Growth-Dependent Catalase Localization in Exiguobacterium oxidotolerans T-2-2T Reflected by Catalase Activity of Cells

A psychrotolerant and H₂O₂-resistant bacterium, Exiguobacterium oxidotolerans T-2-2^T, exhibits extraordinary H₂O₂ resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.     

Populations of follicles in F344/N rats during aging

Follicular populations were investigated in female F344/N rats to better understand the aging process of the rat ovary. Ovaries dissected at various ages (spanning 1–36 months old) were submitted for histological examination. The total number of primordial, growing (primary and secondary), tertiary, and atretic follicles as well as corpora lutea (CL) were counted in hematoxylin–eosin- and azocarmine–aniline-blue-stained ovarian sections. The number of healthy follicles including primordial, growing and tertiary follicles decreased rapidly between the first and third months and gradually thereafter. CL were found in 3-month-old rats, and their number remained unchanged until 18 months of age, at which point it decreased. The number of atretic follicles started to increase in rats older than 18 months, which corresponded to the cessation of estrous cyclicity. Several healthy follicles and CL were observed even in 36-month-old rats.     

Kallikrein-related Peptidase 5 Functions in Proteolytic Processing of Profilaggrin in Cultured Human Keratinocytes

Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage.     

Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics

NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited k_cat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.     

Near Fixation of 374l Allele Frequencies of the Skin Pigmentation Gene SLC45A2 in Africa

The L374F polymorphism of the SLC45A2 gene, encoding the membrane-associated transporter protein that plays an important role in melanin synthesis, has been suggested to be associated with skin color in human populations. In this study, the detailed distribution of the 374f and 374l alleles has been investigated in 2,581 unrelated subjects from 36 North, East, West, and Central African populations. We found once more the highly significant (p < 0.001) correlation coefficient (r = 0.957) cline of 374f frequencies with degrees of latitude in European and North African populations. Almost all the African populations located below 16° of latitude are fixed for the 374l allele. Peul, Toucouleur, and Soninké populations have 374l allele frequencies of 0.06, 0.03, and 0.03, respectively.     

Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3 h after intravenous injection and rapid clearance from normal tissues within 24 h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3 h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.     

Studies on the Metabolic Products of Oospora sp.

A microorganism was isolated from the air of a patient-room and classified in the genus Oospora. This microorganism was cultured on a malt extract medium, and the mycellium was separated from the culture filtrate. A new compound (O-1), m.p. 129°C, C₁₁H₁₀O₃, and eburicoic acid, m.p. 290°C, C₃₁H₅₀O₃ were obtained from the dried mycellium. Another new compound (O-2), m.p. 176°C, C₁₁H₈O₅ was obtained from the culture filtrate.     

Adenylate Cyclase in Silkworm: Regulation by Calcium and a Calcium-binding Protein

Homogenates of silkworm pupal fat body were separated into particulate and supernatant fractions by centrifugation. The particulate fraction was further washed with EGTA. Adenylate cyclase activity of the washed particulate fraction was stimulated 2-fold by the addition of supernatant fraction in the presence of low concentrations of Ca²⁺. The activating factor in supernatant was heat-stable, non-dialyzable and trypsin-sensitive, and shown to be a Ca²⁺-dependent regulator protein. For the activation of adenylate cyclase by the regulator protein, the optimum concentrations of free Ca²⁺ were in a range of 2 µm, and higher concentrations of Ca²⁺ were inhibitory.