The motor domain of testis-enriched kinesin KIF9 is essential for its localization in the mouse flagellum

Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.     

Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9

CRISPR systems build adaptive immunity against mobile genetic elements by DNA capture and integration catalysed by Cas1–Cas2 protein complexes. Recent studies suggested that CRISPR repeats and adaptation module originated from a novel type of DNA transposons called casposons. Casposons encode a Cas1 homologue called casposase that alone integrates into target molecules single and double-stranded DNA containing terminal inverted repeats (TIRs) from casposons. A recent study showed Methanosarcina mazei casposase is able to integrate random DNA oligonucleotides, followed up in this work using Acidoprofundum boonei casposase, from which we also observe promiscuous substrate integration. Here we first show that the substrate flexibility of Acidoprofundum boonei casposase extends to random integration of DNA without TIRs, including integration of a functional gene. We then used this to investigate targeting of the casposase-catalysed DNA integration reactions to specific DNA sites that would allow insertion of defined DNA payloads. Casposase–Cas9 fusions were engineered that were catalytically proficient in vitro and generated RNA-guided DNA integration products from short synthetic DNA or a gene, with or without TIRs. However, DNA integration could still occur unguided due to the competing background activity of the casposase moiety. Expression of Casposase-dCas9 in Escherichia coli cells effectively targeted chromosomal and plasmid lacZ revealed by reduced β-galactosidase activity but DNA integration was not detected. The promiscuous substrate integration properties of casposases make them potential DNA insertion tools. The Casposase–dCas9 fusion protein may serves as a prototype for development in genetic editing for DNA insertion that is independent of homology-directed DNA repair.     

The dynamic nature of the K-Ras/calmodulin complex can be altered by oncogenic mutations

Oncogenic mutant K-Ras promotes cancer cell proliferation, migration, invasion, and survival by assembling signaling complexes. To date, the functional and structural roles of K-Ras mutations within these complexes are incompletely understood despite their mechanistic and therapeutic significance. Here, we review recent advances in understanding specific binding between K-Ras and the calcium sensor calmodulin. This interaction positively and negatively regulates diverse functions of K-Ras in cancer, suggesting flexibility in K-Ras/calmodulin complex formation. Also, structural data suggest that oncogenic K-Ras likely samples several conformational states, influencing its distinct assemblies with calmodulin and with other proteins. Understanding how K-Ras interacts with calmodulin and with other partners is essential to discovering novel inhibitors of K-Ras in cancer.     

The antineoplastic effect of carnosine is accompanied by induction of PDK4 and can be mimicked by l-histidine

Carnosine (β-alanyl-l-histidine) is a naturally occurring dipeptide that shows antineoplastic effects in cell culture as well as in animal experiments. Since its mode of action and the targets at the molecular level have not yet been elucidated, we performed qRT-PCR experiments with RNA isolated from glioblastoma cell lines treated with carnosine, β-alanine, l-alanine, l-histidine and the dipeptide l-alanine-l-histidine. The experiments identified a strong induction of expression of the gene encoding pyruvate dehydrogenase 4 (PDK4) under the influence of carnosine and l-histidine, but not by the other substances employed. In addition, inhibition of cell viability was only detected in cells treated with carnosine and l-histidine, with the latter showing a significantly stronger effect than carnosine. Since the tumor cells expressed the tissue form of carnosinase (CN2) but almost no serum carnosinase (CN1), we conclude that cleavage by CN2 is a prerequisite for the antineoplastic effect of carnosine. In addition, enhanced expression of PDK4 under the influence of carnosine/l-histidine opens a new perspective for the interpretation of the ergogenic potential of dietary β-alanine supplementation and adds a new contribution to a growing body of evidence that single amino acids can regulate key metabolic pathways important in health and disease.     

PET/CT targeted tissue sampling reveals virus specific dIgA can alter the distribution and localization of HIV after rectal exposure

Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of ⁶⁴Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection.     

S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approximately 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T- and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replication-competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.     

The activities of the Escherichia coli MalK protein in maltose transport, regulation, and inducer exclusion can be separated by mutations

The maltose regulon consists of several genes encoding proteins involved in the uptake and utilization of maltose and maltodextrins. Five proteins make up a periplasmic binding-protein-dependent active transport system. One of these proteins, MalK, contains an ATP-binding site and is thought to couple the hydrolysis of ATP to the accumulation of substrate. Beside its function in transport, MalK has two additional roles: (i) it negatively regulates mal regulon expression and (ii) it serves as the target for regulation of transport activity by enzyme IIIGlc of the phosphotransferase system. To determine whether the three functions of MalK are separable, we have isolated and characterized three classes of malK mutations. The first type (class I) exhibited constitutive mal gene expression but still allowed normal transport of maltose; the second type (class II) lacked the ability to transport maltose but retained the ability to repress the mal genes. Class I mutations were localized in the last third of the gene, at amino acids 267 (Trp to Gly) and 346 (Gly to Ser). Mutations of class II were found at the positions 137 (Gly to Ala), 140 (delta Gln Arg), and 158 (Asp to Asn). These mutations are near or within the region of MalK that exhibits extensive homology to the B site of an ATP-binding fold. In addition, site-directed mutagenesis was used to add or remove one amino acid in the A site of the ATP-binding fold. Plasmids carrying these mutations also behaved as class II mutants. The third class of malK mutations resulted in resistance to the enzyme IIIGlc-mediated inhibitory effects of alpha-methylglucoside. These mutations did not interfere with the regulatory function of MalK. One of these mutations (exchanging a serine at position 282 for leucine) is located in a short stretch of amino acids that exhibits homology to a sequence in the Escherichia coli Lac permease in which alpha-methylglucoside-resistant mutations have been found.     

The in vitro developmental competence of bovine oocytes can be related to the morphology of the ovary

This study was designed to assess whether the developmental potential of bovine cumulus-oocyte complexes (COCs) could be related to the morphology of their originating ovary, providing a simple, noninvasive and objective selection criterion. Ovaries were divided into 3 categories on the basis of: A) presence of a follicle > 10 mm in diameter, B) presence of more than 10 follicles of 2 to 5 mm in diameter and no follicles > 10 mm, and C) presence of less than 10 follicles of 2 to 5 mm in diameter and no follicles > 10 mm. The COCs, isolated from ovaries of Category C, showed lower rates of maturation and blastocyst formation than those from Categories A and B. Moreover, blastocysts derived from Category C ovaries had fewer cells than those derived from the other 2 categories. It is concluded that ovarian morphology is a simple and noninvasive parameter for an effective selection of oocytes with better developmental competence.     

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.     

A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens

The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time. Received: 2 February 1998 / Accepted: 15 October 1998     

Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/- mice analysis.

KIF3A is a classical member of the kinesin superfamily proteins (KIFs), ubiquitously expressed although predominantly in neural tissues, and which forms a heterotrimeric KIF3 complex with KIF3B or KIF3C and an associated protein, KAP3. To elucidate the function of the kif3A gene in vivo, we made kif3A knockout mice. kif3A-/- embryos displayed severe developmental abnormalities characterized by neural tube degeneration and mesodermal and caudal dysgenesis and died during the midgestational period at approximately 10.5 dpc (days post coitum), possibly resulting from cardiovascular insufficiency. Whole mount in situ hybridization of Pax6 revealed a normal pattern while staining by sonic hedgehog (shh) and Brachyury (T) exhibited abnormal patterns in the anterior-posterior (A-P) direction at both mesencephalic and thoracic levels. These results suggest that KIF3A might be involved in mesodermal patterning and in turn neurogenesis.     

Exogenous dihydrosphingosine 1 phosphate mediates collagen synthesis in cardiac fibroblasts through JAK/STAT signalling and regulation of TIMP1

Cardiac fibrosis and myocyte hypertrophy are hallmarks of the cardiac remodelling process in cardiomyopathies such as heart failure (HF). Dyslipidemia or dysregulation of lipids contribute to HF. The dysregulation of high density lipoproteins (HDL) could lead to altered levels of other lipid metabolites that are bound to it such as sphingosine-1- phosphate (S1P). Recently, it has been shown that S1P and its analogue dihydrosphingosine-1-phosphate (dhS1P) are bound to HDL in plasma. The effects of dhS1P on cardiac cells have been obscure. In this study, we show that extracellular dhS1P is able to increase collagen synthesis in neonatal rat cardiac fibroblasts (NCFs) and cause hypertrophy of neonatal cardiac myocytes (NCMs). The janus kinase/signal transducer and activator (JAK/STAT) signalling pathway was involved in the increased collagen synthesis by dhS1P, through sustained increase of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). Extracellular dhS1P increased phosphorylation levels of STAT1 and STAT3 proteins, also caused an early increase in gene expression of transforming growth factor-β (TGFβ), and sustained increase in TIMP1. Inhibition of JAKs led to inhibition of TIMP1 and TGFβ gene and protein expression. We also show that dhS1P is able to cause NCM hypertrophy through S1P-receptor-1 (S1PR1) signalling which is opposite to that of its analogue, S1P. Taken together, our results show that dhS1P increases collagen synthesis in cardiac fibroblasts causing fibrosis through dhS1P-JAK/STAT-TIMP1 signalling.     

Effects of flavonoids on MicroRNA 145 regulation through Klf4 and myocardin in neointimal formation in vitro and in vivo

MicroRNA 145 (miR-145) is a critical modulator of vascular smooth muscle cell (VSMC) phenotyping and proliferation. Flavonoids have been studied extensively due to their diverse pharmacological properties, including anti-inflammatory effects. The aims of this study is designed to evaluate the atheroprotective effects on angiotensin II (Ang II)-induced miR-145 and Klf4/myocardin expression in vitro and in vivo of flavonoids, including (−)-epigallocatechin gallate (EGCG), chrysin, wogonin, silibinin, and ferulic acid. Ang II significantly reduced the miR-145 compared with the control VSMC groups; all the tested flavonoids increased miR-145 in the 100 nM concentration. Among the test compounds, EGCG showed the strongest augmenting effect on miR-145 and myocardin, however, it also abolished Ang II-induced Klf4. A [³H]-thymidine incorporation proliferation assay demonstrated that EGCG inhibited Ang II-induced VSMC proliferation, and Klf4 siRNA presented with the similar results. Immunohistochemical analysis and confocal microscopy demonstrated increased Klf4 expression and the arterial lumen was narrowed after balloon injury 14 days. With the addition of EGCG (50 mg/kg) and Klf4 siRNA, neointimal formation was reduced by 40.7% and 50.5% compared with balloon injury 14 days; Klf4 expression also was attenuated. This study demonstrated EGCG increased miR-145 and attenuated Klf4, and ameliorated neointimal formation in vitro and in vivo. The novel suppressive effect was mediated through the miR-145 and Klf4/myocardin pathways.