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A 65-year-old woman was admitted to our hospital with heart failure secondary to aortic regurgitation and paroxysmal atrial fibrillation. During surgery for valve-sparing aortic root replacement, a biopsy was obtained from the right atrium because of paroxysmal AF and mild LV hypokinesis. A histopathological examination revealed atrial natriuretic peptide amyloidosis, and the patient was clinically diagnosed with isolated atrial amyloidosis (IAA). Although IAA is rarely diagnosed clinically, it should be considered in patients with AF and heart failure.

Copper participates in a range of critical functions in the nervous system and human brain. Disturbances in brain copper content is strongly associated with neurological diseases. For example, changes in the level and distribution of copper are reported in neuroblastoma, Alzheimer's disease, and Lewy body disorders, such as Parkinson disease and dementia with Lewy bodies (DLB). There is a need for more sensitive techniques to measure intracellular copper levels to have a better understanding of the role of copper homeostasis in neuronal disorders. Here, we report a reaction-based near-infrared (NIR) ratiometric fluorescent probe CyCu1 for imaging Cu 2+ in biological samples. High stability and selectivity of CyCu1 enabled the probe to be deployed as a sensor in a range of systems, including SH-SY5Y cells and neuroblastoma tumors. Furthermore, it can be used in plant cells, reporting on copper added to Arabidopsis roots. We also used CyCu1 to explore Cu 2+ levels and distribution in post-mortem brain tissues from patients with DLB. We found significant decreases in Cu 2+ content in the cytoplasm, neurons, and extraneuronal space in the degenerating substantia nigra in DLB compared with healthy age-matched control tissues. These findings enhance our understanding of Cu 2+ dysregulation in Lewy body disorders. Our probe also shows promise as a photoacoustic imaging agent, with potential for applications in bimodal imaging.

Arabidopsis microRNA165a (miR165a) targets Class III Homeodomain Leucine-Zipper (HD-ZIPIII) transcription factors to regulate various aspects of plant development and stress response. Over-expression of miR165a mimics the loss-of-function phenotype of HD-ZIPIII genes and leading to ectopic organ formation, shoot apical meristem (SAM) termination, loss of leaf polarity, and defective vasculature development. However, the molecular mechanisms underlying these phenotypes remain unresolved. Here, we over-expressed miR165a in a dexamethasone inducible manner and identified differentially expressed genes in the SAM through RNA-Seq. Simultaneously, using multi-channel FACS combined with RNA-Seq approach, we characterized global transcriptome patterns in miR165a expressing cell-types compared to HD-ZIPIII expressing cell-types and other cell-types in SAM. By integrating our results we identified sets of genes which are up-regulated by miR165a as well have enriched expression in miR165a cell-types, and vice-versa. Known plant development related genes such as HD-ZIPIII and their targets LITTLE ZIPPERs, Like AUXIN RESISTANT 2, BEL1-like homeodomain 6, ROTUNDIFOLIA like 16 were found to be down-regulated. Among the up-regulated genes, GIBBERELLIN 2-OXIDASEs, various elemental transporters (YSL3, ZIFL1, SULTR), and other transporter genes were prominent. Thus, the genes identified in this study help to unravel the molecular mechanism of miR165a and HD-ZIPIII regulated plant development and stress response., (© 2023. The Author(s).)

We evaluated the effectiveness of a high-intensity preoperative resistance and endurance training program in improving physical function among patients scheduled for total knee arthroplasty. This non-randomized controlled trial included 33 knee osteoarthritis patients scheduled to undergo total knee arthroplasty at a tertiary public medical university hospital. Fourteen and nineteen patients were non-randomly assigned to intervention and control groups, respectively. All patients underwent total knee arthroplasty and a postoperative rehabilitation program. The intervention group participated in a preoperative rehabilitation program comprising high-intensity resistance and endurance training exercises to increase lower limb muscle strength and endurance capacity. The control group received only exercise instruction. The primary outcome was the 6-min walking distance, which was significantly higher in the intervention group (399 ± 59.8 m) than in the control group (348 ± 75.1 m) 3 months post-surgery. There were no significant differences between the groups 3 months post-surgery in muscle strength, visual analog scale, WOMAC-Pain, range of motion of knee flexion, and extension. A 3-week preoperative rehabilitation program combining muscle strengthening and endurance training improved endurance 3 months after total knee arthroplasty. Thus, preoperative rehabilitation is important for improving postoperative activity.

Background: We hypothesized that 3 weeks of preoperative rehabilitation could improve postoperative pain in patients undergoing total knee arthroplasty (TKA). Aim: This study aimed to evaluate the effects of 3 weeks of preoperative rehabilitation on postoperative pain after TKA., Methods: This prospective cohort study included 29 subjects (41 knees) divided into two groups: the preoperative rehabilitation group included 14 subjects (20 knees) and the control group included 15 subjects (21 knees). All subjects were scheduled for unilateral or bilateral TKA. The preoperative rehabilitation group completed a 90-min rehabilitation program 3 days per week for 3 weeks before their TKA. The rehabilitation included body weight exercise, resistance exercise, and cycle ergometer exercise. The control group did not undergo any rehabilitation prior to TKA. We assessed the patients using Western Ontario and McMaster Universities' Osteoarthritis Index (WOMAC) and recorded their physical activity of walking, standing, sitting, and lying down at study entry and/or before TKA and 1 month after TKA., Results: The WOMAC total and WOMAC pain scores were significantly lower after 3-weeks of rehabilitation, but before TKA and 1 month after surgery were significantly lower in the preoperative rehabilitation group than in the control group. The time spent walking, standing, sitting, and lying down for 12 h did not change after TKA in the preoperative rehabilitation group. In contrast, in the control group, the time spent in walking and standing positions decreased and the time in the sitting position increased after TKA (p < 0.05). Conclusion: We found that 3-week preoperative training reduced knee pain and helped maintain physical activity after surgery in patients with severe osteoarthritis who underwent TKA., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

At the Arabidopsis shoot apex, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis. While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined. Here, we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighboring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighboring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH) are characterized by elevated pulmonary arterial pressure resulting in right heart failure. Right ventricular (RV) dyssynchrony may be associated with early-stage RV dysfunction; however, the differences in RV dyssynchrony between CTEPH and PAH and the factors contributing to RV dyssynchrony remain unclear., Methods and results: Forty-four patients (CTEPH, 26; PAH, 18) were enrolled in this study. RV dyssynchrony was assessed by determining the standard deviation of the intervals from the peak QRS to peak systolic strain for 6 segments of the RV free and septal wall by using 2-dimensional speckle-tracking echocardiography (RV-6SD). The RV-6SD, pulmonary hemodynamics, echocardiographic findings, and patient demographics in CTEPH and PAH patients were compared and their correlations with RV-6SD were investigated. CTEPH patients were older and had significantly higher pulse pressure of the pulmonary artery (PP), tricuspid valve regurgitation pressure gradient, and RV-6SD, and lower pulmonary arterial compliance (PAC), despite showing comparable pulmonary arterial pressures. Age-adjusted multiple logistic analysis showed that RV-6SD and PAC were predictors of CTEPH rather than PAH. RV-SD6 was positively correlated with PP and RV dimension and negatively correlated with PAC., Conclusions: CTEPH patients showed more evident RV dyssynchrony than PAH patients. Low PAC and a widened PP may delay RV free wall motion and cause RV dyssynchrony.

Plants develop lateral organs such as leaves and flowers throughout their post-embryonic life from a structure called the shoot apical meristem (SAM), located at the plant shoot apex. This process is highly dynamic, and therefore in order to understand meristem and organ development, it is critical to be able to analyze these processes with high temporal and spatial resolution. Although several protocols have been published for imaging the Arabidopsis inflorescence meristem, gaining access to the vegetative meristem for imaging has been considered more difficult. Here we describe a method to dissect young Arabidopsis seedlings in order to obtain a clear view of the vegetative meristem and young leaf primordia using confocal microscopy.

In the Arabidopsis thaliana shoot apical meristem (SAM) the expression domains of Class III Homeodomain Leucine Zipper (HD-ZIPIII) and KANADI (KAN) genes are separated by a narrow boundary region from which new organs are initiated. Disruption of this boundary through either loss of function or ectopic expression of HD-ZIPIII and KAN causes ectopic or suppression of organ formation respectively, raising the question of how these transcription factors regulate organogenesis at a molecular level. In this study we develop a multi-channel FACS/RNA-seq approach to characterize global patterns of gene expression across the HD-ZIPIII-KAN1 SAM boundary. We then combine FACS, RNA-seq and perturbations of HD-ZIPIII and KAN expression to identify genes that are both responsive to REV and KAN1 and normally expressed in patterns that correlate with REV and KAN1. Our data reveal that a significant number of genes responsive to REV are regulated in opposite ways depending on time after induction, with genes associated with auxin response and synthesis upregulated initially, but later repressed. We also characterize the cell type specific expression patterns of auxin responsive genes and identify a set of genes involved in organogenesis repressed by both REV and KAN1., Competing Interests: The authors have declared that no competing interests exist.

Voltage-dependent inactivation of ion channels contributes to the regulation of the membrane potential of excitable cells. Mouse polycystic kidney disease 2-like 1 (PKD2L1) forms voltage-dependent nonselective cation channels, which are activated but subsequently inactivated in response to membrane depolarization. Here, we found that the mutation of an asparagine 533 residue (N533Q) in the outer pore loop region of PKD2L1 caused a marked increase in outward currents induced by depolarization. In addition, the tail current analysis demonstrated that the N533Q mutants are activated during depolarization but the subsequent inactivation does not occur. Interestingly, the N533Q mutants lacked the channel activation triggered by the removal of stimuli such as extracellular alkalization and heating. Our findings suggest that the N533 residue in the outer pore loop region of PKD2L1 has a key role in the voltage-dependent channel inactivation.

The periodic formation of plant organs such as leaves and flowers gives rise to intricate patterns that have fascinated biologists and mathematicians alike for hundreds of years [1]. The plant hormone auxin plays a central role in establishing these patterns by promoting organ formation at sites where it accumulates due to its polar, cell-to-cell transport [2-6]. Although experimental evidence as well as modeling suggest that feedback from auxin to its transport direction may help specify phyllotactic patterns [7-12], the nature of this feedback remains unclear [13]. Here we reveal that polarization of the auxin efflux carrier PIN-FORMED 1 (PIN1) is regulated by the auxin response transcription factor MONOPTEROS (MP) [14]. We find that in the shoot, cell polarity patterns follow MP expression, which in turn follows auxin distribution patterns. By perturbing MP activity both globally and locally, we show that localized MP activity is necessary for the generation of polarity convergence patterns and that localized MP expression is sufficient to instruct PIN1 polarity directions non-cell autonomously, toward MP-expressing cells. By expressing MP in the epidermis of mp mutants, we further show that although MP activity in a single-cell layer is sufficient to promote polarity convergence patterns, MP in sub-epidermal tissues helps anchor these polarity patterns to the underlying cells. Overall, our findings reveal a patterning module in plants that determines organ position by orienting transport of the hormone auxin toward cells with high levels of MP-mediated auxin signaling. We propose that this feedback process acts broadly to generate periodic plant architectures., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial-abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets., Competing Interests: The authors declare no conflict of interest.

Regenerative therapy with stem cell transplantation is used to treat various diseases such as coronary syndrome and Buerger's disease. For instance, stem-cell transplantation into the infarcted myocardium is an innovative and promising strategy for treating heart failure due to ischemic heart disease. Basic studies using small animals have shown that transplanted cells improve blood flow in the infarcted region. Magnetic resonance imaging (MRI) can noninvasively identify and track transplanted cells labeled with superparamagnetic iron oxide (SPIO). Although clinical regenerative therapies have been clinically applied to patients, the fate of implanted cells remains unknown. In addition, follow-up studies have shown that some adverse events can occur after recovery. Therefore, the present study evaluated the ability of MRI using a 3T scanner to track implanted peripheral blood mononuclear cells labeled with SPIO on days 0 and 7 after intramuscular (i.m.) and intravenous (i.v.) injection into a cynomolgus monkey. Labeled cells were visualized at the liver and triceps surae muscle on MR images using T1- and T2-weighted sequences and histologically localized by Prussian blue staining. The transplanted cells were tracked without abnormal clinical manifestations throughout this study. Hence, MRI of cynomolgus monkey transplanted SPIO-labeled cells is a safe and efficient method of tracking labeled cells that could help to determine the mechanisms involved in regenerative therapy.

Shoot branching requires the establishment of new meristems harboring stem cells; this phenomenon raises questions about the precise regulation of meristematic fate. In seed plants, these new meristems initiate in leaf axils to enable lateral shoot branching. Using live-cell imaging of leaf axil cells, we show that the initiation of axillary meristems requires a meristematic cell population continuously expressing the meristem marker SHOOT MERISTEMLESS (STM). The maintenance of STM expression depends on the leaf axil auxin minimum. Ectopic expression of STM is insufficient to activate axillary buds formation from plants that have lost leaf axil STM expressing cells. This suggests that some cells undergo irreversible commitment to a developmental fate. In more mature leaves, REVOLUTA (REV) directly up-regulates STM expression in leaf axil meristematic cells, but not in differentiated cells, to establish axillary meristems. Cell type-specific binding of REV to the STM region correlates with epigenetic modifications. Our data favor a threshold model for axillary meristem initiation, in which low levels of STM maintain meristematic competence and high levels of STM lead to meristem initiation.

Two interrelated problems in biology are understanding the regulatory logic and predictability of morphological evolution. Here, we studied these problems by comparing Arabidopsis thaliana, which has simple leaves, and its relative, Cardamine hirsuta, which has dissected leaves comprising leaflets. By transferring genes between the two species, we provide evidence for an inverse relationship between the pleiotropy of SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) homeobox genes and their ability to modify leaf form. We further show that cis-regulatory divergence of BP results in two alternative configurations of the genetic networks controlling leaf development. In C. hirsuta, ChBP is repressed by the microRNA164A (MIR164A)/ChCUP-SHAPED COTYLEDON (ChCUC) module and ChASYMMETRIC LEAVES1 (ChAS1), thus creating cross-talk between MIR164A/CUC and AS1 that does not occur in A. thaliana. These different genetic architectures lead to divergent interactions of network components and growth regulation in each species. We suggest that certain regulatory genes with low pleiotropy are predisposed to readily integrate into or disengage from conserved genetic networks influencing organ geometry, thus rapidly altering their properties and contributing to morphological divergence., (© 2015 Rast-Somssich et al.; Published by Cold Spring Harbor Laboratory Press.)

The serum levels of several metabolites are significantly altered in schizophrenia patients. In this study, we performed a targeted analysis of 34 candidate metabolites in schizophrenia patients (n = 25) and compared them with those in age- and gender-matched healthy subjects (n = 27). Orthogonal partial least square-discriminant analysis revealed that complete separation between controls and patients was achieved based on these metabolites. We found that the levels of γ-glutamylcysteine (γ-GluCys), linoleic acid, arachidonic acid, D-serine, 3-hydroxybutyrate, glutathione (GSH), 5-hydroxytryptamine, threonine, and tyrosine were significantly lower, while D-lactate, tryptophan, kynurenine, and glutamate levels were significantly higher in schizophrenia patients compared to controls. Using receiver operating characteristics (ROC) curve analysis, the sensitivity, specificity, and the area under curve of γ-GluCys, a precursor of GSH, and D-lactate, a terminal metabolite of methylglyoxal, were 88.00%, 81.48%, and 0.8874, and 88.00%, 77.78%, and 0.8415, respectively. In addition, serum levels of D-lactate were negatively correlated with γ-GluCys levels in patients, but not in controls. The present results suggest that oxidative stress-induced damage may be involved in the pathogenesis of schizophrenia.

The aboveground tissues of higher plants are derived from a small population of stem cells located at the shoot apex within a structure called the shoot apical meristem (SAM). The SAM not only includes the stem cells but also incorporates a region from which lateral organs arise. The SAM is therefore of prime interest for understanding plant growth and development. In this chapter we outline methods for using confocal microscopy to image the Arabidopsis inflorescence SAM. This method enables detailed examination of cell division and growth patterns (Reddy et al., Development 131:4225-4237, 2004) as well as gene expression and protein localization patterns over time (Heisler et al. Curr Biol 15:1899-1911, 2005). When combined with perturbation approaches, the method offers an extremely powerful system for investigating SAM function in great detail.

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

Background: Cell numbers in above-ground meristems of plants are thought to be maintained by a feedback loop driven by perception of the glycopeptide ligand CLAVATA3 (CLV3) by the CLAVATA1 (CLV1) receptor kinase and the CLV2/CORYNE (CRN) receptor-like complex. CLV3 produced in the stem cells at the meristem apex limits the expression level of the stem cell-promoting homeodomain protein WUSCHEL (WUS) in the cells beneath, where CLV1 and WUS RNA are localized. WUS downregulation nonautonomously reduces stem cell proliferation. Overexpression of CLV3 eliminates the stem cells, causing meristem termination, and loss of CLV3 function allows meristem overproliferation. There are many questions regarding the CLV3/CLV1 interaction, including where in the meristem it occurs, how it is regulated, and how it is that a large range of CLV3 concentrations gives no meristem size phenotype., Results: Here we use genetics and live imaging to examine the cell biology of CLV1 in Arabidopsis meristematic tissue. We demonstrate that plasma membrane-localized CLV1 is reduced in concentration by CLV3, which causes trafficking of CLV1 to lytic vacuoles. We find that changes in CLV2 activity have no detectable effects on CLV1 levels. We also find that CLV3 appears to diffuse broadly in meristems, contrary to a recent sequestration model., Conclusions: This study provides a new model for CLV1 function in plant stem cell maintenance and suggests that downregulation of plasma membrane-localized CLV1 by its CLV3 ligand can account for the buffering of CLV3 signaling in the maintenance of stem cell pools in plants., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis., Competing Interests: The authors have declared that no competing interests exist.

Water-insoluble fragmin/protamine microparticles of about 0.5-1 mum in diameter were prepared by simple mixing of low-molecular-weight heparin (fragmin) with protamine. We investigated the capability of these microparticles to immobilize fibroblast growth factor (FGF)-2, to protect FGF-2 against degradation, to enhance FGF-2 activity, and to facilitate controlled release of FGF-2. FGF-2 bound to the fragmin/protamine microparticles with high affinity (Kd = 2.08 x 10(-9) M) and the half-life of FGF-2-activity was prolonged substantially through binding of FGF-2 to the microparticles, by protection of FGF-2 from inactivation by heat and proteolysis. After subcutaneous injection into the back of mice, the fragmin/protamine microparticles underwent biodegradation and disappeared in about 2 weeks. A similar injection of FGF-2-containing microparticles resulted in significant neovascularization and fibrous tissue formation near the injection site after 1 week. These results indicate that controlled release of biologically active FGF-2 occurs through both slow diffusion and biodegradation of the microparticles, with subsequent induction of neovascularization. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009.

A central unanswered question in stem cell biology, both in plants and in animals, is how the spatial organization of stem cell niches are maintained as cells move through them. We address this question for the shoot apical meristem (SAM) which harbors pluripotent stem cells responsible for growth of above-ground tissues in flowering plants. We find that localized perception of the plant hormone cytokinin establishes a spatial domain in which cell fate is respecified through induction of the master regulator WUSCHEL as cells are displaced during growth. Cytokinin-induced WUSCHEL expression occurs through both CLAVATA-dependent and CLAVATA-independent pathways. Computational analysis shows that feedback between cytokinin response and genetic regulators predicts their relative patterning, which we confirm experimentally. Our results also may explain how increasing cytokinin concentration leads to the first steps in reestablishing the shoot stem cell niche in vitro.

To clarify the significance of expression of system L amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in the developing intestine, immunohistochemical investigation and molecular analysis were performed in the human embryonic and/or fetal intestines, ranging from 28-30 days to 34-35 weeks gestation. The molecular analysis for the expression of LAT1 and 4F2hc mRNAs was done in the pure epithelial cell samples prepared after laser assisted microdissection. The immunoreactivities against LAT1 and 4F2hc were detected along the basolateral cell membrane of the primitive gut epithelium at 28-30 days gestation. According to advance in gestational age of up to 24-25 weeks gestation, the immunoreactivity of LAT1 was predominantly observed in the supranuclear cytoplasmic localization with a granular or dot-like staining pattern. Up to 8-9 weeks gestation, the immunoreactivity of 4F2hc showed almost the same as that of LAT1. However, after the age of 12-13 weeks gestation, the immunoreactivity of 4F2hc was predominantly localized along the cell membrane of apical surface of the epithelial cells. No apical and linear membranous localization of LAT1 was observed until nearly 20 weeks gestation. In the late gestational stage, both the immunoreactivities against LAT1 and 4F2hc were localized along the apical surface of the epithelial cells. In conclusion, the expression of LAT1 and 4F2hc in early developing intestine suggests they have a more important role in cell proliferation rather than functional differentiation. The predominant cytoplasmic localization of LAT1 during mid-fetal life seems to be largely inactive as amino acid transporter. On the other hand, the apical and linear membranous co-localization of LAT1 and 4F2hc in the late fetal life suggests that these molecules may play a role as a functional amino acid transporter in the fetal intestinal epithelium.

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca(2+) and incubated overnight at 4 degrees C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca(2+)-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.