Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

Glutathione (GSH) is synthesized by gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed gamma-GCS-GS catalyzing both gamma-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the gamma-GCS activity, S. agalactiae gamma-GCS-GS had different substrate specificities from those of Escherichia coli gamma-GCS. Furthermore, S. agalactiae gamma-GCS-GS synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-X(aa)-Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding gamma-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae gamma-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed gamma-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-Cys-X(aa). Whereas the substrate specificities of gamma-GCS domain protein and GS domain protein of S. agalactiae gamma-GCS-GS were the same as those of S. agalactiae gamma-GCS-GS.     

Production of emetic alkaloid by in vitro culture of cephaelis ipecacuanha A. Richard

Callus and adventitious roots were induced on leaf segments from shoot culture of Cephaelis ipecacuanha A. Richard on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, 1-naphthaleneacetic acid and kinetin. The contents of emetic alkaloids in calli, roots and root suspension cultures were quantified by HPLC. Roots cultured in solid and liquid Murashige-Skoog media yielded emetine and cephaeline. The amount of the two alkaloids in the root suspension culture was very similar to that of roots from ipecac mother plant grown in a greenhouse. In contrast, calli subcultured on Murashige-Skoog media containing combinations of 2,4-dichlorophenoxyacetic acid and kinetin produced only trace amounts of emetic alkaloids.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA l-naphthaleneacetic acid - Kin kinetin - MS Murashige-Skoog - EM emetine - CP cephaeline - DW dry weight.     

Postoperative Structural Brain Changes and Cognitive Dysfunction in Patients with Breast Cancer

Objective

The primary purpose of this study was to clarify the influence of the early response to surgery on brain structure and cognitive function in patients with breast cancer. It was hypothesized that the structure of the thalamus would change during the early response after surgery due to the effects of anesthesia and would represent one aspect of an intermediate phenotype of postoperative cognitive dysfunction (POCD).

Methods

We examined 32 postmenopausal females with breast cancer and 20 age-matched controls. We assessed their cognitive function (attention, memory, and executive function), and performed brain structural MRI 1.5 ± 0.5 days before and 5.6 ± 1.2 days after surgery.

Results

We found a significant interaction between regional grey matter volume (rGMV) in the thalamus (P < 0.05, familywise error (FWE), small volume correction (SVC)) and one attention domain subtest (P = 0.001, Bonferroni correction) after surgery in the patient group compared with the control group. Furthermore, the changes in attention were significantly associated with sevoflurane anesthetic dose (r ² = 0.247, β = ‒0.471, P = 0.032) and marginally associated with rGMV changes in the thalamus (P = 0.07, FWE, SVC) in the Pt group.

Conclusion

Our findings suggest that alterations in brain structure, particularly in the thalamus, may occur shortly after surgery and may be associated with attentional dysfunction. This early postoperative response to anesthesia may represent an intermediate phenotype of POCD. It was assumed that patients experiencing other risk factors of POCD, such as the severity of surgery, the occurrence of complications, and pre-existing cognitive impairments, would develop clinical POCD with broad and multiple types of cognitive dysfunction.     

Induction of ovulation in <Emphasis Type="Italic">Xenopus</Emphasis>without hCG injection: the effect of adding steroids into the aquatic environment

Background  

The African clawed frog, Xenopus laevis, is widely used in studies of oogenesis, meiotic cell cycle and early embryonic development. However, in order to perform such studies, eggs are normally collected after the injection of hCG into the dorsal lymph sac of fully-grown female frogs following pre-injection of PMSF. Although this protocol is established and used as standard laboratory approach, there are some concerns over whether the injections could cause the transmission of deleterious microorganisms. Moreover, these injection protocols require a competent skilled worker to carry out the procedure efficiently.     

Regional Gray Matter Density Associated with Cognitive Reflectivity–Impulsivity: Evidence from Voxel-Based Morphometry

When faced with a problem or choice, humans can use two different strategies: “cognitive reflectivity,” which involves slow responses and fewer mistakes, or “cognitive impulsivity,” which comprises of quick responses and more mistakes. Different individuals use these two strategies differently. To our knowledge, no study has directly investigated the brain regions involved in reflectivity–impulsivity; therefore, this study focused on associations between these cognitive strategies and the gray matter structure of several brain regions. In order to accomplish this, we enrolled 776 healthy, right-handed individuals (432 men and 344 women; 20.7 ± 1.8 years) and used voxel-based morphometry with administration of a cognitive reflectivity–impulsivity questionnaire. We found that high cognitive reflectivity was associated with greater regional gray matter density in the ventral medial prefrontal cortex. Our finding suggests that this area plays an important role in defining an individual’s trait associated with reflectivity and impulsivity.     

Heat shock factor binds to heat shock elements upstream of heat shock protein 70a and Samui genes to confer transcriptional activity in Bombyx mori diapause eggs exposed to 5°C

To understand the molecular mechanisms of how 5 °C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5′-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (−95 to −58) and -2 (−145 to −121), and SamuiHSE-1 (−84 to −55) and -2 (−304 to −290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5 °C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6–8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5 °C.     

The stabilization of beta-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression

Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.