Metabolic analysis of reepithelializing rabbit cornea using phosphorus-31 nuclear magnetic resonance spectroscopy

To investigate metabolic differences between the central and peripheral cornea the latter including the limbal area, corneas were dissected and examined using phosphorus-31 (31P) nuclear magnetic resonance spectroscopy. Since most 31P signals originate from the epithelium, 31P spectra of the cornea primarily represent the metabolic state of the epithelium. The spectra of the peripheral cornea showed all phosphorus resonances detected in the whole cornea; in contrast, the central cornea showed no phosphocreatine and glycerophosrylethanolamine, and only low levels of ATP. These results indicate that there is a higher metabolic activity in the peripheral epithelium, especially in the limbal area, than in the central epithelium. To evaluate the metabolic state of corneal epithelium during regeneration, we also examined corneas reepithelializing after 7 mm of central epithelial tissue had been removed by mechanical scraping. Rabbits were killed 24 and 48 h after scraping. The reepithelializing corneas clearly showed an increase in ATP, phosphocreatine, and sugar phosphates with time, although phosphorylcholine remained depressed. These findings suggest that the reepithelializing cornea has an elevated level of energy production and that it may have reached a higher steady state, thereby indicating accelerated metabolism of the epithelium during regeneration.     

Effect of maternal exercise with graded treadmill on fetal heart rate]

The effect of maternal exercise on the fetal and maternal heart rate was studied in 10 normal pregnant women in their third trimester. They participated in 15 minute graded treadmill exercise. Fetal heart rate (FHR), uterine contraction, maternal blood pressure, electrocardiogram, and oxygen consumption were monitored, before, during, and after exercise. FHR could be monitored in 7 women. Maternal maximal heart rate ranged from 143 to 168 bpm which approximated 61% to 84% training intensity. FHR showed a significant rise in 4 women during exercise. Of four cases which showed more than 70% training intensity, 3 demonstrated fetal tachycardia over 160 bpm and one demonstrated fetal bradycardia under 120 bpm. The baseline fetal heart rate remained from 120 bpm to 160 bpm in cases under 70% training intensity. There was no pathological deceleration and the variability was preserved in all cases. It is suggested that maternal exercise intensity should be less than 70% of the patient's maximal capacity, which approximates maternal heart rate of about 150 bpm.     

One-step sandwich enzyme-linked immunosorbent assay of human tissue plasminogen activator using monoclonal antibodies

Five monoclonal antibodies to human tissue plasminogen activator (t-PA) were obtained. Three of them recognized a determinant designated alpha, and other two, another determinant named beta. A t-PA molecule was found to bear one each of determinants alpha and beta arranged in such a manner that the binding of alpha with one of its corresponding antibodies did not interfere with the binding of beta with one of its corresponding antibodies and vice versa. This allowed the development of a one-step sandwich enzyme-linked immunosorbent assay (ELISA) in which the antibody to beta was immobilized on wells of a microtiter plate and the bound t-PA was detected with the enzyme-conjugated antibody to alpha. The ELISA detected both one- and two-chain t-PA with a high sensitivity. We also discuss the determinants, alpha and beta.     

Functional overlap between RecA and MgsA (RarA) in the rescue of stalled replication forks in Escherichia coli

Escherichia coli RecA protein plays a role in DNA homologous recombination, recombination repair, and the rescue of stalled or collapsed replication forks. The mgsA (rarA) gene encodes a highly conserved DNA-dependent ATPase, whose yeast orthologue, MGS1, plays a role in maintaining genomic stability. In this study, we show a functional relationship between mgsA and recA during DNA replication. The mgsA recA double mutant grows more slowly and has lower viability than a recA single mutant, but they are equally sensitive to UV-induced DNA damage. Mutations in mgsA and recA cause lethality in DNA polymerase I deficient cells, and suppress the temperature-dependent growth defect of dnaE486 (Pol III alpha-catalytic subunit). Moreover, recAS25P, a novel recA allele identified in this work, does not complement the slow growth of DeltamgsA DeltarecA cells or the lethality of polA12 DeltarecA, but is proficient in DNA repair, homologous recombination, SOS mutagenesis and SOS induction. These results suggest that RecA and MgsA are functionally redundant in rescuing stalled replication forks, and that the DNA repair and homologous recombination functions of RecA are separated from its function to maintain progression of replication fork.     

Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization

Here we report the generation and characterization of a monoclonal antibody, mAb 5H7-G1, which recognizes egg antigens in the animal cortex of fertilized, but not unfertilized, Xenopus eggs. The mAb 5H7-G1 was generated by subtractive immunization of mice: primary immunization with unfertilized egg extract followed by immunosuppression treatment with cyclophosphamide and repeated immunization with fertilized egg extract. In immunoblotting analysis, mAb 5H7-G1 recognizes multiple protein bands of fertilized (but not unfertilized or the ionophore-activated) Xenopus eggs. N-linked polysaccharide is most likely the target of mAb 5H7-G1 because immunoreactivity of mAb 5H7-G1 is effectively diminished when protein samples are treated with N-glycosidase F. Moreover, mAb 5H7-G1 recognizes some, but not all, tyrosine-phosphorylated proteins in eggs treated with H2O2, an artificial activator of the egg tyrosine kinase Src, suggesting that these proteins also contain N-linked sugars. When microinjected into fertilized Xenopus embryos, mAb 5H7-G1 causes a retardation or complete inhibition of first cell cleavage, suggesting that the mAb 5H7-G1-reactive antigens play an important role in this event. These results demonstrate that mAb 5H7-G1 is useful to analyze differential proteome display during fertilization and early development. More generally, subtractive immunization may work as a strategy to uncover cellular events that operate during different cellular conditions of interest.     

Phospholipid polymer surfaces reduce bacteria and leukocyte adhesion under dynamic flow conditions

Persistence of infection can occur when the host immune response is compromised because of the presence of a foreign implant. Surface modification of biomaterials with phospholipid polymers may enhance biocompatibility and reduce incidence of infection by impeding bacterial and leukocyte adhesion. A rotating disk model, which generates shear stress from 0 to 18 dynes/cm(2), was used to characterize adhesion of neutrophils, monocytes, and bacteria in phosphate-buffered saline (PBS) or 25% human serum on polyethylene terephthalate surfaces coated with a phospholipid polymer, poly[omega-methacryloyloxyalkyl phosphorylcholine (MAPC)-co-n-butyl methacrylate (BMA)]. The material designated PMB30 contains a methylene chain length, (CH(2))(n), of n = 2, whereas PMHB30 contains a chain length of n = 6. In PBS, bacterial adhesion was shear stress dependent with the lowest bacterial density observed on PMB30. However, the presence of serum proteins eliminated shear stress and surface chemistry effects in addition to bacterial adhesion reduced to <10% of adhesion in PBS. Trends for leukocyte adhesion in serum demonstrated shear dependence with PMB30 exhibiting the lowest cell density throughout the range of shear stresses. In conclusion, modification of the polyethylene terephthalate surfaces with phospholipid polymers resulted in reduced bacterial and leukocyte adhesion. Furthermore, shortening the methylene chain length of the MAPC copolymer most effectively reduced adhesion.     

Amelotin--a Novel Secreted, Ameloblast-specific Protein

We aimed to analyze the differential gene expression in various murine dental tissues, expecting to find novel factors that are involved in tooth formation. We here describe the identification of a novel ameloblast-specific gene, amelotin (AMTN), by differential display polymerase chain-reaction (DD-PCR) analysis of microdissected ameloblasts, odontoblasts, dental pulp, and alveolar bone cells of 10-day-old mouse incisors. The conceptually translated protein sequence was unique and showed significant homology only with its human orthologue. The amelotin genes from mouse and human displayed a similar exon-intron structure and were expressed from loci on chromosomes 5 and 4, respectively, which have been associated with various forms of amelogenesis imperfecta. Expression of amelotin mRNA was restricted to maturation-stage ameloblasts in developing murine molars and incisors. Amelotin protein was efficiently secreted from transfected cells in culture. Taken together, our findings suggest that amelotin is a novel factor produced by ameloblasts that plays a critical role in the formation of dental enamel.     

Role of dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine against non-small cell lung cancer--in correlation with the tumoral expression of thymidylate synthase and dihydropyrimidine dehydrogenase

5-Fluorouracil (5-FU) and its derivatives have been used worldwide for the treatment of several malignancies in solid organs. The effectiveness of these drugs is well proven in gastrointestinal malignancy, and has been reported upon the inverse correlation with the tumoral expression of dihydropyrimidine dehydrogenase (DPD). However, the significance of DPD expression in 5-FU based chemotherapy has not been well investigated in non-small cell lung cancer (NSCLC). We examined enzymatic activities and immunohistochemical expression of thymidylate synthase (TS) and DPD in 84 cases of NSCLC. In vitro sensitivity for 5-FU was tested in 53 cases of them to evaluate these predictive values for effectiveness of 5-FU. Efficacy of 5-chloro-2,4-dihydroxypyridine (CDHP), potent DPD inhibitor, was also examined in 27 cases of them. There was a reversal correlation between protein expression of DPD and sensitivity to 5-FU (r = -0.65; p < 0.001). Six (33.3%) of 18 cases with strong expression of DPD showed 10% or more increment of the anti-tumor effect by adding CDHP to 5-FU. DPD inhibitory fluoropyrimidine and examination of the tumoral expression of DPD might be a promising chemotherapeutic strategy in NSCLC.