lymphoid cells, and anti-galactosyl ceramide antibodies consistently failed to inhibit HIV infection of these cells. These data suggest that galactosyl ceramide (or a derivative) is an essential component of the receptor for HIV on the surface of HT29 cells.