Ultrastructural and Biochemical Analysis of In Vivo and In Vitro Capsid Assembly of the Alkaliphilic Phage Φ1N2-2 for Applications in Nanomedicine

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

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Biochemical analysis of programmed cell death during premeiotic stages of spermatogenesis in vivo and in vitro

Developmental Genetics ◽

10.1002/dvg.1020160207 ◽

1995 ◽

Vol 16 (2) ◽

pp. 140-147 ◽

Cited By ~ 29

Author(s):

Gloria V. Callard ◽

Joan C. Jorgensen ◽

J. Michael Redding

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Biochemical Analysis

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Functional Analysis of the RdxA and RdxB Nitroreductases of Campylobacter jejuni Reveals that Mutations in rdxA Confer Metronidazole Resistance

Journal of Bacteriology ◽

10.1128/jb.01638-09 ◽

2010 ◽

Vol 192 (7) ◽

pp. 1890-1901 ◽

Cited By ~ 5

Author(s):

Deborah A. Ribardo ◽

Lacey K. Bingham-Ramos ◽

David R. Hendrixson

Keyword(s):

Campylobacter Jejuni ◽

Biochemical Analysis ◽

Normal Growth ◽

Deletion Mutations ◽

Replication Errors ◽

Metronidazole Resistance ◽

H Pylori ◽

Dna Replication Errors

ABSTRACT Campylobacter jejuni is a leading cause of gastroenteritis in humans and a commensal bacterium of the intestinal tracts of many wild and agriculturally significant animals. We identified and characterized a locus, which we annotated as rdxAB, encoding two nitroreductases. RdxA was found to be responsible for sensitivity to metronidazole (Mtz), a common therapeutic agent for another epsilonproteobacterium, Helicobacter pylori. Multiple, independently derived mutations in rdxA but not rdxB resulted in resistance to Mtz (Mtzr), suggesting that, unlike the case in H. pylori, Mtzr might not be a polygenic trait. Similarly, Mtzr C. jejuni was isolated after both in vitro and in vivo growth in the absence of selection that contained frameshift, point, insertion, or deletion mutations within rdxA, possibly revealing genetic variability of this trait in C. jejuni due to spontaneous DNA replication errors occurring during normal growth of the bacterium. Similar to previous findings with H. pylori RdxA, biochemical analysis of C. jejuni RdxA showed strong oxidase activity, with reduction of Mtz occurring only under anaerobic conditions. RdxB showed similar characteristics but at levels lower than those for RdxA. Genetic analysis confirmed that rdxA and rdxB are cotranscribed and induced during in vivo growth in the chick intestinal tract, but an absence of these genes did not strongly impair C. jejuni for commensal colonization. Further studies indicate that rdxA is a convenient locus for complementation of mutants in cis. Our work contributes to the growing knowledge of determinants contributing to susceptibility to Mtz (Mtzs) and supports previous observations of the fundamental differences in the activities of nitroreductases from epsilonproteobacteria.

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Possible Role for Cellular Karyopherins in Regulating Polyomavirus and Papillomavirus Capsid Assembly

Journal of Virology ◽

10.1128/jvi.01221-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 9848-9857 ◽

Cited By ~ 21

Author(s):

Gregory Bird ◽

Malinda O'Donnell ◽

Junona Moroianu ◽

Robert L. Garcea

Keyword(s):

Dna Binding ◽

Capsid Assembly ◽

Dependent Manner ◽

Cellular Mechanisms ◽

Virion Assembly ◽

Localization Signal ◽

Murine Polyomavirus ◽

Cytoplasmic Assembly

ABSTRACT Polyomavirus and papillomavirus (papovavirus) capsids are composed of 72 capsomeres of their major capsid proteins, VP1 and L1, respectively. After translation in the cytoplasm, L1 and VP1 pentamerize into capsomeres and are then imported into the nucleus using the cellular α and β karyopherins. Virion assembly only occurs in the nucleus, and cellular mechanisms exist to prevent premature capsid assembly in the cytosol. We have identified the karyopherin family of nuclear import factors as possible “chaperones” in preventing the cytoplasmic assembly of papovavirus capsomeres. Recombinant murine polyomavirus (mPy) VP1 and human papillomavirus type 11 (HPV11) L1 capsomeres bound the karyopherin heterodimer α2β1 in vitro in a nuclear localization signal (NLS)-dependent manner. Because the amino acid sequence comprising the NLS of VP1 and L1 overlaps the previously identified DNA binding domain, we examined the relationship between karyopherin and DNA binding of both mPy VP1 and HPV11 L1. Capsomeres of L1, but not VP1, bound by karyopherin α2β1 or β1 alone were unable to bind DNA. VP1 and L1 capsomeres could bind both karyopherin α2 and DNA simultaneously. Both VP1 and L1 capsomeres bound by karyopherin α2β1 were unable to assemble into capsids, as shown by in vitro assembly reactions. These results support a role for karyopherins as chaperones in the in vivo regulation of viral capsid assembly.

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Repeated Neonatal Sevoflurane Induced Neurocognitive Impairment Through NF-κB-Mediated Pyroptosis

10.21203/rs.3.rs-404817/v1 ◽

2021 ◽

Author(s):

Jing Dai ◽

Xue Li ◽

Cai Wang ◽

Shuxin Gu ◽

Lei Dai ◽

...

Keyword(s):

Hippocampal Neurons ◽

Biochemical Analysis ◽

Neurological Diseases ◽

Neurocognitive Impairment ◽

Postnatal Period ◽

Neuronal Morphology ◽

Clinical Settings

Abstract Background: Exposure to general anesthesia (GA) during the postnatal period is associated with neuroinflammation and long-term neurocognitive impairment in preclinical and clinical settings. Pyroptosis, a novel type of programmed cell death that along with inflammation, plays an important role in the mechanism of diverse neurological diseases. Nevertheless, its role in GA-induced neuroinflammation and neurocognitive impairment in developing brain has not been investigated. Methods: Rats at postnatal day 6 or primary hippocampal neurons at 9 days in vitro, received 3% sevoflurane for 2 hours daily for three consecutive days. A pharmacological inhibitor of nuclear factor (NF)‑κB (BAY 11-7082) was administered to suppress NF-κB activation. Histological and biochemical analysis were performed to assess the pyroptosis and neuronal and synaptic damages both in vivo and in vitro. In addition, behavioral tests were performed to evaluate the neurocognitive ability in rats.Results: Repeated sevoflurane exposures activated NF-κB-mediated pyroptosis and neuroinflammation in the hippocampus of developing rats, caused damages in neuronal morphology and synaptic integrity, and induced neurocognitive impairment in rats. BAY 11-7082, the inhibitor of NF-κB, suppressed the activation of pyroptosis, attenuated the neuronal and synaptic damages, and ameliorated the neurocognitive impairment induced by repeated sevoflurane in the developing rats.Conclusions: These results demonstrated that repeated sevoflurane GA might induce neuroinflammation and cognitive impairment in developing rats via activation of NF-κB-mediated pyroptosis. Our findings characterize a novel role for pyroptosis as a potential therapeutic target in neuroinflammation to repeated neonatal GA.

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Injection of antibodies into the nucleus of amphibian oocytes: an experimental means of interfering with gene expression in the living cell

Development ◽

10.1242/dev.97.supplement.223 ◽

1986 ◽

Vol 97 (Supplement) ◽

pp. 223-242

Author(s):

Ulrich Scheer

Keyword(s):

Living Cell ◽

Biochemical Analysis ◽

A Priori ◽

In Vitro Assays ◽

Active Chromatin ◽

Nuclear Antigens ◽

Experimental Approaches ◽

Active Genes

The chromatin constituents associated with transcriptionally active genes can be studied by a variety of experimental approaches. Most of the methods used involve the isolation of active chromatin fractions followed by biochemical analysis, or are based on in vitro assays with the ultimate goal of reconstituting faithful transcription by adding purified and defined components to the transcriptional machinery (for reviews see Mathis, Oudet & Chambon, 1980; Rose, Stetler & Jacob, 1983a; Beroldingen et al. 1984; Reeves, 1984; Dynan & Tjian, 1985; Sentenac, 1985). Since these approaches require isolation of chromatin or of individual components therefrom, possible structural rearrangements or selective losses of certain constituents cannot a priori be excluded. Therefore it often remains questionable whether the results obtained in vitro reflect the actual situation in the living cell. In order to examine the nature of transcriptionally active chromatin in the living cell and to decipher the in vivo function of nuclear proteins in transcriptional processes, we have microinjected antibodies to various nuclear antigens directly into the nucleus of living amphibian oocytes.

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Recent Advances in Understanding and Engineering Polyketide Synthesis

F1000Research ◽

10.12688/f1000research.7326.1 ◽

2016 ◽

Vol 5 ◽

pp. 208 ◽

Cited By ~ 6

Author(s):

Wenjun Zhang ◽

Joyce Liu

Keyword(s):

Biochemical Analysis ◽

Polyketide Synthase ◽

Bioactive Molecules ◽

New Developments ◽

Recent Advances ◽

Fundamental Understanding ◽

Block Formation ◽

Enzymatic Machinery

Polyketides are a diverse group of natural products that form the basis of many important drugs. The engineering of the polyketide synthase (PKS) enzymes responsible for the formation of these compounds has long been considered to have great potential for producing new bioactive molecules. Recent advances in this field have contributed to the understanding of this powerful and complex enzymatic machinery, particularly with regard to domain activity and engineering, unique building block formation and incorporation, and programming rules and limitations. New developments in tools for in vitro biochemical analysis, full-length megasynthase structural studies, and in vivo heterologous expression will continue to improve our fundamental understanding of polyketide synthesis as well as our ability to engineer the production of polyketides.

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Synthesis of repressible acid phosphatase in Saccharomyces cerevisiae under conditions of enzyme instability

Molecular and Cellular Biology ◽

10.1128/mcb.2.1.1-10.1982 ◽

1982 ◽

Vol 2 (1) ◽

pp. 1-10

Author(s):

K A Bostian ◽

J M Lemire ◽

H O Halvorson

Keyword(s):

Saccharomyces Cerevisiae ◽

Acid Phosphatase ◽

Structural Gene ◽

Gene Transcription ◽

Biochemical Analysis ◽

Growth Conditions ◽

Regulatory Model ◽

Produce Acid

The synthesis of repressible acid phosphatase in Saccharomyces cerevisiae was examined under conditions of blocked derepression as described by Toh-e et al. (Mol. Gen. Genet. 162:139-149, 1978). Based on a genetic and biochemical analysis of the phenomenon these authors proposed a new regulatory model for acid phosphatase expression involving a simultaneous interaction of regulatory factors in the control of structural gene transcription. We demonstrate here that under growth conditions that fail to produce acid phosphatase the enzyme is readily inactivated. Furthermore, we demonstrate under these conditions the production of acid phosphatase mRNA which is active both in vitro and in vivo in the synthesis of enzyme. This eliminates any step prior to translation of acid phosphatase polypeptide as an explanation for the phenomenon. We interpret our results for the block in appearance of acid phosphatase as a result of both deaccelerated growth and cellular biosynthesis during derepression, accompanied by an enhanced instability of the enzyme.

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HS6ST1 Insufficiency Causes Self-Limited Delayed Puberty in Contrast With Other GnRH Deficiency Genes

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2018-00646 ◽

2018 ◽

Vol 103 (9) ◽

pp. 3420-3429 ◽

Cited By ~ 10

Author(s):

Sasha R Howard ◽

Roberto Oleari ◽

Ariel Poliandri ◽

Vasiliki Chantzara ◽

Alessandro Fantin ◽

...

Keyword(s):

Biochemical Analysis ◽

Hypogonadotropic Hypogonadism ◽

Wild Type Mouse ◽

Tissue Expression ◽

Postnatal Growth ◽

Delayed Puberty ◽

Gene Variant ◽

Whole Exome

Abstract Context Self-limited delayed puberty (DP) segregates in an autosomal-dominant pattern, but the genetic basis is largely unknown. Although DP is sometimes seen in relatives of patients with hypogonadotropic hypogonadism (HH), mutations in genes known to cause HH that segregate with the trait of familial self-limited DP have not yet been identified. Objective To assess the contribution of mutations in genes known to cause HH to the phenotype of self-limited DP. Design, Patients, and Setting We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited DP, validated the pathogenicity of the identified gene variant in vitro, and examined the tissue expression and functional requirement of the mouse homolog in vivo. Results A potentially pathogenic gene variant segregating with DP was identified in 1 of 28 known HH genes examined. This pathogenic variant occurred in HS6ST1 in one pedigree and segregated with the trait in the six affected members with heterozygous transmission (P = 3.01 × 10−5). Biochemical analysis showed that this mutation reduced sulfotransferase activity in vitro. Hs6st1 mRNA was expressed in peripubertal wild-type mouse hypothalamus. GnRH neuron counts were similar in Hs6st1+/− and Hs6st1+/+ mice, but vaginal opening was delayed in Hs6st1+/− mice despite normal postnatal growth. Conclusions We have linked a deleterious mutation in HS6ST1 to familial self-limited DP and show that heterozygous Hs6st1 loss causes DP in mice. In this study, the observed overlap in potentially pathogenic mutations contributing to the phenotypes of self-limited DP and HH was limited to this one gene.

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