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Abnormal photoreceptor outer segment development and early retinal degeneration in kif3a mutant zebrafish
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Reem H. Alhasani Xinzhi Zhou Margaret Mullin James Reilly Wenchang Li Mugen Liu Xinhua Shu 《Cell biochemistry and function》2016,34(6):429-440
Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A (kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport. 相似文献
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Background
Spermatogenesis represents the transformation process at the level of cellular development. KIF3A and KIF3B are believed to play some roles in the assembly and maintenance of flagella, intracellular transport of materials including organelles and proteins, and other unknown functions during this process. During spermatogenesis in Eriocheir sinensis, if the sperm shaping machinery is dependent on KIF3A and KIF3B remains unknown.Methodology/Principal Findings
The cDNA of KIF3A and KIF3B were obtained by designing degenerate primers, 3′RACE, and 5′RACE. We detected the genetic presence of kif3a and kif3b in the heart, muscle, liver, gill, and testis of E. sinensis through RT-PCR. By western blot analysis, the protein presence of KIF3A and KIF3B in heart, muscle, gill, and testis reflected the content in protein level. Using in situ hybridization and immunofluorescence, we could track the dynamic location of KIF3A and KIF3B during different developmental phases of sperm. KIF3A and KIF3B were found surrounding the nucleus in early spermatids. In intermediate spermatids, these proteins expressed at high levels around the nucleus and extended to the final phase. During the nuclear shaping period, KIF3A and KIF3B reached their maximum in the late spermatids and were located around the nucleus and concentrated in the acrosome to some extent.Conclusions/Significance
Our results revealed that KIF3A and KIF3B were involved in the nuclear and cellular morphogenesis at the levels of mRNA and protein. These proteins can potentially facilitate the intracellular transport of organelles, proteins, and other cargoes. The results represent the functions of KIF3A and KIF3B in the spermatogenesis of Crustacea and clarify phylogenetic relationships among the Decapoda. 相似文献4.
Zhangji Dong Shuwen Wu Chenwen Zhu Xueting Wang Yuanyuan Li Xu Chen Dong Liu Liang Qiang Peter W. Baas Mei Liu 《Traffic (Copenhagen, Denmark)》2019,20(1):71-81
KIF15, the vertebrate kinesin‐12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin‐5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice‐blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9‐based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP‐alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon‐Beard (R‐B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R‐B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons. 相似文献
5.
T. M. Fahima Azira A. A. Nursolehah Y. Norhayati M. I. A. Majid A. A. Amirul 《World journal of microbiology & biotechnology》2011,27(10):2287-2295
A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was found capable of producing terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] using γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol as the carbon source. The present of
3HB, 3HV and 4HB monomers were confirmed by gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. PHA concentration
of 1.9 g/l was the highest value obtained using the combination of 1,4-butanediol and 1-pentanol through one-step cultivation
process. PHA concentration obtained through two-step cultivation process was higher for all the combinations and the highest
value achieved was 2.5 g/l using γ-butyrolactone and 1-pentanol as carbon source. Various molar fractions of 4HB and 3HV ranging
from 6 to 14 mol% and 39 to 87 mol%, respectively were produced through two-step cultivation process by manipulating the concentration
of γ-butyrolactone. As the culture aeration was reduced, the molar fraction of 3HV and 4HB increased from 40 to 67 mol% and
10 to 24 mol%, respectively while the dry cell weight and PHA content decreased. The terpolymer produced was characterized
using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The number-average molecular weight
(M
n) and the melting temperature (T
m)) of the terpolymer were in the range of 177–484 kDa and 160–164°C, respectively. 相似文献
6.
Prohibitin plays a key role in maintaining mitochondrial membrane integrity and retaining its normal function. We have initially
cloned and sequenced the cDNA of prohibitin from testis of the crab Eriocheir sinensis. The 1,357 bp Prohibitin cDNA comprises a 105 bp 5′ untranslated region, a 427 bp 3′ untranslated region and a 825 bp open
reading frame. Protein alignment substantiates that the Prohibitin has 70.2, 69.8, 70.5, 70.9, 72.4, 70.6 and 74.9% identity
with its homologues in Mus musculus, Homo sapiens, Gallus gallus, Danio rerio, Xenopus tropicalis, Drosophila mojavensis and Aedes aegypti, respectively. In situ hybridization revealed that the Prohibitin mRNA was mainly localized around the proacrosomal vesicle
and nucleus membrane in early-stage spermatid. In the following middle stage, Prohibitin mRNA was situated inside the invaginated
region of half-moon-like nucleus and surrounded the proacrosomal vesicle. In late-stage spermatid, the mRNA was aggregated
in the acrosomal tubule, the band between the acrosome and cup-like nucleus, remanent cytoplasm as well. In the mature sperm,
mRNA was only found in the acrosomal tubule and the limited space between the nucleus and acrosome. Therefore, we presume
that Prohibitin may fulfill critical functions in the spermiogenesis of Eriocheir sinensis. 相似文献
7.
Aslı Ozdilek Burcu Cengel Gaye Kandemir Yasemin Tayanc Ercan Velioglu Zeki Kaya 《Plant Systematics and Evolution》2012,298(2):337-349
The genetic diversity and evolutionary divergence in Liquidambar species and Liquidambar orientalis varieties were compared with respect to the matK gene. A total of 66 genotypes from 18 different populations were sampled in southwestern Turkey. The matK region, which is about 1,512 bp in length, was sequenced and studied. L. orientalis, L. styraciflua, and L. formosana had similar magnitude of nucleotide diversity, while L. styraciflua and L. acalycina possessed higher evolutionary divergence. The highest evolutionary divergence was found between L. styraciflua and eastern Asian Liquidambar species (0.0102). However, the evolutionary divergence between L. orientalis and other species was of a similar magnitude. The maximum-parsimony phylogenetic tree showed that L. styraciflua and L. orientalis formed a closer clade while East Asian species were in a separate clade. This suggests that the North Atlantic Land Bridge
through southern Greenland may have facilitated continuous distribution of Liquidambar species from southeastern Europe to eastern North America in early Tertiary period. The maximum-parsimony tree with only
18 Oriental sweetgum populations indicated that there were two main clusters: one with mainly L. orientalis var. integriloba and the other with var. orientalis and undetermined populations. High nucleotide diversity (0.0028) and divergence (0.00072) were found in L. orientalis var. integriloba populations and Muğla-1 geographical region. This region could be considered as the major refugium and genetic diversity
center for the species. The low genetic diversity and divergence at intraspecies level suggest that L. orientalis populations in Turkey share an ancestral polymorphism from which two varieties may have evolved. 相似文献
8.
A full-length metallothionein-1(MT-1) cDNA was cloned from the Chinese mitten crab, Eriocheir sinensis, based upon the hepatopancreas cDNA library. The full-length cDNA contained a single 180 bp open reading frame that encoded
a 59 amino acid protein. The deduced amino acid sequence was cysteine (Cys)-rich, with residues observed in patterns characteristic
of other reported MTs: Cys–X–Cys, Cys–X–X–Cys, or Cys–X–X–X–Cys. Gene structure obtained via PCR yielded a 3816 bp gene, which
was comprised of three exons and two introns arranged in a “3 + 2” pattern. The cloned 5′flanking region (1,735 bp) contained
several predicted binding sites, which included MREs, AP-1, SP1, USF, GATA, HNF-1, and HSF. MT-1 mRNA expression analysis
revealed that while levels were highest in the hepatopancreas, expression was abundant in testis and thoracic ganglia, moderate
in intestine (P < 0.05), and weak in other tissues (P < 0.05). MT-1 mRNA expression exhibited reproductive variation in the male, with levels approximately tenfold greater in
August, during seasonal gonadal maturation, compared to other times of the year. Cu2+ exposure via tank water (0–1 mg/l for 7 days) resulted in a dose-dependent bell curve response in MT-1 mRNA expression, with
peak expression observed after exposure to 0.1 mg/l Cu2+. A time course experiment (0.1 mg/l Cu2+ over 9 days) revealed MT-1 mRNA expression peaked sharply on day 5 before gradually decreasing with prolonged exposure. In
the present report, we provide sequence analysis of the first MT-1 gene cloned in E. sinensis, and evidence that its physiological and toxicological regulation is evolutionary conserved. 相似文献
9.
Chanprateep S Katakura Y Visetkoop S Shimizu H Kulpreecha S Shioya S 《Journal of industrial microbiology & biotechnology》2008,35(11):1205-1215
A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically
and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole
ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was
200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value,
whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained.
Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB
was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally
as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies,
a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating
variable, and was successfully proved by the experiments.
The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession
number EF988626. 相似文献
10.
(S)-Tetrahydroberberine [(S)-THB] oxidase is the last enzyme of benzylisoquinoline alkaloids pathway which catalyzes the dehydrogenation of four hydrogen
atoms of (S)-THB to produce berberine, the final step of berberine biosynthesis. A (S)-THB gene, designated as Cs(S)-THBO (Genbank accession No. HQ393909), was cloned from a Corydalis saxicola cDNA library by rapid amplification of cDNA ends. The full-length of cDNA of Cs(S)-THBO was 1127 bp with an open reading frame of 699 bp that predicted to encode a 232-amino acid polypeptide, with a predicted
molecular mass of 25.20 kDa. Cs(S)-THBO was the first (S)-THBO gene found in C. saxicola. Real-time quantitative PCR analysis indicated that Cs(S)-THBO was constitutively expressed in roots, stems, leaves and flowers of C. saxicola, and with the highest expression level in roots. The results of treatment experiment for plant defense responses revealed
that expression of Cs(S)-THBO had a prominent diversity. Recombinant Cs(S)-THBO protein expressed in Escherichia coli strain BL21 (DE3) was active. The results of feeding experiment and HPLC–DAD–ESI–MSn analysis showed that Cs(S)-THBO had the function of catalyzing (S)-tetrahydroberberine to berberine. 相似文献
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A chalcone reductase (CHR) gene was isolated from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). The full-length cDNA of A. mongholicus CHR, designated as Amchr (GenBank accession No. HM357239), was 1196 bp long. It had a 957 bp open reading frame encoding a 318-amino acid protein
of 35 kDa, a 67 bp 5′ non-coding region and a 172 bp 3′-untranslated region. The putative AmCHR protein showed striking similarity
to CHR from other leguminous species. Two-dimensional structure modeling showed that AmCHR consisted of 45.28% α-helix, 10.38%
extended strand and 44.34% random coil. Prediction showed that three-dimensional AmCHR was a global protein containing an
aldo-ket-red domain, with a putative Asp-Tyr-Lys-His catalytic tetrad in the center. The AmCHR gene was 1251 bp long, consisting
of three exons and two introns. Intron I was 125 bp and intron II was 169 bp long. Southern blot analysis indicated that Amchr belonged to a small multigene family. Under natural conditions, Amchr was expressed differentially in the root, stem and leaf tissues of A. mongholicus, with a preferential expression in the root. The recombinant AmCHR protein was successfully expressed in Escherichia coli strain BL21 with pET42a vector. The result showed that the expressed AmCHR protein had molecular weight of about 35 kDa,
which matched the size of the predicted protein by bioinformatic analysis. This study opened avenues towards understanding
of the function of AmCHR protein and the role of the Amchr gene in the calycosin-7-O-β-d-glucoside branch pathway in A. mongholicus. 相似文献
13.
Homo- and heterodimers of Kif5 proteins form the motor domain of Kinesin-1, a major plus-end directed microtubule motor. Kif5s have been implicated in the intracellular transport of organelles, vesicles, proteins, and RNAs in many cell types. There are three mammalian KIF5s. KIF5A and KIF5C proteins are strictly neural in mouse whereas, KIF5B is ubiquitously expressed. Mouse knockouts indicate crucial roles for KIF5 in development and human mutations in KIF5A lead to the neurodegenerative disease Hereditary Spastic Paraplegia. However, the developmental functions and the extent to which individual kif5 functions overlap have not been elucidated. Zebrafish possess five kif5 genes: kif5Aa, kif5Ab, kif5Ba, kif5Bb, and kif5C. Here we report their tissue specific expression patterns in embryonic and larval stages. Specifically, we find that kif5As are strictly zygotic and exhibit neural-specific expression. In contrast, kif5Bs exhibit strong maternal contribution and are ubiquitously expressed. Lastly, kif5C exhibits weak maternal expression followed by enrichment in neural populations. In addition, kif5s show distinct expression domains in the larval retina. 相似文献
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15.
The complete mitochondrial genome 15,650 bp in size of the Deracantha onos has been determined. The gene content, base composition and codon usage of D. onos are coincident to typical hexapods mitochondrial genomes. Genes arrangement of D. onos is identical to Gryllotalpa orientalis, Ruspolia dubia and Anabrus simplex, in that the relative locations of tRNALys and tRNAAsp was different to that of Locusta migratoria. All tRNAs could be folded into the typical cloverleaf secondary structure, excluding tRNASer(AGN) which forms another structure according to the Steinberg–Cedergren tertiary structure. Sequence analysis of the A + T-rich
region with Dot-plot did not find any conspicuous repeat clusters. Two poly-thymine (poly-T) nucleotide stretches of 20 bp
and 11 bp in size, which may involved in the recognition of replication origin, were found on the H-strand and L-strand in
the A + T-rich region of the D. onos mitogenome, respectively. One open reading frame (ORF) 87 amino acids in size was found on the H-strand, but Protein Blast
searches analysis indicated that it was a nonfunctional ORF. 相似文献
16.
We describe the complete mitochondrial genomes of the green lacewing species Chrysoperla nipponensis (Okamoto, 1914) and Apochrysa matsumurae Okamoto 1912 (Neuroptera: Chrysopidae). The genomes were 16,057 and 16,214 bp in size, respectively, and comprised 37 genes
(13 protein coding genes, 22 tRNA genes and two rRNA genes). A major noncoding (control) region was 1,244 bp in C. nipponensis and 1,407 in A. matsumurae, and the structure was simpler than that reported in other Neuroptera, lacking conserved blocks or long tandem repeats. The
overall arrangement of genes was almost the same as that found in most arthropod mitochondrial genomes, with the one exception
of a tRNA rearrangement to tRNA-Cys–tRNA-Trp–tRNA-Tyr, rather than the plesiomorphic tRNA-Trp–tRNA-Cys–tRNA-Tyr. A high A + T
content (78.89 and 79.02%, respectively), A + T-rich codon bias, and a mismatch between the most-used codon and its corresponding
tRNA anticodon were observed as a typical feature of the insect mitochondrial genome. 相似文献
17.
Miklós Takó Adél Tóth László G. Nagy Judit Krisch Csaba Vágv?lgyi Tamás Papp 《Antonie van Leeuwenhoek》2010,97(1):1-10
In this study, a β-glucosidase coding gene (bgl) of the zygomycete fungus Rhizomucor miehei has been cloned and characterized. The gene comprises a total of 2,826 bp including the coding sequence of a 717 amino acids
length putative protein and 10 introns dispersed in the whole coding region. The putative N-and C-terminal catalytic domains
(aa 68 to aa 274 and aa 358–601, respectively) were identified; the two domains are connected with a 84-amino-acids linker.
The catalytic region showed an extensive sequence homology with other fungal β-glucosidases classified as family 3 glycoside
hydrolases. The isolated Rhizomucor gene was expressed in the related fungus Mucor circinelloides. Transformant Mucor strains maintained the introduced plasmid in an autoreplicative manner and showed significantly higher cellobiase activity
than the recipient strain. 相似文献
18.
Caixia Yuan Liping Lu Xiaoli Gao Yanbo Wu Maolin Guo Ying Li Xueqi Fu Miaoli Zhu 《Journal of biological inorganic chemistry》2009,14(6):841-851
Abstract A series of oxovanadium complexes with mixed ligands, a tridentate ONO-donor Schiff base ligand [viz., salicylidene anthranilic
acid (SAA)], and a bidentate NN ligand [viz., 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq), dipyrido[3,2-a:2′,3′-c]phenazine (dppz), or 7-methyldipyrido[3,2-a:2′,3′-c]phenazine (dppm)], have been synthesized and characterized by elemental analysis, electrospray ionization mass spectrometry,
UV–vis spectroscopy, Fourier transform IR spectroscopy, EPR spectroscopy, and X-ray crystallography. Crystal structures of
both complexes, [VIVO(SAA)(bpy)]·0.25bpy and [VIVO(SAA)(phen)]·0.33H2O, reveal that oxovanadium(IV) is coordinated with one nitrogen and two oxygen atoms from the Schiff base and two nitrogen
atoms from the bidentate planar ligands, in a distorted octahedral geometry (VO3N3). The oxidation state of V(IV) with d
1 configuration was confirmed by EPR spectroscopy. The speciation of VO–SAA–bpy in aqueous solution was investigated by potentiomtreic
pH titrations, and the results revealed that the main species are two ternary complexes at a pH range of 7.0–7.4, and one
is the isolated crystalline complex. The complexes have been found to be potent inhibitors against human protein tyrosine
phosphatase 1B (PTP1B) (IC50 approximately 30–61 nM), T-cell protein tyrosine phosphatase (TCPTP), and Src homology phosphatase 1 (SHP-1) in vitro. Interestingly,
the [VIVO(SAA)(bpy)] complex selectively inhibits PTP1B over the other two phosphatases (approximate ninefold selectivity against
SHP-1 and about twofold selectivity against TCPTP). Kinetics assays suggest that the complexes inhibit PTP1B in a competitive
and reversible manner. These suggest that the complexes may be promising candidates as novel antidiabetic agents.
Graphical Abstract
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
19.
Zhiyong Chen Toshiaki Nakajima Nobuhiro Tanabe Kunihiko Hinohara Seiichiro Sakao Yasunori Kasahara Koichiro Tatsumi Yoshinori Inoue Akinori Kimura 《Human genetics》2010,128(4):443-452
A deletion/insertion (Del/Ins) polymorphism of 28 base pairs (bp) in the 3′ untranslated region (UTR) of fibrinogen alpha
gene (FGA) was associated with thromboembolic diseases, but the underlying mechanisms remain unknown. Computational predication reveals
that the 28 bp polymorphic fragment is complementary to the sequence of a microRNA, miR-759. In this study, we aim to investigate the association and implicated mechanisms between FGA polymorphisms and the susceptibility to chronic thromboembolic pulmonary hypertension (CTEPH). The Del/Ins polymorphism was
analyzed in 190 patients with CTEPH and 628 controls. The FGA 3′UTR and miR-759 interaction was investigated using luciferase assay and quantitative RT-PCR method. Expression of miR-759 and FGA in human tissues was investigated by RT-PCR. The results reveal that the allele frequency of Ins was significantly higher
in the patients than in the controls (55.8 vs. 47.1%, P = 0.003, odds ratio = 1.42, 95% confidence interval: 1.13–1.79). Both miR-759 and FGA were expressed in human liver. Co-transfection of miR-759 decreased the expression and mRNA stability of reporter gene containing the FGA 3′UTR. The effect of miR-759 was stronger on the Ins allele than on the Del allele. These observations suggest that the expression of FGA was regulated by miR-759 through its interaction at the polymorphic 3′UTR sequence, which was associated with the susceptibility to CTEPH. 相似文献
20.
In this study, vegetative insecticidal proteins vip3 genes from Bacillus thuringiensis strains were detected based on polymerase chain reaction–high resolution melt (PCR–HRM) analysis. A pair of primers was designed
according to the conservative sequences in 150 bp region of the known vip3 subfamily. The 150 bp regions of difference vip3 genes have only a few nucleotide difference vip3 genes were detected in 8 of 11 standard B. thuringiensis strains, and vip3Aa genes, vip3Af genes and vip3Ba gene can be distinguished as different melting curves by this method. The results demonstrate the utility of the HRM assay
for mutant screening using vip3 gene. The PCR–HRM method may be a valuable and reliable tool for specific detection and identification of vip3 genes. 相似文献