Kinetic analysis methods applied to single motor protein trajectories.

Molecular motors convert chemical or electrical energy into mechanical displacement, either linear or rotary. Under ideal circumstances, single-molecule measurements can spatially and temporally resolve individual steps of the motor, revealing important properties of the underlying mechanochemical process. Unfortunately, steps are often hard to resolve, as they are masked by thermal noise. In such cases, details of the mechanochemistry can nonetheless be recovered by analyzing the fluctuations in the recorded traces. Here, we expand upon existing statistical analysis methods, providing two new avenues to extract the motor step size, the effective number of rate-limiting chemical states per translocation step, and the compliance of the link between the motor and the probe particle. We first demonstrate the power and limitations of these methods using simulated molecular motor trajectories, and we then apply these methods to experimental data of kinesin, the bacterial flagellar motor, and F1-ATPase.

Acid secretion by the boring organ of the burrowing giant clam, Tridacna crocea.

The giant clam Tridacna crocea, native to Indo-Pacific coral reefs, is noted for its unique ability to bore fully into coral rock and is a major agent of reef bioerosion. However, T. crocea's mechanism of boring has remained a mystery despite decades of research. By exploiting a new, two-dimensional pH-sensing technology and manipulating clams to press their presumptive boring tissue (the pedal mantle) against pH-sensing foils, we show that this tissue lowers the pH of surfaces it contacts by greater than or equal to 2 pH units below seawater pH day and night. Acid secretion is likely mediated by vacuolar-type H+-ATPase, which we demonstrate (by immunofluorescence) is abundant in the pedal mantle outer epithelium. Our discovery of acid secretion solves this decades-old mystery and reveals that, during bioerosion, T. crocea can liberate reef constituents directly to the soluble phase, rather than producing sediment alone as earlier assumed.

Selective inhibition of tumor cell associated Vacuolar-ATPase 'a2' isoform overcomes cisplatin resistance in ovarian cancer cells.

Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo-resistance. These pH alterations are mediated by aberrant activation of key multi-subunit proton pumps, Vacuolar-ATPases (V-ATPases). In tumor cells, its 'a2' isoform (V-ATPase-V0a2) is a component of functional plasma-membrane complex and promotes tumor invasion through tumor-acidification and immuno-modulation. Its involvement in chemo-resistance has not been studied. Here, we show that V-ATPase-V0a2 is over-expressed in acquired-cisplatin resistant OVCA cells (cis-A2780/cis-TOV112D). Of all the 'a' subunit isoforms, V-ATPase-V0a2 exhibited an elevated expression on plasma membrane of cisplatin-resistant cells compared to sensitive counterparts. Immuno-histochemistry revealed V-ATPase-V0a2 expression in both low grade (highly drug-resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V-ATPase-V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin-DNA-adduct formation and suppressed DNA-repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum-associated DNA-damage. As an indicator of reduced metastasis and chemo-resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2-knockdown resistant cells. Interestingly, pre-treatment with monoclonal V0a2-inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 could serve as a therapeutic strategy for chemo-resistant ovarian carcinoma and improve efficacy of platinum drugs.

Identification of the mitochondrially encoded subunit 6 of F1FO ATPase in Trypanosoma brucei.

Kinetoplast maxicircle DNA of trypanosomatids encodes eighteen proteins. RNA editing is required to confer translatability to mRNA for twelve of these. Sequence conservation of the predicted hydrophobic polypeptides indicates that they represent functional components of the respiratory chain. Yet, so far only two of those, cytochrome c oxidase subunit I and apocytochrome b of cytochrome c reductase, have been identified with biochemical methods. Here we report on identification of A6 subunit of F1FO ATPase encoded by a pan-edited mRNA in Trypanosoma brucei. The polypeptide was present among the (35)S-labeled mitochondrial translation products characterized by anomalous migration in denaturing 2D gels. It was identified as an ATPase subunit by co-migration with this complex in Blue Native 2D gels. A partial N-terminal sequence of the corresponding polypeptide present in the gel-purified ATPase complex from Leishmania tarentolae was consistent with the predicted A6 sequence.

High-speed angle-resolved imaging of a single gold nanorod with microsecond temporal resolution and one-degree angle precision.

We developed two types of high-speed angle-resolved imaging methods for single gold nanorods (SAuNRs) using objective-type vertical illumination dark-field microscopy and a high-speed CMOS camera to achieve microsecond temporal and one-degree angle resolution. These methods are based on: (i) an intensity analysis of focused images of SAuNR split into two orthogonally polarized components and (ii) the analysis of defocused SAuNR images. We determined the angle precision (statistical error) and accuracy (systematic error) of the resultant SAuNR (80 nm × 40 nm) images projected onto a substrate surface (azimuthal angle) in both methods. Although both methods showed a similar precision of ∼1° for the azimuthal angle at a 10 µs temporal resolution, the defocused image analysis showed a superior angle accuracy of ∼5°. In addition, the polar angle was also determined from the defocused SAuNR images with a precision of ∼1°, by fitting with simulated images. By taking advantage of the defocused image method's full revolution measurement range in the azimuthal angle, the rotation of the rotary molecular motor, F1-ATPase, was measured with 3.3 µs temporal resolution. The time constants of the pauses waiting for the elementary steps of the ATP hydrolysis reaction and the torque generated in the mechanical steps have been successfully estimated. The high-speed angle-resolved SAuNR imaging methods will be applicable to the monitoring of the fast conformational changes of many biological molecular machines.

Cellular fatty acid profile and H(+)-ATPase activity to assess acid tolerance of Bacillus sp. for potential probiotic functional attributes.

The present study has been focused widely on comparative account of probiotic qualities of Bacillus spp. for safer usage. Initially, 170 heat resistant flora were isolated and selected for non-pathogenic cultures devoid of cytK, hblD, and nhe1 virulence genes. Subsequently, through biochemical tests along with 16S rRNA gene sequencing and fatty acid profiling, the cultures were identified as Bacillus megaterium (AR-S4), Bacillus subtilis (HR-S1), Bacillus licheniformis (Csm1-1a and HN-S1), and Bacillus flexus (CDM4-3c and CDM3-1). The selected cultures showed 70-80 % survival under simulated gastrointestinal condition which was also confirmed through H(+)-ATPase production. The amount of H(+)-ATPase increased by more than 2-fold when grown at pH 2 which support for the acid tolerance ability of Bacillus isolates. The study also examined the influence of acidic pH on cellular fatty acid composition of Bacillus spp. A remarkable shift in the fatty acid profile was observed at acidic pH through an increased amount of even numbered fatty acid (C16 and C18) in comparison with odd numbered (C15 and C17). Additionally, the cultures exhibited various probiotic functional properties. Overall, the study increases our understanding of Bacillus spp. and will allow both industries and consumers to choose for well-defined probiotic with possible health benefits.

Expression of bone markers and micro-CT analysis of alveolar bone during orthodontic relapse.

OBJECTIVES: To investigate biological changes in alveolar bone occurring during orthodontic relapse. MATERIALS AND METHODS: Rat maxillary first molars were moved mesially for 10 days. After orthodontic tooth movement (OTM), appliances were removed, and the molars were allowed to relapse for one, three, five, seven, 14 or 21 days. Changes in 3D morphometric parameters of bone located mesial to the first molars were evaluated by micro-CT. Total RNA was isolated from the same bone site, and real-time RT-PCR was used to measure the expression of bone formation and resorption markers. RESULTS: One day after appliance removal, the molars relapsed to a mean 73% of the achieved OTM and then steadily relapsed to 93% at 21 days. Tissue mineral density and per cent bone volume increased over the experimental period. Inversely, there was a decrease in total porosity. Gene expression of OCN, Coll-I and ALP decreased during OTM, whilst as the molars relapsed showed tended to increase. Gene expression of RANKL and TRAP increased during OTM. Changes in mRNA expression of H(+)-ATPase were minor. By 21 days post-appliance removal, the remodelling process in rats appeared to have returned to control levels. CONCLUSIONS: Bone tissue reactions on a molecular level are similar during OTM and orthodontic relapse. These findings validate the importance of immediate retention following active OTM.

Characterization of osmotically induced filaments of Salmonella enterica.

Salmonella enterica forms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were active in vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F(0)F(1)-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2 × Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.

A novel biosensor regulated by the rotator of F0F1-ATPase to detect deoxynivalenol rapidly.

A novel biosensor (immuno-rotary biosensor) was developed by conjugating deoxynivalenol (DON) monoclonal antibodies with the "rotator" ε-subunit of F(0)F(1)-ATPase within chromatophores with an ε-subunit monoclonal antibody-biotin-avidin-biotin linker to capture DON residues. The conjugation conditions were then optimized. The capture of DON was based on the antibody-antigen reaction and it is indicated by the change in ATP synthetic activity of F(0)F(1)-ATPase, which is measured via chemiluminescence using the luciferin-luciferase system with a computerized microplate luminometer analyzer. 10(-7)mg/ml of DON can be detected. The whole detection process requires only about 20min. This method has promising applications in the detection of small molecular compounds because of its rapidity, simplicity, and sensitivity.

Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy.

High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein-nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F(1)-ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10-15 h, depending mainly on the substrate surface to be used.

Biopsy-Proven Type 1 Renal Tubular Acidosis in a Patient with Metabolic Acidosis

No abstract available.

alphaENaC-mediated lithium absorption promotes nephrogenic diabetes insipidus.

Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.

Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection.

Crystals of biological macromolecules often exhibit considerable inter-crystal and intra-crystal variation in diffraction quality. This requires the evaluation of many samples prior to data collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron-based facilities optimized for high-throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample-containing loop for microcrystals using an X-ray beam of 5 microm diameter and selecting the most ordered regions of relatively large crystals using X-ray beams of 5-50 microm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction-quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRF's structural biology beamlines is also presented.

Sequential processing from cell lysis to protein assay on a chip enabling the optimization of an F(1)-ATPase single molecule assay condition.

We developed an integrated protein assay device, "Single Molecule MicroTAS (SMM)," which enables cell lysis, protein extraction, purification, and activity assay. The assay was achieved at the single-molecule scale for a genetically engineered protein, F(1)-ATPase, which is the smallest known rotary motor. A cell lysis condition, with a wide range of applied voltages (50-250 V) and other optimized values (pulse width: 50 micros; duty: 0.01%; electrode gap: 25 microm; total flow rate: 5 microL min(-1)) provided a high enough protein concentration for the assay. Successively, the protein was extracted and purified by specific binding in a microfluidic channel. During the assay process, the diffusion effect of lysate between a two-phase laminar flow contributes to optimizing the single-molecule assay condition, because the concentration of the original lysate from the E. coli solution is too high to assay. To achieve the most efficient assay condition, the protein diffusion effect on the assay was experimentally and numerically evaluated. The results reveal that, in our experimental conditions, concentrations of F(1) and other contaminated effluents are optimized for the F(1) rotational assay at a channel position. The adenosine triphosphate (ATP)-driven rotation speed measured in the SMM was compatible with that obtained by conventional purification and assay. Such a sequential process from cell lysis to assay proves that the SMM is an example of a sample-in-answer-out system for F(1) protein evaluation.

Comparative proteomic analysis of mouse melanoma cell line B16, a metastatic descendant B16F10, and B16 overexpressing the metastasis-associated tyrosine phosphatase PRL-3.

Metastasis is a complex, multistep process by which a cancer cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary cancer. A deeper understanding of the molecular events underlying metastasis will provide information that will be useful for the development of new diagnostic and therapeutic strategies. The B16 and B16F10 mouse melanoma cell lines are widely used as model system for studying many aspects of cancer biology including metastasis. Compared with B16, which has a low metastatic potential, the highly metastatic cell line B16F10 displayed a higher metastatic ability along with higher expression levels of the metastasis-associated phosphatase of regenerating liver-3 (PRL-3). B16 cells transfected with PRL-3 cDNA (B16-PRL3) had metastatic abilities comparable to those of Bl16F10 cells. To study the molecular mechanisms that underlie metastasis, the proteomes of the B16, B16F10, and B16-PRL3 cell lines were compared using two-dimensional differential in-gel electrophoresis. Proteins that varied significantly in levels between these cell lines were selected and identified using mass spectrometry. Interestingly, many proteins, especially those present in membrane fractions, were similarly up- or downregulated in both the Bl16F10 and B16-PRL3 cells lines compared to B16 cell lines. The list of similarly regulated proteins included heat shock protein 70, fascin-1, septin-6, ATP synthase beta subunit, and bone morphogenic protein receptor type IB. These proteins may play a causal role in PRL-3-mediated metastasis. These investigations open an avenue for the further characterization of the molecular mechanisms that underlie metastasis.

Comparative proteomic analysis of osteosarcoma cell and human primary cultured osteoblastic cell.

To identify new biomarkers that facilitate the accurate early diagnosis of osteosarcoma and that may possibly include novel therapeutic candidates, we performed a proteomic approach to compare osteosarcoma cells and human primary cultured osteoblastic cells. Image analysis of silver-stained 2-DE gels revealed that the level of 12 protein spots was significantly different between the two groups of samples (p < .004). After mass spectroscopic identification and database searches, we found that in osteosarcoma cells, the level of HSP70, actin capping protein, ATP synthase, Mthsp75, UQCRC1, Ras-related nuclear protein, UCH-L1, and PRDX4 was elevated. However, the level of pyruvate dehydrogenase E1, Prohibitin, and Annexin V was decreased. Subsequent Western blot analyses of UQCRC1, UCH-L1, and PRDX4 in osteosarcoma tissues confirmed the results obtained by the proteomic analyses. These identified proteins may be potential molecular targets for osteosarcomatous diagnostics and therapeutics.

Expression of Atp8b3 in murine testis and its characterization as a testis specific P-type ATPase.

Spermatogenesis is a complex process that produces haploid motile sperms from diploid spermatogonia through dramatic morphological and biochemical changes. P-type ATPases, which support a variety of cellular processes, have been shown to play a role in the functioning of sperm. In this study, we isolated one putative androgen-regulated gene, which is the previously reported sperm-specific aminophospholipid transporter (Atp8b3, previously known as Saplt), and explored its expression pattern in murine testis and its biochemical characteristics as a P-type ATPase. Atp8b3 is exclusively expressed in the testis and its expression is developmentally regulated during testicular development. Immunohistochemistry of the testis reveals that Atp8b3 is expressed only in germ cells, especially haploid spermatids, and the protein is localized in developing acrosomes. As expected, from its primary amino acid sequence, ATP8B3 has an ATPase activity and is phosphorylated by an ATP-producing acylphosphate intermediate, which is a signature property of the P-Type ATPases. Together, ATP8B3 may play a role in acrosome development and/or in sperm function during fertilization.

Larval anopheline mosquito recta exhibit a dramatic change in localization patterns of ion transport proteins in response to shifting salinity: a comparison between anopheline and culicine larvae.

Mosquito larvae live in dynamic aqueous environments, which can fluctuate drastically in salinity due to environmental events such as rainfall and evaporation. Larval survival depends upon the ability to regulate hemolymph osmolarity by absorbing and excreting ions. A major organ involved in ion regulation is the rectum, the last region for modification of the primary urine before excretion. The ultrastructure and function of culicine larval recta have been studied extensively; however, very little published data exist on the recta of anopheline larvae. To gain insight into the structure and functions of this organ in anopheline species, we used immunohistochemistry to compare the localization of three proteins [carbonic anhydrase (CA9), Na+/K+ P-ATPase and H+ V-ATPase] in the recta of anopheline larvae reared in freshwater and saline water with the localization of the same proteins in culicine larvae reared under similar conditions. Based on the following key points, we concluded that anophelines differ from culicines in larval rectal structure and in regulation of protein expression: (1) despite the fact that obligate freshwater and saline-tolerant culicines have structurally distinct recta, all anophelines examined (regardless of saline-tolerance) have a structurally similar rectum consisting of distinct DAR (dorsal anterior rectal) cells and non-DAR cells; (2) anopheline larvae undergo a dramatic shift in rectal Na+/K+-ATPase localization when reared in freshwater vs saline water. This shift is not seen in any culicine larvae examined. Additionally, we use these immunohistochemical analyses to suggest possible functions for the DAR and non-DAR cells of anopheline larvae in freshwater and saline conditions.

All Trypanosoma cruzi developmental forms present lysosome-related organelles.

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.