Aberrant glycosylation based on the neo-expression of poly- N-acetyllactosamine structures in squamous cell carcinomas of the head and neck

An immuno- and lectin-histochemical study was performed to investigate the aberrant expression of blood group-related antigens and poly-N- acetyllactosamine structures in squamous cell carcinomas of the maxillary sinus, the larynx, the apipharynx, the hypopharynx, the oral cavity, the parotid gland and the tonsil from 52 patients using monoclonal antibodies against A, B and H antigens, and six lectins, UEA-I, PNA, VVA-B4, PWM, LEA and DSA. In addition, GSA- II staining following endo-·-galactosidase digestion procedure was also applied. A, B and H antigens were expressed in most normal epithelial cells of head and neck organs, and depended on the patient blood type. However, in squamous cell carcinoma, A antigen was not detected in eight out of 25 individuals of blood groups A and AB, although B antigen was consistently expressed in carcinoma cells from all the B and AB individuals. On the other hand, H antigen was expressed in carcinoma cells not only from all blood group O individuals, but from 32 out of 35 individuals of blood groups A, B and AB. T and Tn antigens, which are recognized by PNA and VVA-B4, were strongly expressed in carcinoma cells from 40 and 42 out of 52 individuals respectively. Reactivity with GSA-II staining following endo-·-galactosidase digestion, which recognizes linear poly-N-acetyllactosamine structures, was found in a few malignant cells from 21 individuals. Staining with anti-A, -B and -H monoclonal antibodies and UEA-I lectin was diminished after endo-·-galactosidase digestion in some cases. Lectins specific for poly-N-acetyllactosamine, such as PWM, LEA and DSA, exhibited reactivity in some malignant cells from 30, 22 and 32 out of 52 individuals respectively. These results suggested that the expression of the blood group-related antigens is suppressed and immature carbohydrate chains, that is H, T and Tn antigens, are accumulated in squamous cell carcinomas of the head and neck. The results further suggested that poly-N-acetyllactosamine structures are simultaneously synthesized along with the deletion of A antigen and the accumulation of precursors This revised version was published online in November 2006 with corrections to the Cover Date.     

Exon Redefinition by a Point Mutation within Exon 5 of the Glucose-6-Phosphatase Gene is the Major Cause of Glycogen Storage Disease Type 1a in Japan

Glycogen storage disease (GSD) type 1a (von Gierke disease) is an autosomal recessive disorder caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase). We have identified a novel mutation in the G6Pase gene of a individual with GSD type 1a. The cDNA from the patient's liver revealed a 91-nt deletion in exon 5. The genomic DNA from the patient's white blood cells revealed no deletion or mutation at the splicing junction of intron 4 and exon 5. The 3' splicing occurred 91 bp from the 5' site of exon 5 (at position 732 in the coding region), causing a substitution of a single nucleotide (G to T) at position 727 in the coding region. Further confirmation of the missplicing was obtained by transient expression of allelic minigene constructs into animal cells. Another eight unrelated families of nine Japanese patients were all found to have this mutation. This mutation is a new type of splicing mutation in the G6Pase gene, and 91% of patients and carriers suffering from GSD1a in Japan are detectable with this splicing mutation.     

Microsomal prostaglandin E synthase-1 enhances bone cancer growth and bone cancer-related pain behaviors in mice

AimsNonsteroidal anti-inflammatory drugs are a therapeutic modality for chronic cancer pain arising from bone metastases. Chronic administration of a cyclooxygenase (COX)-2 inhibitor is effective to bone cancer-related pain. However, adverse cardiovascular effects have limited COX-2 inhibitor therapy, and elucidation of better targets for blocking prostaglandin (PG) biosynthesis is necessary. Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that catalyzes isomerization of the endoperoxide PGH₂ to PGE₂. To investigate the validity of mPGES-1 as a therapeutic target, we evaluated bone cancer pain-related behaviors in mPGES-1 knockout (PGES-1?/?) mice.Main methodsLewis lung carcinoma cells (LLCCs) were injected into the intramedullary space of the femur of wild-type (WT) and PGES-1?/? mice. Pain-related behaviors were evaluated.Key findingsPGES-1?/? mice exhibited reduced tumor growth in bone marrow compared to WT. The expression of pro-calcitonin gene-related peptide (CGPR) in the dorsal root ganglia of L_1–5 was significantly higher in WT mice at day 14, whereas it was unchanged in mPGES-1 mice. In the observation of pain-related behaviors, mPGES-1?/? mice exhibited significantly fewer spontaneous flinches and their onset was several days later than WT. The appearance of other pain-related behaviors in mPGES-1?/? mice was also delayed as compared to WT. LLCC-injected WT mice treated with a COX-2 inhibitor, celecoxib, exhibited similar temporal changes to mPGES1?/?.SignificanceThe present results suggest that mPGES-1 plays a crucial role in the enhancement of bone cancer growth and bone cancer pain, and that inhibition of mPGES-1 may have clinical utility in the management of bone cancer pain.     

β₃-Adrenergic activation of sequential Ca(2+) release from mitochondria and the endoplasmic reticulum and the subsequent Ca(2+) entry in rodent brown adipocytes

We studied how mitochondrial uncoupling by β(3)-adrenergic stimulation elicits Ca(2+) signals in rodent brown adipocytes by fluorometry of Ca(2+) concentrations ([Ca(2+)](i), [Ca(2+)](m) and [Ca(2+)](ER)) in the cytoplasm, mitochondria and the endoplasmic reticulum (ER), respectively, and mitochondrial membrane potential, using fura-2, rhod-5N, cameleon and rhodamine 123. Immunoblotting demonstrated α(1A)- and β(3)-adrenergic receptor and UCP1 in adipocytes, while RT-PCR revealed the mRNA of type 3, 7 and 9 adenylate cyclase, UCP1, UCP2, UCP3 and type 1 and 2 inositoltrisphosphate receptors. Isoproterenol and BRL37344, β-agonist, caused triphasic rises in [Ca(2+)](i) (β-responses) with mitochondrial depolarization in adipocytes. BRL37344 transiently decreased [Ca(2+)](m). β-Responses were blocked by propranolol, β-antagonist, H-89, protein kinase A blocker, and knockout of UCP1 gene. The late phase of β-responses was depressed by a Ca(2+) free, EGTA solution, U73122, a phospholipase C blocker, and thapsigargin, ER-Ca(2+) pump blocker, and by transfecting siRNA for type 2 IP(3)R. Intracellular loading of BAPTA/AM depressed the late phase more strongly than the initial phase. β-Agonists, phenylephrine, α-agonist, and cyclopiazonic acid, ER-Ca(2+) pump blocker, decreased [Ca(2+)](ER). Thus, the mitochondrial uncoupling by β(3)-adrenergic activation causes Ca(2+) release from mitochondria and subsequently from the ER and further evokes plasmalemmal Ca(2+) entries, including the store-operated Ca(2+) entry.     

A theory on auto-oscillation and contraction in striated muscle

It is widely accepted that muscle cells take either force-generating or relaxing state in an all-or-none fashion through the so-called excitation–contraction coupling. On the other hand, the membrane-less contractile apparatus takes the third state, i.e., the auto-oscillation (SPOC) state, at the activation level that is intermediate between full activation and relaxation. Here, to explain the dynamics of all three states of muscle, we construct a novel theoretical model based on the balance of forces not only parallel but also perpendicular to the long axis of myofibrils, taking into account the experimental fact that the spacing of myofilament lattice changes with sarcomere length and upon contraction. This theory presents a phase diagram composed of several states of the contractile apparatus and explains the dynamic behavior of SPOC, e.g., periodical changes in sarcomere length with the saw-tooth waveform. The appropriate selection of the constant of the molecular friction due to the cross-bridge formation can explain the difference in the SPOC periods observed under various activating conditions and in different muscle types, i.e., skeletal and cardiac. The theory also predicts the existence of a weak oscillation state at the boundary between SPOC and relaxation regions in the phase diagram. Thus, the present theory comprehensively explains the characteristics of auto-oscillation and contraction in the contractile system of striated muscle.     

Male Courtship Behavior and Weapon Trait as Indicators of Indirect Benefit in the Bean Bug,Riptortus pedestris

Females prefer male traits that are associated with direct and/or indirect benefits to themselves. Male–male competition also drives evolution of male traits that represent competitive ability. Because female choice and male–male competition rarely act independently, exploring how these two mechanisms interact is necessary for integrative understanding of the evolution of sexually selected traits. Here, we focused on direct and indirect benefits to females from male attractiveness, courtship, and weapon characters in the armed bug Riptortus pedestris. The males use their hind legs to fight other males over territory and perform courtship displays for successful copulation. Females of R. pedestris receive no direct benefit from mating with attractive males. On the other hand, we found that male attractiveness, courtship rate, and weapon size were significantly heritable and that male attractiveness had positive genetic covariances with both courtship rate and weapon traits. Thus, females obtain indirect benefits from mating with attractive males by producing sons with high courtship success rates and high competitive ability. Moreover, it is evident that courtship rate and hind leg length act as evaluative cues of female choice. Therefore, female mate choice and male–male competition may facilitate each other in R. pedestris. This is consistent with current basic concepts of sexual selection.     

Sip1, an AP-1 Accessory Protein in Fission Yeast,Is Required for Localization of Rho3 GTPase

Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3 ⁺ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi/endosomal trafficking in fission yeast.