On the Role of Myosin-II in Cytokinesis: Division of Dictyostelium Cells under Adhesive and Nonadhesive Conditions

We have investigated the role of myosin in cytokinesis in Dictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (ΔBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing ΔBLCBS-myosin were previously shown to divide in suspension (Uyeda et al., 1996). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, ΔBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a ΔBLCBS-myosin is similar to that in wild-type cells.

Electrostatic interactions between rotor and stator in the bacterial flagellar motor

Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor–stator interface.

Reciprocal electromechanical properties of rat prestin: The motor molecule from rat outer hair cells

Cochlear outer hair cells (OHCs) are responsible for the exquisite sensitivity, dynamic range, and frequency-resolving capacity of the mammalian hearing organ. These unique cells respond to an electrical stimulus with a cycle-by-cycle change in cell length that is mediated by molecular motors in the cells' basolateral membrane. Recent work identified prestin, a protein with similarity to pendrin-related anion transporters, as the OHC motor molecule. Here we show that heterologously expressed prestin from rat OHCs (rprestin) exhibits reciprocal electromechanical properties as known for the OHC motor protein. Upon electrical stimulation in the microchamber configuration, rprestin generates mechanical force with constant amplitude and phase up to a stimulus frequency of at least 20 kHz. Mechanical stimulation of rprestin in excised outside-out patches shifts the voltage dependence of the nonlinear capacitance characterizing the electrical properties of the molecule. The results indicate that rprestin is a molecular motor that displays reciprocal electromechanical properties over the entire frequency range relevant for mammalian hearing.

Autoimmunity to βIV spectrin in paraneoplastic lower motor neuron syndrome

Paraneoplastic neurological disorders may result from autoimmunity directed against antigens shared by the affected neurons and the associated cancer cells. We have recently reported the case of a woman with breast cancer and paraneoplastic lower motor neuron syndrome whose serum contained autoantibodies directed against axon initial segments and nodes of Ranvier of myelinated axons, including the axons of motoneurons. Here, we show that major targets of the autoantibodies of this patient are βIVΣ1 spectrin and βIV spectrin 140, two isoforms of the novel βIV spectrin gene, as well as a neuronal surface epitope yet to be identified. Partial improvement of the neurological symptoms following cancer removal was associated with a drastic reduction in the titer of the autoantibodies against βIV spectrin and nodal antigens in general, consistent with the autoimmune pathogenesis of the paraneoplastic lower motor neuron syndrome. The identification of βIV spectrin isoforms and surface nodal antigens as novel autoimmune targets in lower motor neuron syndrome provide new insights into the pathogenesis of this severe neurological disease.

Kinesin molecular motors: Transport pathways, receptors, and human disease

Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.

Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules.

Kinesin and ncd motor proteins are homologous in sequence yet move in opposite directions along microtubules. We have previously shown that monomeric kinesin and ncd bind in the same orientation on equivalent sites relative to the ends of tubulin sheets of known polarity. We now report cryoelectron microscope images of 16-protofilament microtubules decorated with both single- and double-headed kinesin and double-headed ncd. Three-dimensional density maps and difference maps show that, in adenosine 5'-[beta,gamma-imido]triphosphate, both dimeric motors bind tightly to microtubules via one head, leaving the other free, though apparently in a fixed position. The attached heads of dimers bind to tubulin in the same way as single kinesin heads. The second heads are connected to the tops of the first but, whereas the second kinesin head is closely associated with the first, pairs of ncd heads are splayed apart. There is also a distinct difference in orientation: the second kinesin head is tilted toward the microtubule plus end, while the second head of ncd points toward the minus end.

The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.

ncd and kinesin motor domains interact with both alpha- and beta-tubulin.

Motor domains of the Drosophila minus-end-directed microtubule (MT) motor protein ncd, were found to saturate microtubule binding sites at a stoichiometry of approximately one motor domain per tubulin dimer. To determine the tubulin subunit(s) involved in binding to ncd, mixtures of ncd motor domain and MTs were treated with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide) (EDC). EDC treatment generated covalently cross-linked products of ncd and alpha-tubulin and of ncd and beta-tubulin, indicating that the ncd motor domain interacts with both alpha- and beta-tubulin. When the Drosophila kinesin motor domain protein was substituted for the ncd motor domain, cross-linked products of kinesin and alpha-tubulin and of kinesin and beta-tubulin were produced. EDC treatment of mixtures of ncd motor domain and unassembled tubulin dimers or of kinesin motor domain and unassembled tubulin dimers produced the same motor-tubulin products generated in the presence of MTs. These results indicate that kinesin family motors of opposite polarity interact with both tubulin monomers and support a model in which some portion of each protein's motor domain overlaps adjacent alpha- and beta-tubulin subunits.

Fluctuation analysis of rotational speeds of the bacterial flagellar motor.

We measured the dependence of the variance in the rotation rate of tethered cells of Escherichia coli on the mean rotation rate over a regime in which the motor generates constant torque. This dependence was compared with that of broken motors. In either case, motor torque was augmented with externally applied torque. We show that, in contrast to broken motors, functioning motors in this regime do not freely rotationally diffuse and that the variance measurements are consistent with the predicted values of a stepping mechanism with exponentially distributed waiting times (a Poisson stepper) that steps approximately 400 times per revolution.

Aplicação de técnicas inteligentes com análise no domínio do tempo para reconhecimento de defeitos em motores de indução trifásicos

Os motores de indução trifásicos são os principais elementos de conversão de energia elétrica em mecânica motriz aplicados em vários setores produtivos. Identificar um defeito no motor em operação pode fornecer, antes que ele falhe, maior segurança no processo de tomada de decisão sobre a manutenção da máquina, redução de custos e aumento de disponibilidade. Nesta tese são apresentas inicialmente uma revisão bibliográfica e a metodologia geral para a reprodução dos defeitos nos motores e a aplicação da técnica de discretização dos sinais de correntes e tensões no domínio do tempo. É também desenvolvido um estudo comparativo entre métodos de classificação de padrões para a identificação de defeitos nestas máquinas, tais como: Naive Bayes, k-Nearest Neighbor, Support Vector Machine (Sequential Minimal Optimization), Rede Neural Artificial (Perceptron Multicamadas), Repeated Incremental Pruning to Produce Error Reduction e C4.5 Decision Tree. Também aplicou-se o conceito de Sistemas Multiagentes (SMA) para suportar a utilização de múltiplos métodos concorrentes de forma distribuída para reconhecimento de padrões de defeitos em rolamentos defeituosos, quebras nas barras da gaiola de esquilo do rotor e curto-circuito entre as bobinas do enrolamento do estator de motores de indução trifásicos. Complementarmente, algumas estratégias para a definição da severidade dos defeitos supracitados em motores foram exploradas, fazendo inclusive uma averiguação da influência do desequilíbrio de tensão na alimentação da máquina para a determinação destas anomalias. Os dados experimentais foram adquiridos por meio de uma bancada experimental em laboratório com motores de potência de 1 e 2 cv acionados diretamente na rede elétrica, operando em várias condições de desequilíbrio das tensões e variações da carga mecânica aplicada ao eixo do motor.

Three Dichotomies in Lawyers’ Ethics (With Particular Attention to the Corporation as Client)

Three usually unexpressed, and too often unnoticed, conceptual dichotomies underlie our perception and understanding of lawyers’ ethics. First, the existence of a special body of professional ethics and professional regulation presupposes some special need or risk. Criminal and civil law are apparently insufficient. Ordinary day-to-day morality and ordinary ethics, likewise, are not considered to be enough. What is the risk entailed by the notion of a profession that is special; who needs protection, and from what? Two quite different possible answers to this question provide the first of the three dichotomies examined in this article: one can understand the risk as primarily to a vulnerable client from a powerful professional; or, to the contrary, from a powerful client-lawyer combination toward vulnerable others. Second, what is the foundational orientation of lawyers? Are lawyers serving primarily their particular clients, and those clients’ preferences, choices and autonomy? Or is the primary allegiance of lawyers to some community or collective goal or interest distinct from the particular goals or interests of the client? The third dichotomy concerns not the substance of the risk, or the primary orientation, but the appropriate means of responding to that risk or that fundamental obligation. Should professional ethics be implemented primarily through rules? Or, should we rely on character and the discretion of lawyers to make a thought out, all things considered, decision? Each of these three presents a fundamental difference in how we perceive and address issues of lawyers’ ethics. Each affects our understanding and analysis on multiple levels, from (1) determining the appropriate or requisite conduct in a particular situation, to (2) framing a specific rule or approach for a particular category of situations, to (3) more general or abstract theory or policy. A person’s inclinations in regard to the dichotomies affects the conclusions that person will reach on each of those levels of analysis, yet those inclinations and assumptions are frequently unexamined and unarticulated. One’s position on each of the dichotomies tends to structure the approach and outcome without the issues and choice having been explicitly addressed or possibly even noticed. This article is an effort to ameliorate that problem. Part I addresses the question of what is the risk in the work of lawyers, or the function of lawyers, for which professional ethics is the answer. The concluding section focuses on the particular problem of the corporation as client. Part II then asks the related and possibly consequent question of what is the foundational orientation or allegiance of the lawyer? Is it to the individual client? Or is it to some larger community interest? Again, the concluding section focuses on the corporation. Part III turns to the means or method for addressing the obligations and possible problems of the professional ethics of lawyers. Should lawyers’ ethics guide and confine the conduct of lawyers primarily through rules? Or should it function primarily through reliance on the knowledge, judgment and character of lawyers? If the latter were the guide, ethical decisions would be made on a situation by situation basis under the discretion of each lawyer. Toward the end of each discussion possibilities for bridging the dichotomy are considered (and with such bridges each dichotomy may come to look more like a spectrum or continuum.). At several points after its introduction in Parts I and II, the special problem of the corporation as client is revisited and possible solutions suggested. Illustrating the usefulness of keeping the dichotomies in view, Part IV applies them to several exemplary situations of ethical difficulty in actual lawyer practice. For readers finding it difficult to envision the consequences of these distinctions, turning ahead to Part IV may be useful in making the discussion more concrete. Some commonalities across the dichotomies and connections among them are then developed in the concluding section, Part V.

Three Dichotomies in Lawyers’ Ethics (With Particular Attention to the Corporation as Client)

Three usually unexpressed, and too often unnoticed, conceptual dichotomies underlie our perception and understanding of lawyers’ ethics. First, the existence of a special body of professional ethics and professional regulation presupposes some special need or risk. Criminal and civil law are apparently insufficient. Ordinary day-to-day morality and ordinary ethics, likewise, are not considered to be enough. What is the risk entailed by the notion of a profession that is special; who needs protection, and from what? Two quite different possible answers to this question provide the first of the three dichotomies examined in this article: one can understand the risk as primarily to a vulnerable client from a powerful professional; or, to the contrary, from a powerful client-lawyer combination toward vulnerable others. Second, what is the foundational orientation of lawyers? Are lawyers serving primarily their particular clients, and those clients’ preferences, choices and autonomy? Or is the primary allegiance of lawyers to some community or collective goal or interest distinct from the particular goals or interests of the client? The third dichotomy concerns not the substance of therisk, or the primary orientation, but the appropriate means of responding to that risk or that fundamental obligation. Should professional ethics be implemented primarily through rules? Or, should we rely on character and the discretion of lawyers to make a thought out, all things considered, decision? Each of these three presents a fundamental difference in how we perceive and address issues of lawyers’ ethics. Each affects our understanding and analysis on multiple levels, from (1) determining the appropriate or requisite conduct in aparticular situation, to (2) framing a specific rule or approach for a particular category of situations, to (3) more general or abstract theory or policy. A person’s inclinations in regard to the dichotomies affects the conclusions that person will reach on each of those levels of analysis, yet those inclinations and assumptions are frequently unexamined and unarticulated. One’s position on each of the dichotomies tends to structure the approach and outcome without the issues and choice having been explicitly addressed or possibly even noticed. This article is an effort to ameliorate that problem. Part I addresses the question of what is the risk in the work of lawyers, or the function of lawyers, for which professional ethics is the answer. The concluding section focuses on the particular problem of the corporation as client. Part II then asks the related and possibly consequent question of what is the foundational orientation or allegiance of the lawyer? Is it to the individual client? Or is it to some larger community interest? Again, the concluding section focuses on thecorporation. Part III turns to the means or method for addressing the obligations and possible problems of the professional ethics of lawyers. Should lawyers’ ethics guide and confine the conduct of lawyers primarily through rules? Or should it function primarily through reliance on the knowledge, judgment and character of lawyers? If the latter were the guide, ethical decisions would be made on a situation by situation basis under the discretion of each lawyer. Toward the end of each discussion possibilities for bridging the dichotomy are considered (and with such bridges each dichotomy may come to look more like a spectrum or continuum.). At several points after its introduction in Parts I and II, the special problem of the corporation as client is revisited and possible solutions suggested. Illustrating the usefulness of keeping the dichotomies in view, Part IV applies them to several exemplary situations of ethical difficulty in actual lawyer practice. For readers finding it difficult to envision the consequences of these distinctions, turning ahead to Part IV may be useful in making the discussion more concrete. Some commonalities across the dichotomies and connections among them are then developed in the concluding section, Part V.

Trends in mortality due to motor vehicle traffic accident injuries between 1987 and 2011 in a Spanish region (Comunitat Valenciana)

Objective: To analyse the time evolution of the rates of mortality due to motor vehicle traffic accidents (MVTA) injuries that occurred among the general population of Comunitat Valenciana between 1987 and 2011, as well as to identify trend changes by sex and age group. Methods: An observational study of annual mortality trends between 1987 and 2011. We studied all deaths due to MVTA injuries that occurred during this period of time among the non-institutionalised population residing in Comunitat Valenciana (a Spanish Mediterranean region that had a population of 5,117,190 inhabitants in 2011). The rates of mortality due to MVTA injuries were calculated for each sex and year studied. These rates were standardised by age for the total population and for specific age groups using the direct method (age-standardised rate – ASR). Joinpoint regression models were used in order to detect significant trend changes. Additionally, the annual percentage change (APC) of the ASRs was calculated for each trend segment, which is reflected in statistically significant joinpoints. Results: For all ages, ASRs decrease greatly in both men and women (70% decrease between 1990 and 2011). In 1990 and 2011, men have rates of 36.5 and 5.2 per 100,000 men/year, respectively. In the same years, women have rates of 8.0 and 0.9 per 100,000 women/year, respectively. This decrease reaches up to 90% in the age group 15–34 years in both men and women. ASR ratios for men and women increased over time for all ages: this ratio was 3.9 in 1987; 4.6 in 1990; and 5.8 in 2011. For both men and women, there is a first significant segment (p < 0.05) with an increasing trend between 1987 and 1989–1990. After 1990, there are 3 segments with a significant decreasing APC (1990–1993, 1993–2005 and 2005–2011, in the case of men; and 1989–1996, 1999–2007 and 2007–2011, in the case of women). Conclusion: The risk of death due to motor vehicle traffic accidents injuries has decreased significantly, especially in the case of women, for the last 25 years in Comunitat Valenciana, mainly as of 2006. This may be a consequence of the road-safety measures that have been implemented in Spain and in Comunitat Valenciana since 2004. The economic crisis that this country has undergone since 2008 may have also been a contributing factor to this decrease. Despite the decrease, ASR ratios for men and women increased over time and it is still a high-risk cause of death among young men. It is thus important that the measures that helped decrease the risk of death are maintained and improved over time.

Petition of Sever and Welsteed to the General Court (manuscript copy), [1723 June-August]

Two folio-sized leaves containing a two-page handwritten copy of the June 21, 1723 petition of Sever and Welsteed requesting the question of non-resident Fellows in the Corporation be readdressed by the General Court, and a one-page handwritten copy of the answer to the petition passed by the General Court on August 7, 1723. The text of the petition begins "Sheweth that we have formerly represented to the Overseers of the College the Difficulty we were under in the business of it by reason of our not being vested with the power of its Charter..." The petition is incorrectly dated as "June 24th 1722" instead of 1723.

Memorial of the President and Fellows of Harvard College to the General Court for the enlargement of their power to hold real estate. Copy, [1797 July 15]

Four folio-sized leaves containing a handwritten copy of a petition to the Massachusetts General Court from the Harvard Corporation requesting the College's amount of tax exempt real estate be enlarged.