Studies on the endogenous L-selectin ligands: systematic and highly efficient total synthetic routes to lactamized-sialyl 6-O-sulfo Lewis X and other novel gangliosides containing lactamized neuraminic acid

Systematic syntheses of lactamized neuraminic acid-containing gangliosides GM4, sulfated sialylparagloboside, and sulfated/nonsulfated sialyl Lewis X are described. The highly efficient, one-step lactamization of neuraminic acid was accomplished by treatment of the N-deacetylated sialic acid (neuraminic acid)-containing gangliosides with HBTU and HOBt in DMF at 65 degrees C. Both the lactamized neuraminic acid residue and the sulfate group at O-6 of the GlcNAc residue were found to be involved in the antigenic determinant defined by G159 monoclonal antibody, while the fucose residue may not be critical for the recognition by G159 mAb.     

Cloning and embryonic expression of six wnt genes in the medaka (Oryzias latipes) with special reference to expression of wnt5a in the pectoral fin buds

WNTs are secreted signaling molecules which control cell differentiation and proliferation. They are known to play essential roles in various developmental processes. Wnt genes have been identified in a variety of animals, and it has been shown that their amino acid sequences are highly conserved throughout evolution. To investigate the role of wnt genes during fish development from the evolutionary viewpoint, six medaka wnt genes (wnt4, wnt5a, wnt6, wnt7b, wnt8b and wnt8-like) were isolated and their embryonic expression was examined. These wnt genes were expressed in various tissues during embryonic development, and most of their expression patterns were conserved or comparable to those of other vertebrates. Thus, these wnt genes may be useful as molecular markers to investigate development and organogenesis using the medaka. Focus was on wnt5a, which was expressed in the pectoral fin buds, because its expression pattern was particularly comparable to that in tetrapod limbs. Its detailed expression pattern was further examined during pectoral fin bud development. The conservation and diversification of Wnt5a expression through the evolutionary transition from fish fins to tetrapod limbs is discussed.     

Crystal structure of hyperthermophilic archaeal initiation factor 5A: a homologue of eukaryotic initiation factor 5A (eIF-5A)

Eukaryotic initiation factor 5A (eIF-5A) is ubiquitous in eukaryotes and archaebacteria and is essential for cell proliferation and survival. The crystal structure of the eIF-5A homologue (PhoIF-5A) from a hyperthermophilic archaebacterium Pyrococcus horikoshii OT3 was determined at 2.0 A resolution by the molecular replacement method. PhoIF-5A is predominantly composed of beta-strands comprising two distinct folding domains, an N-domain (residues 1-69) and a C-domain (residues 72-138), connected by a short linker peptide (residues 70-71). The N-domain has an SH3-like barrel, while the C-domain folds in an (oligonucleotide/oligosaccharide binding) OB fold. Comparison of the structure of PhoIF-5A with those of archaeal homologues from Methanococcus jannaschii and Pyrobaculum aerophilum showed that the N-domains could be superimposed with root mean square deviation (rmsd) values of 0.679 and 0.624 A, while the C-domains gave higher values of 1.824 and 1.329 A, respectively. Several lines of evidence suggest that eIF-5A functions as a biomodular protein capable of interacting with protein and nucleic acid. The surface representation of electrostatic potential shows that PhoIF-5A has a concave surface with positively charged residues between the N- and C-domains. In addition, a flexible long hairpin loop, L1 (residues 33-41), with a hypusine modification site is positively charged, protruding from the N-domain. In contrast, the opposite side of the concave surface at the C-domain is mostly negatively charged. These findings led to the speculation that the concave surface and loop L1 at the N-domain may be involved in RNA binding, while the opposite side of the concave surface in the C-domain may be involved in protein interaction.     

Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes

We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.     

Expression of Hex homeobox gene during skin development: Increase in epidermal cell proliferation by transfecting the Hex to the dermis

A number of homeobox genes have been found to be expressed in skin and its appendages, such as scale and feather, and appear to be candidates for the regulation of the development of these tissues. We report that the proline-rich divergent homeobox gene Hex is expressed during development of chick embryonic skin and its appendages (scale and feather). In situ hybridization analysis revealed that, during development of the skin, a transient expression of the Hex gene was observed. While the expression of Hex in the dermis was closely correlated with proliferation activity of epidermal basal cells, that in the epidermis was related to a suppression of epidermal differentiation. When dermal fibroblasts were transfected with Hex, stimulation of both DNA synthesis and proliferation of the epidermal cells followed by two-fold scale ridge elongation and increase in epidermal area was observed during culture of the skin, whereas epidemal keratinization was not affected. This is the first study to demonstrate that Hex is expressed during development of the skin and its appendages and that its expression in the dermal cells regulates epidermal cell proliferation through epithelial mesenchymal interaction.     

Imprint cytology of epithelioid angiomyolipoma in a patient with tuberous sclerosis. A case report

BACKGROUND: Angiomyolipoma composed predominantly of epithelioid cells has been referred to as epithelioid angiomyolipoma. As this subtype shows considerable cellular atypia, it may be erroneously diagnosed as malignant epithelioid tumor, such as renal cell carcinoma and hepatocellular carcinoma. So far, only one report describing the cytologic findings of epithelioid angiomyolipoma has been documented, and epithelioid angiomyolipoma occurring in the peritoneal cavity has not been reported. CASE: Eleven years after resection of a renal epithelioid angiomyolipoma in a 34-year-old male with tuberous sclerosis, a tumor appeared in the peritoneal cavity and three masses in the liver. The intraoperative smears imprinted from part of the peritoneal mass revealed many large, atypical cells. The well-preserved atypical cells showed abundant, round to polyhedral, granular cytoplasm. Bizarre, giant nuclei with hyperchromasia and huge nucleoli were occasionally seen. Intranuclear cytoplasmic inclusions and mitotic figures were occasionally observed. As the epithelioid cells were markedly pleomorphic, we could not rule out hepatocellular carcinoma, cytologically and histologically, in the intraoperative consultation. In permanent sections the tumor was composed predominantly of epithelioid cells showing an alveolar pattern or sheetlike arrangement. Mitotic counts were zero to one per 10 high-power fields. Immunohistochemically, the epithelioid tumor cells were positive for vimentin, alpha-smooth muscle actin and HMB-45, consistent with epithelioid angiomyolipoma. MIB-1-labeling index was 1.6%. CONCLUSION: When one sees atypical epithelioid tumor cells in a tuberous sclerosis patient during an intraoperative consultation, one must consider epithelioid angiomyolipoma.     

Rush and grab strategies in foraging marine endotherms: the case for haste in penguins

The speed at which air-breathing marine predators that forage by diving should swim is likely to depend on a variety of factors that differ substantially from those relevant in animals for which access to oxygen is unlimited. We used loggers attached to free-living penguins to examine the speed at which three species swam during periods searching for prey and compared this to their speeds during actual prey pursuit. All penguin species appeared to travel at similar speeds around 2 m/s during normal commuting between the surface and feeding depths, which accords closely with minimum costs of transport. However, Adélie penguins, Pygoscelis adeliae, slowed down to feed, Magellanic penguins, Spheniscus magellanicus, speeded up and king penguins, Aptenodytes patagonicus, travelled at a variety of speeds, although mean speed did not change from normal commuting. Since energy expenditure, and therefore oxygen usage, in swimming animals increases with the cube of the speed, we hypothesized that prey escape speed (a function of prey size) and prey density would prove critical in determining optimum pursuit speeds in predators. Simple models of this type help explain why it is that some penguin species apparently benefit by increasing speed to capture prey while others benefit by decreasing speed.     

Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.     

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.