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Background: Lactocaseibacillus paracasei subsp. paracasei NTU 101 (NTU101) is a well-known commercial probiotic with multiple health beneficial effects. In this study, the gut microbiota modulation effect of an NTU 101 product, Vigiis 101-LAB, on healthy human was investigated in a randomized, double-blind, placebo-controlled human trial., Results: Vigiis 101-LAB significantly modulated human gut microbiota at fourth and sixth weeks of trial (anosim analysis, P = 0.001). It also significantly improved peristalsis (P = 0.003) and shortened defecation interval of subjects. The shift of gut microbiota is significantly fit with defecation interval (P = 0.009) and stool shape (P = 0.001) of subjects., Conclusion: Our results suggest that Vigiis 101-LAB promotes human intestinal health with improvement of peristalsis and fecal quality. The gut modulation effects of Vigiis 101-LAB subsequently raised the abundance of vitamin B7, vitamin K, pyrimidine and purine biosynthesis pathways. Vigiis 101-LAB may promote peristalsis via purinergic pathway and possibly conferring prophylactic benefits against irritable bowel syndrome with constipation. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Probiotics, as defined by the World Health Organization, are live microorganisms that, when consumed in sufficient quantities, provide health benefits to the host. Although some countries have approved specific probiotic species for use in food, safety concerns may still arise with individual strains. Lacticaseibacillus paracasei subsp. paracasei NTU 101 (NTU 101), isolated from the gut of healthy infants, has demonstrated various probiotic effects and shown safety in a prior 28-day animal feeding study. To further verify its safety and mitigate potential risks, we performed a comprehensive genotypic and phenotypic safety evaluation in accordance with the European Food Safety Authority guidelines for safety assessment through whole genome sequencing and related literature. In this research, minimum inhibitory concentration testing identified NTU 101's resistance to chloramphenicol; however, subsequent gene analysis confirmed no associated risk of resistance. Assessments of safety, including biogenic amine content, hemolytic activity, mucin degradation, and D-lactic acid production, indicated a low level of risk. Additionally, a repeated-dose 90-day oral toxicity study in Sprague-Dawley rats revealed no toxicity at a dose of 2000 mg/kg body weight, further supporting the strain's safety for consumption. Based on these comprehensive analyses, NTU 101 is considered safe for regular consumption as a health supplement.

This study assessed the anti-obesity effects of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) both in vitro and in vivo. Initially, the cytotoxicity and lipid accumulation inhibitory effects of NTU 101 on 3T3-L1 cells were evaluated using the MTT assay and oil red O assay, respectively. Subsequently, the anti-obesity effects of NTU 101 were investigated in high-fat diet-induced obese rat. Moreover, western blotting was performed to measure the obesity-related protein expression of PPARα, PPARβ, PPARγ, C/EBPα, C/EBPβ, ATGL, p-p38 MAPK, p-ERK1/2, p-AMPK and CPT-1 in both 3T3-L1 adipocytes and adipose and liver tissues. Treatment with 16 × 10 8  CFU/mL NTU 101 reduced lipid accumulation in 3T3-L1 adipocytes by more than 50 %. Oral administration of NTU 101 significantly attenuated body weight gain, as well as adipose tissue weight. NTU 101 administration enhanced fatty acid oxidation increasing expression levels of PPARα, CPT-1, and p-AMPK proteins in liver tissue, while simultaneously inhibited adipogenesis by reducing PPARγ and C/EBPα proteins in adipose tissue. Furthermore, NTU 101 supplementation positively modulated the composition of gut microbiota, notably increasing the abundance of Akkermansiaceae. This present study suggests that NTU 101 exerts anti-obesity effects by regulating gut microbiota, fatty acid oxidation, lipolysis and adipogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Chronic allergic skin conditions, including atopic dermatitis (AD), are characterized by pruritus, erythema, xerosis, desquamation, and inflammation, significantly impacting quality of life. Long-term steroid use, while common in treatment, carries the risk of adverse effects. Previous studies have demonstrated the potential of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) in alleviating AD symptoms from a preventive perspective. This study, however, focuses on exploring NTU 101's therapeutic potential by investigating its effects on regulatory T cell (Treg) maturation and Th1/Th2 balance. The results revealed that NTU 101 administration effectively reduced serum IgE levels and inflammatory cell infiltration in the skin, leading to a significant improvement in both epidermal and dermal thickness in the AD model. Additionally, NTU 101 modulated the immune response by increasing the proportion of CD4+/IL-4+ (Th2) cells in the spleen and concurrently enhancing FOXP3 expression in CD4+/CD25+ cells, which is critical for Treg cell development. This immune modulation was further associated with a rebalancing of the Th1/Th2 ratio, achieved by increasing the proportion of CD4+/IFN-γ+ (Th1) cells. Moreover, NTU 101 influenced the proportion of CD4+IL-17+ (Th17) cells, thereby supporting neutrophil maturation and promoting allergen clearance, ultimately mitigating AD symptoms. These findings underscore the potential of NTU 101 not only in managing AD symptoms but also in modulating key immune pathways involved in the pathogenesis of the disease, offering a promising alternative or adjunct to conventional steroid therapies.

Alzheimer's disease (AD) is the most common neurodegenerative disease, with symptoms appearing in the cerebral cortex and hippocampus. amyloid β peptide (Aβ) has been shown to deposit in the brain, causing oxidative stress and inflammation, leading to impaired memory and learning. Lactocaseibacillus fermentation can produce deglycosylated isoflavones with high physiological activity, which can scavenge free radicals, enhance total antioxidant capacity and inhibit oxidative inflammatory responses. Therefore, in this study, Lactocaseibacillus paracasei subsp. paracasei NTU101 (NTU101) fermented soybean milk and its extracts were used as test substances, and AD model rats were established by infusion of Aβ40 in the brain for 28 days, and the preventive and ameliorating effects of NTU 101 fermented soymilk were discussed. Effects of soymilk and unfermented soymilk on AD, and explore its effects on AD. Main functional ingredients. The results showed that deglycosylated isoflavones in NTU101 fermented soybean milk improved AD symptoms. Mechanisms of actions include the inhibition of oxidative inflammation; reduction in the expression of risk factors for tau protein and apo E protein production, the deposition of Aβ40 around the hippocampus, and the expression of TLR-2 and RAGE proteins in astrocytes and microglia; and improvement in the memory and learning ability., (© 2024. The Author(s).)

Atopic dermatitis (AD) is a recurrent allergic disease characterized by symptoms such as itching, redness, swelling, dryness, scaling skin, inflammation, and tissue damage. The underlying pathogenesis of AD remains unclear. Steroid drugs are commonly used in the clinical treatment of AD; however, their long-term use may lead to associated complications. Numerous studies have indicated that probiotics could modulate the immune system, enhance immune function, or suppress excessive immune responses. In this study, Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) was orally administered for a duration of 4 weeks, followed by the induction of AD using ovalbumin (OVA) in a mouse model. The skin condition of the stimulated site was observed during the induction period. Subsequently, the serum immunoglobulin E (IgE) content, splenocyte T cell typing, and skin histological interpretation were examined to evaluate the efficacy of NTU 101 in alleviating AD symptoms in allergen-exposed animals. The findings indicated that administering NTU 101 beforehand effectively alleviated skin symptoms in animals with AD. It reduced the infiltration of inflammatory cells in skin tissue sections, and compared to the OVA group, there was a significant reduction in the thickening of the epidermal cell layer (decreased from 89.0 ± 20.2 µM to 48.6 ± 16.0 µM) and dermis layer (decreased from 310.3 ± 69.0 µM to 209.7 ± 55.5 µM). Moreover, the proportion of regulatory T (Treg) cells and T helper 2 (Th2) cells in splenocytes significantly increased, while the proportions of T helper 1 (Th1) and T helper 17 (Th17) cells did not differ. It is speculated that the potential mechanism by which NTU 101 prevents AD involves increasing the expression of Forkhead box protein P3 (FOXP3) and promoting Treg cell maturation, thereby alleviating allergic reaction symptoms associated with AD.

Background/aim: PHD and RING finger domain-containing protein 1 (PHRF1) ubiquitinates TGIP (TG-interacting protein) and redistributes cPML (cytoplasmic variant of PML) to the cytoplasm to enhance TGF-β signaling by. It is unclear whether PHRF1 affects invasion and survival when both mutations of the activated oncogene Kras and inactivation of the tumor suppressor p53 are present., Materials and Methods: We knockout PHRF1 expression using Crispr-Cas9 editing in HCT116-p53 -/- (KrasG13D/p53 -/- ) cells and analyzed the expression profile in HCT116-p53 -/- PHRF1 -/- cells., Results: In contrast to lung cancer A549 (KrasG12S/p53wt) cells, the expression of Zeb1, a transcription factor for epidermal-mesenchymal transition (EMT), was not affected in PHRF1-knockout HCT116 p53 -/- cells. Instead, SOX4 displayed a significant contribution to the impaired invasion in HCT116-p53 -/- PHRF1 -/- cells. Mechanistically, the C-terminal SRI domain of PHRF1 was required for both transwell invasion and SOX4 expression. The reintroduction of SOX4 into HCT116-p53 -/- PHRF1 -/- cells partially restored their invasive capability., Conclusion: This study sheds light on the role of PHRF1 in the invasion of colorectal cancer HCT116-p53 -/- cells, which harbor the oncogenic KrasG13D mutation and lack p53. These findings provide novel insights regarding the role of PHRF1 in invasion by modulating SOX4 expression in colorectal cancer HCT116-p53 -/- cells., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Hepatoma-derived growth factor (HDGF) overexpression and uncontrolled reactive oxygen species (ROS) accumulation are involved in malignant transformation and poor prognosis in various types of cancer. However, the interplay between HDGF and ROS generation has not been elucidated in hepatocellular carcinoma. Here, we first analyzed the profile of HDGF expression and ROS production in newly generated orthotopic hepatomas by ultrasound-guided implantation. In situ superoxide detection showed that HDGF-overexpressing hepatomas had significantly elevated ROS levels compared with adjacent nontumor tissues. Consistently, liver tissues from HDGF-deficient mice exhibited lower ROS fluorescence than those from age- and sex-matched WT mice. ROS-detecting fluorescent dyes and flow cytometry revealed that recombinant HDGF (rHDGF) stimulated the production of superoxide anion, hydrogen peroxide, and mitochondrial ROS generation in cultured hepatoma cells in a dose-dependent manner. In contrast, the inactive Ser103Ala rHDGF mutant failed to promote ROS generation or oncogenic behaviors. Seahorse metabolic flux assays revealed that rHDGF dose dependently upregulated bioenergetics through enhanced basal and total oxygen consumption rate, extracellular acidification rate, and oxidative phosphorylation in hepatoma cells. Moreover, antioxidants of N-acetyl cysteine and MitoQ treatment significantly inhibited HDGF-mediated cell proliferation and invasive capacity. Genetic silencing of superoxide dismutase 2 augmented the HDGF-induced ROS generation and oncogenic behaviors of hepatoma cells. Finally, genetic knockdown nucleolin (NCL) and antibody neutralization of surface NCL, the HDGF receptor, abolished the HDGF-induced increase in ROS and mitochondrial energetics. In conclusion, this study has demonstrated for the first time that the HDGF/NCL signaling axis induces ROS generation by elevating ROS generation in mitochondria, thereby stimulating liver carcinogenesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Studies conducted in the same seas or even study sites nearby each other, showed very different community structure, implying the patchiness of free-living marine nematodes which may be related to the sedimentary environment such as sediment type and food availability of the study area. This study was motivated by the concerns about the impacts of high level of anthropogenic activities on Chaojing Park (gazetted as Wanghaixiang's Chao-Jing Bay Resource Conservation Area (WCJBRA) in 2016). The present study provides baseline knowledge of free-living marine nematode community structure in WCJBRA and identify potential marine nematodes as bioindicators to indicate possible impacts of the anthropogenic activities to the Chaojing Park. A total of 15 stations were selected in the subtidal zones of WCJBRA and its adjacent area. Marine nematode sample collection was carried out on the 13th and 14th of September 2019 using SCUBA diving technique. Results showed positive correlation between nematode density and medium sand (500μm-1.0mm). Presence of certain species such as Daptonema sp., Pomponema sp. and Innocuonema sp. indicates presence of disturbances in S12 and S13. Several species also showed potential to be introduced as indicator for healthy environment subjected to further studies on nematode-pollutants relationship, particularly on autecology as per se. Higher species diversity, H' index of S1-S8 and S11 was categorised as Good Condition; followed by station with moderate species diversity index (S9, S10, S14 -Moderate Condition) zone; and lastly S12, S13 and S15 (Poor Condition)., Competing Interests: The authors have declared that no competing interests exist.

Human PUF-A/PUM3 is a RNA and DNA binding protein participating in the nucleolar processing of 7S to 5.8S rRNA. The nucleolar localization of PUF-A redistributes to the nucleoplasm upon the exposure to genotoxic agents in cells. However, little is known regarding the roles of PUF-A in tumor progression. Phosphoprotein database analysis revealed that Y259 phosphorylation of PUF-A is the most prevalent residue modified. Here, we reported the importance of PUF-A's phosphorylation on Y259 in tumorigenesis. PUF-A gene was knocked out by the Crispr/Cas9 method in human cervix epithelial HeLa cells. Loss of PUF-A in HeLa cells resulted in reduced clonogenic and lower transwell invasion capacity. Introduction of PUF-AY259F to PUF-A deficient HeLa cells was unable to restore colony formation. In addition, the unphosphorylated mutant of PUF-A, PUF-AY259F, attenuated PUF-A protein stability. Our results suggest the important role of Y259 phosphorylation of PUF-A in cell proliferation., Competing Interests: The authors have declared that no competing interests exist.

Ordered arrays of polymer nanostructures have been widely investigated because of their promising applications such as solar-cell devices, sensors, and supercapacitors. It remains a great challenge, however, to manipulate the shapes of individual nanostructures in arrays for tailoring specific properties. In this study, an effective strategy to prepare anisotropic polymer nanopillar arrays via photo-fluidization is presented. Azobenzene-containing polymers (azopolymers) are first infiltrated into the nanopores of ordered anodic aluminum oxide (AAO) templates. After the removal of the AAO templates using weak bases, azopolymer nanopillar arrays can be prepared. Upon exposure of linearly polarized lights, azobenzene groups in the azopolymers undergo trans-cis-trans photoisomerization, causing mass migration and elongation of the nanopillar along with the polarization directions. As a result, anisotropic nanopillar arrays can be fabricated, of which the deformation degrees are controlled by the illumination times. Furthermore, patterned nanopillar arrays can also be constructed with designed photomasks. This work presents a practical and versatile strategy to fabricate arrays of anisotropic nanostructures for future technical applications., (© 2021 Wiley-VCH GmbH.)

PHRF1 is involved in transforming growth factor β (TGF-β) signaling to constrain the formation of acute promyelocytic leukemia (APL) in mouse APL models. PHRF1 also participates in modulating non-homologous end-joining. However, the role of PHRF1 in mammalian dendrite architecture and synaptic plasticity is unclear. Here, we investigated the role of PHRF1 in dendritic formation in the murine hippocampus using Camk2a promoter driven-iCre recombinase to conduct a PHRF1 conditional knockout, namely PHRF1 Δ/Δ , in the forebrain region. PHRF1 Δ/Δ mice developed normally, but exhibited anxiety-like behaviors and displayed defective spatial memory. Alterations of dendritic complexity in apical and basal dendrites of pyramidal neurons were noticed in PHRF1 Δ/Δ mutants. Furthermore, electrical stimulation in the hippocampal CA1 region after the TGF-β1 treatment showed a reduced synaptic plasticity in PHRF1 Δ/Δ mice. Immunoblotting analysis indicated that PHRF1 ablation affected the TGF-β signaling. Collectively, our results demonstrate that PHRF1 is important for the dendritic architecture and required for spatial memory formation in the hippocampus.

The morphology of first stage zoea larvae of three pandalid shrimps Plesionika crosnieri Chan Yu, 1991, P. ortmanni Doflein, 1902, and P. semilaevis Bate, 1888 are described for the first time from larvae obtained in laboratory culture. The zoea I is compared amongst those species of the genus Plesionika with larval morphology known. It is found that the length of the rostrum, the number of anteroventral denticles on the carapace, presence of exopodal setae in the maxillule, and setation pattern of the peduncle of antennule, endopod of antenna, and coxa/basis of maxillipeds are useful characters in distinguishing these larvae.

Background/purposes: Routine cell number determination for specific Lactobacillus strain by cultivation requires at least 4-7 days. Thus rapid and specific cell number determine methods such as strain-specific quantitative PCR (qPCR) are valuable. However, qPCR method is vulnerable to difficult PCR target such as dimer/secondary structure forming sequence., Methods: In this study, a two-component, "Ct contrast" approach was applied to strain-specific qPCR system following the development of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) strain-specific PCR with random amplification of polymorphic DNA (RAPD)-derived strain-specific sequences., Results: The quantitative range of the NTU 101 strain-specific qPCR system was 3.0 × 10 1 to 3.0 × 10 5 copies for pure cultures, and 3.0 × 10 2 to 3.0 × 10 5 copies for multi-strain or unknown food samples. The results of spike in test and real sample testing suggested that non-specific weak background signals did not compromise test specificity, and demonstrated the potential of the NTU 101 strain-specific qPCR system in food samples., Conclusion: The two-component, "Ct contrast" approach is useful for qPCR discrimination when no ideal PCR target is available or the variance of the target site is unpredictable. The Ct contrast approach might provide a simple and robust solution for other challenging qPCR targets., (Copyright © 2017. Published by Elsevier B.V.)

The larvae of the deep-sea pandalid shrimp Plesionika grandis Doflein, 1902 were successfully reared in the laboratory for the first time. The larvae reached the eighth zoeal stage in 36 days, both of which are longest records for the genus. Early larval stages of P. grandis bear the general characters of pandalid shrimps and differ from the other two species of Plesionika with larval morphology known in the number of spines on the anteroventral margin of carapace, number of tubercles on antennule, endopod segmentation in antenna, and third maxilliped setation. Although members in Plesionika are often separated into species groups, members of the same species group do not necessarily have similar early larval morphology. Since the zoea VIII of P. grandis still lacks pleopods and fifth pereiopod, this shrimp likely has at least 12 zoeal stages and a larval development of 120 days.

Parinya Limviriyakul, Li-Chun Tseng, Jiang-Shiou Hwang, and Tung-Wei Shih (2016) Symbiotic anomuran and brachyuran crabs were identified after extensive surveys of reef zones, especially from sponges, scleractinian corals, crinoids, and sea urchins, in the coastal areas of the southern East China Sea. Twenty-nine species belonging to 17 genera, 9 families, and 2 infraorders were identified (3 were identified to the generic level). More crabs belonged to the infraorder Brachyura (82.8%) than to Anomura (17.2%). Two anomuran symbionts ( Allogalathea elegans and Petrolisthes virgatus ) and 5 brachyuran symbionts ( Tetralia glaberrima , Tetralia rubridactyla , Trapezia cymodoce , Trapezia septata , and Cymo melanodactylus ) are common in this area. Two species of Anomura ( Lauriea simulata , Petrolisthes virgatus ) and 3 of Brachyura ( Gonatonotus nasutus , Tetralia aurantistellata and Tetralia nigrolineata ) were identified for the first time from waters adjacent to Taiwan. These records represent the northernmost recorded of L. simulata and T. aurantistellata . The occurrence of P. virgatus is the second in the western Pacific Ocean. This study revealed the geospatial distribution of symbiotic crabs, which connects the region from the southern Ryukyu arc to the Coral Triangle, and provides the supporting taxonomic account of symbiotic anomuran and brachyuran crab fauna inhabiting the reef zone in northern Taiwan.

The first four larval stages of the pandalid shrimp Chlorotocus crassicornis (A. Costa, 1871) are described and illustrated from laboratory-reared material obtained from ovigerous females collected in the southwestern Spain and south Taiwan. The second to fourth larval stages of this species are reported for the first time to science. Detailed examination of the first larval stages reveals that previous description misidentified some key larval characters which have prevented its identification in plankton samples. It is found that the zoeal morphology of Chlorotocus is not very different from other pandalid larvae, and in fact closely resembles Plesionika and Heterocarpus.

The bonding properties and the potential energy surfaces for the chemical reactions of doubly bonded compounds that have the >E13═E15< pattern are studied using density functional theory (M06-2X/Def2-SVPD). Nine molecules, >E13═P< (E13 = B, Al, Ga, In, and Tl) and >B═E15< (E15 = N, P, As, Sb, and Bi), are used as model reactants in this work. Four types of chemical reactions, H2 addition, acetonitrile, benzophenone [2 + 2] cycloadditions, and dimethylbutadiene [4 + 2] cycloaddition, are used to study the chemical reactivity of these inorganic, ethylene-like molecules. The results of these theoretical analyses show that only the >B═P< molecule has a weak B═P double bond, while the >Al═P< , >Ga═P< , >In═P< , >Tl═P< , >B═N< , >B═As<, >B═Sb<, and >B═Bi< compounds are best described as having a strong single σ bond, instead of a traditional p-p π bond. The theoretical results also show that the singlet-triplet energy gap can be used to determine the relative reactivity of these doubly bonded molecules. According to these theoretical investigations, it is predicted that the order of reactivity is as follows: B═P > Al═P > Ga═P > In═P > Tl═P and B═N ≪ B═P < B═As < B═Sb < B═Bi. The conclusions drawn are consistent with the available experimental observations.

The aim of the present work was to assess the genotoxic activity and the potential for toxicity upon repeated dosing of "Vigiis 101" powder, a probiotic consisting of dried bacteria Lactobacillus paracasei subsp. paracasei NTU 101. Results of the Ames test in Salmonella typhimurium strains TA1537, TA98, TA100, TA102, and TA1535 showed that Vigiis 101 (⩽5 mg per plate) was not mutagenic. We used experiments on ICR mice to evaluate the genotoxicity of Vigiis 101. Compared to the control, high-dose Vigiis 101 administration (16.72 g per kg of body weight) did not cause significant changes either in the number of reticulocytes or in the percentage (occurrence) of micronucleated reticulocytes. A mammalian chromosomal aberration test showed that the number of Chinese hamster ovary cells with abnormal chromosomes was <4% after Vigiis 101 treatment (maximal concentration was 5 mg/ml). A 28-day oral toxicity assay in Wistar rats was performed to assess the no-observed-adverse-effect level of Vigiis 101. Compared to the control, high-dose Vigiis 101 administration (5000 mg/kg/day) had no effects on mortality and body weight and did not cause toxicopathological lesions. Taken together, these results show that Vigiis 101 has no significant mutagenic or toxic effects., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Monascus species produce several potent bioactive metabolites through polyketide secondary metabolic pathways; however, little is known of the regulation of metabolic processes in this organism. Therefore, we investigated the effect of extreme growth conditions on the production of secondary metabolites by Monascus purpureus strain NTU 568 and identified pathways that plausibly regulate the polyketide pathways by using proteomic analysis. Citrinin, a type of antibiotic, is synthesized through the polyketide pathway in M. purpureus NTU 568. We hypothesized that production of citrinin might be inhibited by ethanol. When M. purpureus NTU 568 was cultured in the medium containing 4% ethanol, the secretion of secondary metabolites was inhibited, but the dry cell weight was increased. We also found that branched chain amino acid degradation and the expression level of aldehyde dehydrogenase (ALDH) were downregulated, but proteins related to the heat shock response were induced. Furthermore, polyketide synthesis-related proteins, fatty acid synthase, epoxide hydrolase, and proteins involved in the shikimate secondary metabolic pathway were inhibited by ethanol treatment. These results suggested that reduced production of polyketide metabolites resulted not only from the expression levels of proteins in the polyketide synthesis pathway but also from reduction in the concentration of primary metabolism-generated molecules (e.g., acetyl-CoA, fatty acids) that are used as substrates for polyketide syntheses. This study provides insights into the polyketide secondary metabolism of Monascus as well as into improvement of the production of bioactive secondary metabolites in Monascus species., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The eggs of three deep-sea pandalid shrimps Heterocarpus abulbus, H. hayashii and H. sibogae are successfully hatched in the laboratory. The first zoeal stage of these shrimps are described, with those of H. abulbus and H. hayashii being reported for the first time. First zoeae of different Heterocarpus species can be distinguished by the spination at the anteroventral carapace, body size, rostral length and appendage setation.

Error-prone PCR was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant of the α6 helix (lid domain), with an 189E to V substitution at residue 189, lost its thermostability but exhibited higher activity than that of its wild-type predecessor (r03Lip). Site-saturation mutagenesis was used to explore the sequence-function relationship. Five other mutants also lost thermostability (20-40%) but exhibited enhanced thermoactivity (6.3-79-fold). The mutant E189I showed the highest activity retaining 50% activity after maintaining it at 65 °C for 24 h. In comparison to r03Lip, the mutant E189I had a higher affinity for p-nitrophenyl palmitate and p-nitrophenyl stearate (61 and 56% decreased Km) and catalytic efficiency (42-fold and 18-fold increased kcat/Km). The mutant lipase retained its tolerance to n-hexane, but had an improved transesterification activity. The results suggest that residue Glu189 plays a significant role in the thermostability and activity of this thermoalkalophilic lipase., (© Springer Science+Business Media B.V. 2011)

A batch culture of Geobacillus sp. NTU 03 was subjected to a rapid temperature shift for investigating the stress response. Several known heat-shock responses for protein, DNA, and cell membrane recurring were observed on two-dimensional (2D) gels. Heat caused protein and cell membrane disruption greatly affected the electron transport chain. Further, heat caused lower dissolved oxygen (DO) solubility resulting in insufficient oxygen to be electron acceptor, and the NADH could not be reoxidized. Hence, we observed seven dehydrogenase that used NADH as electron donor were downregulated on the 2D gels. In contrast, succinate dehydrogenase that used FADH(2) as electron donor was upregulated. However, this induction may simultaneously increase generation of superoxide; therefore the cellular redox state was imbalanced. We observed that superoxide dismutase (2D gel) and zinc ion ABC transporter (mRNA quantification) were upregulated, whereas ferric ion ABC transporter (2D gel and mRNA quantification) was downregulated. Increase in the reactive oxygen or nitrogen species scavenging activities were also observed. For responding the lower DO solubility, a transient activation of nitrate respiration was observed at transcriptional level. Our results support the view that both heat stress and heat-induced stress should be considered together when investigating the stress responses of thermophiles., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5'-triphosphatase activity that specifically removes the gamma phosphate from the 5' end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m(7)GpppG cap structure was formed at the 5' end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5'-triphosphatase activity.

This study deals with the localization of crustacean hyperglycemic hormone (CHH, Pej-SGPIII) and molt-inhibiting hormone (MIH, Pej-SGP-IV) in the eyestalk of the kuruma prawn Penaeus japonicus using immunohistochemistry. High-titer and highly specific antisera were raised in rabbits against synthetic Pej-SGP-III C-terminal peptide (Glu-Glu-His-Met-Ala-Ala-Met-Gln-Thr-Val-NH2) and Pej-SGP-IV C-terminal peptide (Val-Trp-Ile-Ser-Ile-Leu-Asn-Ala-Gly-Gln-OH), both of which were conjugated with bovine serum albumin by a cross linker. Eyestalks were removed from mature male prawns at the intermolt stage of the molting cycle and fixed in Bouin's solution. Serial sections stained immunohistochemically showed that neurosecretory cells of Pej-SGP-III and Pej-SGP-IV were located in the same cluster of the medulla terminalis ganglionic X-organ (MTGX), and that three kinds of neurosecretory cells, which were stained with anti-PejSGP-III antiserum and/or anti-Pej-SGP-IV antiserum were present. The number of neurosecretory cells which stained with both antisera was much fewer than that of neurosecretory cells which stained with one of the antisera only. The axon and axon terminals in the sinus gland were also stained and the staining density of the sinus gland was always deeper than that of the neurosecretory cells.

Purification and characterization of the female-specific protein (vitellogenin) from the hemolymph of mature female prawn, Macrobrachium rosenbergii, were the objectives of this study. The comparison of biochemical characteristics between vitellogenin and ovarian vitellin was also conducted. Hemolymph vitellogenin was purified with DEAE, hydroxylapatite, and another DEAE chromatographic column. The specific protein (vitellogenin) was shown in the fractions of chromatographic columns on the basis of ELISA, Western blotting, and immunoprecipitation. A purified vitellogenin was obtained with an apparent molecular weight of 700 kDa as determined by PAGE. The purified vitellogenin was considered as a lipoglycoprotein on the basis of staining data. Three subunits (170, 100, and 89 kDa) in purified vitellogenin and two subunits (100 and 89 kDa) in vitellin were detected with SDS-PAGE. Nondisulfide bonds were found in the binding of polypeptide subunits. Only the 89-kDa subunit was a glycopolypeptide in both vitellogenin and vitellin. The amino acid composition of vitellogenin differed from that of vitellin in a few amino acids. Eight amino acid sequences from the N-terminal end of 89- and 100-kDa subunits were determined and they were identical between vitellogenin and vitellin. Seven amino acid sequence from the N-terminal end of the 170-kDa subunit were also identical to the 100-kDa subunit. Purified vitellogenin was more susceptible to precipitation in a solution with low ionic strength than vitellin. This study suggests a close relationship between vitellogenin and vitellin in M. rosenbergii in their biochemical characteristics., (Copyright 1997 Academic Press.)

The domain structures of lipid-free and lipid-bound apolipoprotein A-I (apo A-I) containing reduced and oxidized methionines were analyzed by limited proteolysis. Lipid-free apo A-I is cleaved primarily in the extreme carboxy-terminus and, to a much lesser extent, in the central region of the protein between residues 115 and 136. Oxidation of methionines 112 and 148 to the corresponding sulfoxides in putative amphipathic helices 4 (P99-E120) and 6 (P143-A164), respectively, causes helices 1 (L44-G65), 2 (P66-S87), and 7 (P165-G186) to become susceptible to protease digestion. These results are consistent with a discrete, globular tertiary structure for the lipid-free protein minimally formed from amphipathic helices 1, 2, 4, 6, and 7. In distinct contrast to lipid-free apo A-I, lipid-bound apo A-I is most susceptible to cleavage in the extreme amino-terminus and, to a lesser extent, in both the central and carboxy-terminal regions. The observed cleavage pattern for the reduced lipid-bound protein supports the existence of many of the turns between helices predicted by sequence analysis of the lipid-bound protein. Methionine oxidation of lipid-bound protein results in a decreased protease susceptibility in the extreme amino-terminus and a concomitant increase in protease susceptibility in the central and carboxy-terminal regions. The results from methionine oxidation indicate the oxidation state of the protein is an important determinant in defining the conformation of both lipid-free and lipid-bound apo A-I.

Penclomedine [3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl)pyridine], an antitumor agent, is currently in Phase I clinical trials and is believed to be a prodrug. In these studies, cerebellar effects have been dose limiting. Previous studies identified 4-demethylpenclomedine (4-DM-PEN) as the major plasma metabolite in rodents and humans. 4-DM-PEN was demonstrated to be an antitumor-active metabolite of penclomedine in vivo when evaluated against the penclomedine-sensitive MX-1 human breast tumor xenograft implanted either s.c. or intracerebrally and is believed to be on the metabolic activation pathway of penclomedine. Because earlier studies revealed an absence of neurotoxic cerebellar effects for 4-DM-PEN in contrast to penclomedine in a rat model, this metabolite may be a candidate for an alternative to penclomedine in the clinic for treatment of breast cancer or brain tumors, if the cerebellar effects of penclomedine preclude its further clinical development. Because neither penclomedine nor 4-DM-PEN were very active in vitro, the metabolism of penclomedine was also investigated using rat liver microsomes in an attempt to identify the ultimate active form of the drug. Metabolites and putative metabolites were prepared by chemical synthesis for antitumor evaluation in vitro and in vivo. A reductive metabolite, alpha,alpha-didechloro-PEN, was observed to be much more cytotoxic than penclomedine or 4-DM-PEN in vitro, but evaluation of this and the other metabolites and putative metabolites in vivo against the MX-1 tumor failed to identify any active metabolite among the structures evaluated other than 4-DM-PEN. The limited activity of 4-DM-PEN in vitro indicates that it, like penclomedine, is also a prodrug, demonstrating a need for additional studies on the metabolic activation of penclomedine to identify the ultimate active form of the drug.

Certain thiol-containing compounds catalyze, in a chemical reaction, the isomerization of 9-cis-retinoic acid to a mixture of all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, and 9,13-dicis-retinoic acid. In the presence of such catalysts, all-trans-retinoic acid gives rise to the same mixture. Reactions approaching equilibrium contain more all-trans-retinoic acid than either of the other isomers. Small molecules effective as catalysts are mercaptoethanol, L-cysteine methyl ester, glutathione, and N-acetyl-L-cysteine. Apoferritin (a thiol-containing protein), native microsomes, and, to a lesser extent, boiled microsomes catalyze the reaction. In intact cells, these interconversions also occur in a process inhibited by a sulfhydryl-specific reagent. The thiol-catalyzed isomerization of 9-cis-retinoic acid may be relevant in the biological activity of this compound.

Retinoid beta-glucuronides have emerged as biologically active, water-soluble, natural retinoids with relatively few toxic and teratogenic effects. The mechanism of action of these glucuronides in the control of epithelial differentiation, growth, and tumorigenesis is unknown. Since retinoyl beta-glucuronide (RAG) contains a free carboxyl group, we studied the interactions of RAG with cellular retinoic acid-binding protein (CRABP) and nuclear receptors of retinoic acid (RARs), the possible mediators of the biological action of retinoic acid (RA). RAG did not exhibit any significant affinity to bind either CRABP or RARs. During 24- and 48-hr incubations of RAG in chick cytosol, detectable amounts of RA were generated which interacted with the RA receptors. In chick skin, the biological activity of RAG may be due to this slowly released RA. Other possible modes of action of RAG are suggested.

An NADPH-requiring enzyme present in rat liver microsomes catalyzes the conversion of retinol to retinal; the enzyme activity is induced by 3-methylcholanthrene (MC). The optimum activities for both the constitutive and induced reactions occur within a pH range of 8.2-8.7. For the constitutive enzyme, the KM and Vmax values are 285 microM retinol and 18 nmol of retinal/mg protein/hr, respectively; for the MC-induced activity, the corresponding values are 133 microM retinol and 33 nmol of retinal/mg protein/hr. Neither the constitutive nor the induced activities are detectable in microsomes from seven other tissues. Both hepatic activities are inhibited by citral, ketoconazole, SKF 525-A, and alpha-naphthoflavone; both are stimulated by divalent cations. Neither is inhibited by 3-amino-1,2,4-triazole, pyrazole, or sodium azide or stimulated by monovalent cations. The enzyme appears to be a previously unreported retinol oxidase, distinct from the known cytosolic enzymes, retinal reductase and retinol dehydrogenase.

An enzyme present in rat liver microsomes catalyzes the conversion of retinyl methyl ether (RME) to retinol; NADPH is required for activity. The optimum pH for the reaction is 7.4; the KM and Vmax values are 120 microM RME and 14.3 nmol of retinol/mg protein/hr, respectively. As a substrate, the 2,3,6-trimethyl-4-methoxyphenyl analog of RME is as effective as RME. There is, however, no measurable activity for dealkylation of retinyl ethyl ether or retinyl butyl ether. Hepatic enzyme activity for the metabolism of RME is induced by 3-methylcholanthrene but not by phenobarbital or RME itself. The induced activity also requires NADPH as a cofactor. The optimum pH for the induced enzyme is 8.4; the KM and Vmax values are 50 microM RME and 111 nmol of retinol/mg protein/hr, respectively. For this enzyme, RME is a better substrate than the 2,3,6-trimethyl-4-methoxyphenyl analog of RME; retinyl ethyl ether is less effective; and again, there is no measurable activity with retinyl butyl ether as a substrate. Neither constitutive nor induced activity is detectable in microsomes from lung, spleen, stomach, kidney, small intestine, or large intestine. The enzyme activity that cleaves retinoid ethers appears to be similar to other microsomal NADPH-requiring O-dealkylases and different from a reported tetrahydropteridine-requiring dealkylase.

Enzymatic activity present in liver microsomes from rats slowly hydrolyzed N-(4-hydroxyphenyl)retinamide (4HPR). A product of the reaction was all-trans-retinoic acid. The reaction, which had a pH optimum greater than 8.6, was stimulated by divalent cations, particularly Mn2+. Enzyme activity was highest in liver microsomes but was also present in kidney microsomes, liver cytoplasm, and spleen cytoplasm. Of 10 possible substrates tested, the 13-cis- and all-trans-forms of N-ethylretinamide were most active. The all-trans-form of 4HPR was much more active than the 13-cis-form. Neither 13-cis- nor all-trans-retinoyl leucine was a substrate. Because no detectable [14C]all-trans-retinoic acid could be found in the livers of rats after doses of [14C]4HPR, we conclude that this enzyme is not extensively active in intact animals.

1. Studies on the metabolism of 14C-Michler's ketone (4,4'-bis-(dimethylamino)[carbonyl- 14C]benzophenone) in rats have revealed that this carcinogen is subject to demethylation, ring-hydroxylation and N-acetylation after adjacent methyl groups have been removed. 2 As identified by mass spectral analysis, microsomal metabolites are the mono-, di-, tri- and tetra-demethylated derivatives. 3. The major metabolites appearing in the bile are the di- and tri-demethylated derivatives and the N-acetylated tetra-demethylated compound; minor metabolites, tentatively identified, are the ring-hydroxylated derivatives of di- and tri-demethylated Michler's ketone and of N-acetylated tri- and tetra-demethylated Michler's ketone. 4. The major urinary metabolite is tentatively identified as a ring-hydroxylated derivative of N-acetylated, di-demethylated Michler's ketone; a minor urinary metabolite lacks the hydroxyl group. 5. Injection of 14C-Michler's ketone into rats resulted in the irreversible binding of radioactivity to liver proteins. 6. When the rats were pretreated with phenobarbital, this binding was increased and extended to proteins of some other tissues and to DNA and RNA of liver and DNA of kidney.

Induced and constitutive microsomal enzymes of mouse and hamster lungs catalyze both the hydroxylation of benzo(alpha)pyrene and reactions that lead to its irreversible binding to macromolecules. For mouse and hamster, the induced lung hydroxylases have Km values of 1.10 and 0.52 muM, respectively. The induced hydroxylases are strongly inhibited by 7,8-benzoflavone and are stimulated by cyclohexene oxide, an inhibitor of epoxide hydrase. Formation of the macromolecular product by the induced "binding" enzyme follows. Michaelis-Menten kinetics, except for substrate inhibition, and has Km values of 0.52 and 0.25 muM for lung microsomes from mouse and hamster, respectively. These reactions are also inhibited by 7,8-benzoflavone. The reaction catalyzed by the constitutive hydroxylase of mouse lungs is characterized by a brief lag period but proceeds in a linear fashion after the lag. The enzyme requires 60 muM benzo(alpha)pyrene to achieve maximum reaction velocity. Above this concentration, strong substrate inhibition is observed; accurate values for Vmax and Km cannot be derived. The constitutive hydroxylases are moderately inhibited by butylated hydroxytoluene, retinol, cyclohexene oxide, and 7,8-benzoflavone. The product of the constitutive "binding" enzyme is formed in a reaction that follows Michaelis-Menten kinetics. The Km value for enzymes from mouse and hamster lungs are 11.8 and 4.9 muM, respectively. Formation of this product is strongly inhibited by butylated hydroxytoluene and by retinol but not strongly by 7,8-benzoflavone or cyclohexene oxide. Since other evidence indicates that a constitutive enzyme may be involved in carcinogenesis by benzo(alpha)pyrene and since this reaction is inhibited by two known anticarcinogens, we suggest that it may be involved in this process.

Studies on the in vivo and in vitro disposition of 4,4'-[14C]-methylenebis(N,N-dimethyl)benzamine (reduced Michler's ketone, RMK) were performed. Osborne-Mendel rats retained, after 24 hr, 78% of a p.o. dose of [14C]RMK. At 24 hr after an i.p. dose, fat, liver, and intestine represented major sites for deposition of radioactivity. The major urinary metabolite of RMK, representing 36% of the total radioactivity recovered in the urine, was N,N'-diacetyl-4,4'-(hydroxymethylene)dianiline. In vitro microsomal metabolism of RMK involved demethylation. Products included N,N-dimethyl-4,4'-methylenedianiline, N,N'-dimethyl-4,4'-methylenedianiline, N-methyl-4,4'-methylenedianiline, and 4,4'-methylenedianiline, representing 44.7, 5.3, 11.8, and 6.9%, respectively, of the total radioactivity recovered from the reaction mixture. Although none of the microsomal metabolites was a direct-acting mutagen in the standard Salmonella typhimurium assay, all could be activated to mutagens when incubated with 9000 X g liver supernatants and reduced nicotinamide adenine dinucleotide phosphate. The activation of 4,4'-methylenedianiline to a mutagen suggests that the methyl groups of RMK are not required for the conversion of RMK to a reactive electrophile.

In liver cytosolic preparations containing added glutathione, radioactivity from [14C]1,2-dibromoethane becomes bound preferentially to added polycytidylic acid. When microsomes and NADPH are present, however, the radioactivity becomes bound selectively to microsomal proteins.

Biochemical investigations relating to the mechanism of action and mechanism of activation have been made for the carcinogen, 4-chloro-2-methylaniline. Radioactivity from 4-chloro-2-[methyl-14C]methylaniline became extensively bound to protein, DNA, and RNA of rat liver, but macromolecules of some of the other tissues examined contained little radioactivity. Enzymatic activity dependent upon reduced nicotinamide adenine dinucleotide and leading to irreversible binding to radioactivity from labeled 4-chloro-2-methylaniline to macromolecules in the reaction system was present in microsomes from rat liver. The activity was inducible by phenobarbital. Two soluble products of microsomal enzymes were identified by mass spectral analysis and chemical synthesis as 5-chloro-2-hydroxylaminotoluene and 4,4'-dichloro-2,2'-dimethylazobenzene. The hydroxylamino compound appears to be a more activated form of 4-chloro-2-methylaniline.

Two enzymatic reactions, catalyzed by mouse lung microsomes and distinguishable by selective inhibition and kinetic studies, lead to irreversible binding of benzo[a]pyrene to macromolecules present in vitro reaction systems. One type (low Km) is inducible in the lungs of mice by treatment with benz[a]anthracene and is subject to inhibition by 7,8-benzoflavone. The other type (high Km) is predominant in lungs of untreated mice, but a small amount of low-Km activity is also present. The high-Km activity may be involved in carcinogenesis by benzo[a]pyrene, for it is inhibited by butylated hydroxytoluene, retinol or disulfiram, each of which is reported to have anticarcinogenic activity in intact animals.

Certain thiol-containing compounds catalyze, in a chemical reaction, the isomerization of all-trans-retinoic acid (RA) to 13-cis-RA and of 13-cis-RA to RA. Reactions approaching equilibrium contain more RA than 13-cis-RA. Small molecules effective as catalysts are glutathione, mercaptoethanol, and L-cysteine methyl ester. L-Cysteine is not a catalyst and inhibits the reaction catalyzed by glutathione or mercaptoethanol. Apoferritin (a thiol-containing protein), native microsomes, and, to a lesser extent, boiled microsomes catalyze the reaction, but their activity is reduced or eliminated by prior incubation with iodoacetate. Other cis and trans isomeric retinoids are also substrates for this reaction; the reactions proceed more readily for the cis isomers. The thiol-catalyzed isomerization of RA and 13-cis-RA may account for the observations of both cis and trans forms of retinoids in tissues of animals after administration of either.