The primary structure of human gamma-glutamyl transpeptidase

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.     

Differences in the Optical Response of MSU and CPP Crystals During Magnetic Orientation: Possibility of Diagnosing Gout and Pseudogout

Pseudogout is crystalline arthritis. It has a similar clinical picture to that of gout, and it is difficult to distinguish the two diseases using conventional analysis methods. However, it is important to identify the different crystals responsible for these two cases because the treatment strategies are different. In a previous study, we reported magnetic orientation of monosodium urate (MSU) crystals, which are the causative agent of gout, at the permanent magnet level. In this study, we investigated the effect of an applied magnetic field on calcium pyrophosphate (CPP) crystals, which are the causative agent of pseudogout, and the difference in the magnetic responses of CPP and MSU crystals. We found that the CPP crystals were oriented in a magnetic field on milli-Tesla order because of the anisotropy of the diamagnetic susceptibility. In addition, the CPP crystals exhibited different anisotropic magnetic properties from those of MSU crystals, which led to a characteristic difference between the orientations of the two crystals. That is, we found that the causative agents of gout and pseudogout responded differently to a magnetic field. This report suggests that the discrimination between CPP and MSU by optical measurements is possible by application of magnetic fields appropriately. © 2023 Bioelectromagnetics Society.     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.     

Possible Mechanism of Oxygen Activation in Linoleate Peroxidation by Fusarium Lipoxygenase

The Oxygen activating mechanism of Fusarium lipoxygenase, a heme-containing dioxygenase, was studied. The enzyme did not require any cofactors, such as H₂O₂, however, both superoxide dismutase and catalase inhibited linoleate peroxidation by Fusarium lipoxygenase. A low concentration of H₂O₂ caused a distinct acceleration in enzymatic peroxidation. These results indicate that both O₂? and H₂O₂ are produced as essential intermediates of oxygen activation during formation of linoleate hydroperoxides by Fusarium lipoxygenase. This peroxidation reaction was also prevented by scavengers of singlet oxygen (¹O₂), but not by scavengers of hydroxy 1 radical (OH). Generation of O₂? in the enzyme reaction was detected by its ability to oxidize epinephrine to adrenochrome. Moreover, the rate of peroxide formation was greater in the D₂O than in the H₂O buffer system. These results suggest that the Haber–Weiss reaction (O₂^?+H₂O₂→OH^?+OH^·+¹O₂) is taking part in linoleate peroxidation by Fusarium lipoxygenase, and the ¹O₂ evolved could be responsible for the peroxidation of linoleate. H₂O₂ produced endogenously in the enzyme reaction might act as an activating factor for the enzyme. This possible mechanism of oxygen activation can explain the absence of a need for exogenous cofactors with Fusarium lipoxygenase in contrast to an other heme-containing dioxygenase, tryptophan pyrrolase, which requires an exogenous activating factor, such as H₂O₂.     

Background Factors of Reflux Esophagitis and Non-Erosive Reflux Disease: A Cross-Sectional Study of 10,837 Subjects in Japan

Background

Despite the high prevalence of gastroesophageal reflux disease (GERD), its risk factors are still a subject of controversy. This is probably due to inadequate distinction between reflux esophagitis (RE) and non-erosive reflux disease (NERD), and is also due to inadequate evaluation of adjacent stomach. Our aim is therefore to define background factors of RE and NERD independently, based on the evaluation of Helicobacter pylori infection and gastric atrophy.

Methods

We analyzed 10,837 healthy Japanese subjects (6,332 men and 4,505 women, aged 20–87 years) who underwent upper gastrointestinal endoscopy. RE was diagnosed as the presence of mucosal break, and NERD was diagnosed as the presence of heartburn and/or acid regurgitation in RE-free subjects. Using GERD-free subjects as control, background factors for RE and NERD were separately analyzed using logistic regression to evaluate standardized coefficients (SC), odds ratio (OR), and p-value.

Results

Of the 10,837 study subjects, we diagnosed 733 (6.8%) as RE and 1,722 (15.9%) as NERD. For RE, male gender (SC = 0.557, OR = 1.75), HP non-infection (SC = 0.552, OR = 1.74), higher pepsinogen I/II ratio (SC = 0.496, OR = 1.64), higher BMI (SC = 0.464, OR = 1.60), alcohol drinking (SC = 0.161, OR = 1.17), older age (SC = 0.148, OR = 1.16), and smoking (SC = 0.129, OR = 1.14) are positively correlated factors. For NERD, HP infection (SC = 0.106, OR = 1.11), female gender (SC = 0.099, OR = 1.10), younger age (SC = 0.099, OR = 1.10), higher pepsinogen I/II ratio (SC = 0.099, OR = 1.10), smoking (SC = 0.080, OR = 1.08), higher BMI (SC = 0.078, OR = 1.08), and alcohol drinking (SC = 0.076, OR = 1.08) are positively correlated factors. Prevalence of RE in subjects with chronic HP infection and successful HP eradication denotes significant difference (2.3% and 8.8%; p<0.0001), whereas that of NERD shows no difference (18.2% and 20.8%; p = 0.064).

Conclusions

Significantly associated factors of NERD are considerably different from those of RE, indicating that these two disorders are pathophysiologically distinct. Eradication of Helicobacter pylori may have disadvantageous effects on RE but not on NERD.     

Replication of DNA templates containing 5-formyluracil, a major oxidative lesion of thymine in DNA.

5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template. Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis. Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity. DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair. The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used. These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis.     

Different origins of bird and reptile sex chromosomes inferred from comparative mapping of chicken Z-linked genes

Recent progress of chicken genome projects has revealed that bird ZW and mammalian XY sex chromosomes were derived from different autosomal pairs of the common ancestor; however, the evolutionary relationship between bird and reptilian sex chromosomes is still unclear. The Chinese soft-shelled turtle (Pelodiscus sinensis) exhibits genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes have been identified. In order to investigate this further, we performed molecular cytogenetic analyses of this species, and thereby identified ZZ/ZW-type micro-sex chromosomes. In addition, we cloned reptile homologues of chicken Z-linked genes from three reptilian species, the Chinese soft-shelled turtle and the Japanese four-striped rat snake (Elaphe quadrivirgata), which have heteromorphic sex chromosomes, and the Siam crocodile (Crocodylus siamensis), which exhibits temperature-dependent sex determination and lacks sex chromosomes. We then mapped them to chromosomes of each species using FISH. The linkage of the genes has been highly conserved in all species: the chicken Z chromosome corresponded to the turtle chromosome 6q, snake chromosome 2p and crocodile chromosome 3. The order of the genes was identical among the three species. The absence of homology between the bird Z chromosome and the snake and turtle Z sex chromosomes suggests that the origin of the sex chromosomes and the causative genes of sex determination are different between birds and reptiles.     

Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin

The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation of NBS1 at DNA damage sites.     

Evolutionarily stable stalk to spore ratio in cellular slime molds and the law of equalization in net incomes

The evolutionarily stable stalk ratio (ESSR) in the cellular slime molds is studied when the fruiting body is formed by multiple clones of various size. The survival probability of a spore cell is assumed to depend on the stalk ratio and the fruiting body size. ESSR is obtained as the non-co-operative equilibrium (Nash solution) that maximizes the fitness of each clone. The following two predictions are obtained: (1) the number of spore cells produced by each clone forming a fruiting body tends to be equalized, even if a variation in clone size exists. As a result, the larger clones do not necessarily enjoy higher fitness than the smaller ones. (2) The stalk ratio and the overall fitness of the fruiting body decrease as the genetic diversity in the fruiting body increases. A condition for the stalk to spore ratio to be invariant of overall fruiting body size is also investigated. Finally, "the law of equalization in net incomes" is proposed, extending result (1) into the broader range of resource allocation problems.