Epidermal growth factor receptor (EGFR) at membrane microdomains plays an essential role in the growth control of epidermal cells, including cancer cells derived therefrom. Ligand-dependent activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, but to a much lesser degree by other glycosphingolipids. However, the mechanism of the inhibitory effect of GM3 on EGFR tyrosine kinase has been ambiguous. The mechanism is now defined by binding of N-linked glycan having multiple GlcNAc termini to GM3 through carbohydrate-to-carbohydrate interaction, based on the following data: (i) EGFR (molecular mass, Ϸ170 kDa) has N-linked glycan with GlcNAc termini, as probed by mAb (J1) or lectin (GS-II); (ii) GS-II-bound EGFR also bound to anti-EGFR Ab as well as to GM3-coated beads; (iii) GM3 inhibitory effect on EGFR tyrosine kinase was abrogated in vitro by coincubation with glycan having multiple GlcNAc termini and in cell culture in situ incubated with the same glycan; and (iv) cells treated with swainsonine, which increased expression of complex-type and hybridtype glycans with GlcNAc termini, displayed higher inhibition of EGFR kinase by GM3 than swainsonine-untreated control cells. A similar effect was observed with 1-deoxymannojirimycin, which increased hybrid-type structure in addition to major accumulation of high mannose-type glycan. These findings indicate that N-linked glycan with GlcNAc termini linked to EGFR is the target to interact with GM3, causing inhibition of EGF-induced EGFR tyrosine kinase.carbohydrate-to-carbohydrate interaction ͉ N-linked glycan ͉ glycosphingolipid ͉ ganglioside ͉ oligosaccharide Fr.B

Although seminolipid has long been suspected to play an essential role in spermatogenesis because of its uniquely abundant and temporally regulated expression in the spermatocytes, direct experimental evidence has been lacking. We have tested the hypothesis by examining the testis of the UDP-galactose:ceramide galactosyltransferase-deficient mouse, which is incapable of synthesizing seminolipid. Spermatogenesis in homozygous affected males is arrested at the late pachytene stage and the spermatogenic cells degenerate through the apoptotic process. This stage closely follows the phase of rapid seminolipid synthesis in the wild-type mouse. These observations not only provide the first experimental evidence that seminolipid is indeed essential for normal spermatogenesis but also support the broader concept that cell surface glycolipids are important in cellular differentiation and cell-to-cell interaction.Seminolipid (3-sulfogalactosyl-1-alkyl-2-acyl-sn-glycerol) is the principal glycolipid in spermatozoa of mammals comprising, for example, approximately 3% of total lipids and more than 90% of total glycolipids in boar spermatozoa (1-3). During spermatogenesis, seminolipid is synthesized rapidly in the early phase of spermatocyte development and maintained in subsequent germ cell stages (4 -6). This developmentally regulated rapid synthesis suggested a specific and possibly essential function of seminolipid in spermatogenesis (7) but experimental evidence has been lacking. Firm evidence in support of the speculation would have important bearing to the general concept that cell surface glycoconjugates are important in cellular differentiation, and cell-to-cell interaction (8).Seminolipid is synthesized by sulfation of its precursor, galactosylalkylacylglycerol (GalEAG) 1 . GalEAG is synthesized by UDPgalactose:ceramide galactosyltransferase (CGT, EC 2.4.1.62), which, besides GalEAG, also synthesizes the major myelin galactolipid, galactosylceramide (GalCer), galactosylsphingosine (psychosine), and galactosyldiacylglycerol (GalAAG) (9, 10). The CGTdeficient mice recently generated by gene-targeting do not synthesize any of these products and subsequent derivatives of the products (11)(12)(13)(14). Thus, the CGT-deficient mouse is an ideal experimental model to examine the consequences of lack of seminolipid to spermatogenesis. This report describes the first definitive evidence that deficient seminolipid biosynthesis indeed causes devastating disruption of the normal spermatogenetic process. EXPERIMENTAL PROCEDURESMice-The mice heterozygous for the disrupted Cgt gene (11) were originally supplied by Dr. B. Popko and maintained by backcrossing to C57BL/6N. Genotype was determined according to Coetzee et al. (11). WBB6F1 Kit W/W-v and WBB6F1 Mg f Sl/Sl-d mutant mice were purchased from Japan SLC, Inc., and C57BL/6N inbred mice were purchased from CLEA Japan, Inc. Isolation of Testicular Germ Cells-Testicular germ cells were isolated from decapsulated testes of sexually mature male C57BL/6N mice (15).RT-PCR Analysis-RNA...

Some protein kinases are known to be activated by D-erythro-sphingosine (Sph) or N,N-dimethyl-D-erythrosphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser 60 , Ser 59 , or Ser 58 of 14-3-3␤, 14-3-3 , or 14-3-3 , respectively, was termed "sphingosine-dependent protein kinase-1" (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834 -21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase C␦ (PKC␦) based on the following observations. (i) Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an ϳ40-kDa protein band. This fraction had SDK1 activity ϳ50,000-fold higher than that in the initial extract. (ii) This protein had ϳ53% sequence identity to the Ser/Thr kinase domain of PKC␦ based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii) A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the ϳ40-kDa band is the kinase domain of PKC␦. The kinase activity of PKC␦ did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKC␦ did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKC␦ was incubated with caspase-3, which releases the ϳ40-kDa kinase domain. PKC␦ and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.

The currently used antivirals in the treatment of influenza in Japan include amantadine, oseltamivir, zanamivir, laninamivir, and peramivir. We compared the efficacy of intravenous peramivir with that of other neuraminidase inhibitors for treating pediatric influenza. The present study included 223 influenza patients (≤18 years) who presented at the Hikita Pediatric Clinic between February and April 2011. We compared fever duration after starting treatment with antiviral drugs. Because inhalation drugs are difficult to use in <5-year-old patients and because of the potential adverse effects of oseltamivir in teenagers, we created two different age groups (<10-year-old group and 5–18-year-old group) to evaluate treatment results. In influenza A patients between 5 and 18 years old, the median fever duration after treatment with zanamivir was 2 days, compared with 1 day for peramivir (P = 0.0242). In influenza B patients between 5 and 18 years old, the median fever duration after treatment with laninamivir was 3 days, compared with 1 day for peramivir (P = 0.0097). We found no significant difference for any of the other combinations of drug/disease type/age groups. No adverse effects were observed with the antiviral drugs used. The results suggest that peramivir is very useful in pediatric influenza patients.

We conclude that sumatriptan has potential efficacy in treating of patients with CVS.

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