The role of glycoconjugates in metastatic melanoma blood-borne arrest and cell surface properties

The role of glycoconjugates in cell surface and blood-borne implantation properties of murine metastatic melanoma sublines of low (B16-F1) or high (B 16-F10) potential to colonize lungs was investigated by treating melanoma cells with the antibiotic tunicamycin. This drug prevents glycosylation of glycoproteins by inhibiting the formation of lipid-linked oligosaccharide precursors. The degree of tunicamycin-mediated modifications in glycoproteins was assessed by monitoring the decrease in cell surface sialogalactoproteins by binding of ¹²⁵I-labeled Ricinus communis agglutinin I. Scanning electron microscopy of tunicamycin-treated B16-F1 and B16-F10 cells showed morphologic changes such as cell rounding and formation of numerous surface blebs. Tunicamycin-treated B16-F1 and B16-F10 cells lost their lung colonization abilities when injected intravenously into C57BL/6 mice, concomitant with lowered rates of adhesion to endothelial cell monolayers, endothelial extracellular matrix (basal lamina), and polyvinyl-immobilized fibronectin in vitro, suggesting that this drug inhibits experimental metastasis by modifying the surface glycoproteins involved in determining the adhesive properties of malignant cells.     

The role of fibronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina

Vascular endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma cells, which do not contain significant amounts of FN, attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-F1) or high (B16-F10) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal lamina and completely abolished attachment of B16 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on B16 cells.     

Broadly and narrowly tuned odorant receptors are involved in female sex pheromone reception in Ostrinia moths

Mate-finding communication in many moths is mediated by sex pheromones produced by females. Since the differentiation of sex pheromones is often associated with speciation, it is intriguing to elucidate how the changes in sex pheromones are tracked by the pheromone recognition system of the males. Moths of the genus Ostrinia, which show distinct differentiation in female sex pheromones, are good models to study this. The present study was initiated with the aim of identifying ORs from Ostrinia scapulalis that respond to its own pheromone components, (E)-11- and (Z)-11-tetradecenyl acetates. We isolated six OR gene candidates (OscaOR3–8) from O. scapulalis. The same set of genes homologous to OscaOR3–8 were conserved in all (eight) Ostrinia species examined in addition to the previously reported OscaOR1 (tuned to (E)-11-tetradecenol) and the Or83b homologue OscaOR2. OscaOR3 not only responded to (E)-11- and (Z)-11-tetradecenyl acetates, but also to the pheromone components of the congeners, (Z)-9-, (E)-12-, and (Z)-12-tetradecenyl acetates. OscaOR4 responded with a relatively high specificity to (E)-11-tetradecenyl acetate. While OscaOR5 responded only marginally to a few pheromone components, OscaOR6–8 did not respond to any of the compounds tested. A few conserved ORs, including a unique one with very broad responsiveness, appear to be involved in the sex pheromone reception in O. scapulalis. The findings of the present study are discussed with reference to knowledge on electrophysiological response profiles of olfactory receptor neurons in Ostrinia moths.     

A remembrance of the late Professor Teruo Okuma,M.D., Ph.D

Sleep and Biological Rhythms -     

Rapid genotyping for most common TGFBI mutations with real-time polymerase chain reaction

Recent studies of the corneal dystrophies (CDs) have shown that most cases of granular CD, Avellino CD, and lattice CD type I are caused by mutations in the human transforming growth factor beta-induced (TGFBI) gene. The aim of this study was to develop a rapid diagnostic assay to detect mutations in the TGFBI gene. Sixty-six patients from 64 families with TGFBI-associated CD were studied. A primer probe set was designed to examine the genome from exons 4 and 12 of the TGFBI gene in order to identify mutant and wild-type alleles. A region spanning the mutations was amplified by the polymerase chain reaction (PCR) in a commercial cycler. Mutations were then identified by melting curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. Using this system, we clearly distinguished each CD genotype (homozygous and heterozygous 418GA, heterozygous 417CT, heterozygous 1710CT, and wild-type) of all the patients by means of the clearly distinct melting peaks at different temperatures. One thermal cycling took approximately 54 min, and all results were completely in concordance with the genotypes determined by conventional DNA sequencing. Thus, the technique is accurate and can be used for routine clinical diagnosis. We expect that our new method will help in making precise diagnoses of patients with atypical CDs and aid the revision of the clinical classification of inherited corneal diseases based on the genetic pathogenesis.     

Functional Significance of the Preconditioning-induced Down-regulation of Glutamate Transporter GLT-1 in Neuron/astrocyte Co-cultures

In the brain, prior sublethal ischemia (preconditioning, PC) is known to produce tolerance of neurons to subsequent lethal ischemia. This study aims at elucidating what alterations were induced in neurons and/or astrocytes by PC treatment. The rise in the extracellular concentration of glutamate during ischemia was markedly suppressed by the prior PC treatment. Immunocytochemical and Western blot analyses demonstrated that the expression of the astrocytic glutamate transporter GLT-1 was transiently down-regulated after the PC insult. The PC insult possibly suppressed the neuron-derived factors up-regulating GLT-1. Here we show that PC-induced down-regulation of GLT-1 is crucial for the increased neuronal resistance to subsequent severe ischemic insult.     

Regulation of replication at the R/G chromosomal band boundary and pericentromeric heterochromatin of mammalian cells

Mammalian chromosomes consist of multiple replicons; however, in contrast to yeast, the details of this replication process (origin firing, fork progression and termination) relative to specific chromosomal domains remain unclear. Using direct visualization of DNA fibers, here we show that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. To support this, fluorescence in situ hybridization (FISH)-based replication profiles at the human 1q31.1 (R-band)-32.1 (G-band) regions revealed that replication timing switched around at the putative R/G chromosomal band boundary predicted by marked changes in GC content at the sequence level. Thus, the slowdown of replication fork movement is thought to be the general property of the band boundaries separating the functionally different chromosomal domains. By simultaneous visualization of replication fork movement and pericentromeric heterochromatin sequences on DNA fibers, we observed that this region is duplicated by many replication forks, some of which proceed unidirectionally, that originate from clustered replication origins. We showed that histone hyperacetylation is tightly associated with changes in the replication timing of pericentromeric heterochromatin induced by 5-aza-2'-deoxycytidine treatment. These results suggest that, similar to the yeast system, histone modification is involved in controlling the timing of origin firing in mammals.     

Pumps, paradoxes and ploughshares: mechanism of the MCM2-7 DNA helicase

In eukaryotes, numerous lines of evidence have coalesced into a convincing case that the MCM2-7 complex - a heterohexameric ATPase - is the replicative DNA helicase. However, almost nothing is known about how this enzyme functions in a cellular context. Some models for the mechanism of the MCM2-7 helicase envision that it translocates along single-stranded DNA (ssDNA), whereas, more recently, it is has been suggested that it pumps double-stranded DNA (dsDNA) through its central channel. In particular, one model in which a double hexamer of MCM2-7 pumps dsDNA towards the hexamer interface and extrudes ssDNA laterally as a result of torsional strain is gaining popularity. Here, we discuss existing models and propose a new variation in which a single hexamer is the functional unit of the helicase. Duplex DNA is pumped into MCM2-7 and, as it emerges from the complex, a rigid protein that we term the 'ploughshare' splits the duplex.