Flexibility within the Heads of Muscle Myosin-2 Molecules

We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~ 23 pN·nm/rad2 is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor–lever junction

Dilated cardiomyopathy myosin mutants have reduced force-generating capacity

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human ?-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M.D complex in the steady-state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force holding capacity due to the reduced occupancy of the force-holding state

Διδακτική πρόταση για τη διδασκαλία προγραμματιστικού σχεδίου σε μαθητές Δημοτικού στο ψηφιακό εργαλείο “Χελωνόσφαιρα”: Ιπτάμενα σχήματα.

Αντικείμενο της παρούσας εργασίας είναι η παρουσίαση μιας έρευνας για τη μελέτη του τρόπου λειτουργίας μιας διδακτικής πρότασης για τη διδασκαλία βασικών εννοιών του προγραμματισμού και ενός προγραμματιστικού σχεδίου σε παιδιά δημοτικού. Με αφετηρία την ανάγκη για διδασκαλία σχεδίων και στρατηγικών προγραμματισμού με τρόπο ρητό και σαφή, όπως αυτή υποδεικνύεται από τη βιβλιογραφία, προκειμένου οι αρχάριοι προγραμματιστές να εξοπλιστούν με γνωστικά σχήματα κώδικα που βοηθούν στην κατανόηση και τη δόμηση κώδικα, ο ερευνητής προχώρησε στην ανάπτυξη ενός θεωρητικού σχεδιασμού ως πρόταση διδακτικής τέτοιου είδους προγραμματιστικών σχεδίων. Για τη διερεύνηση της αποτελεσματικότητας του θεωρητικού σχεδιασμού, αλλά και την ανάδειξη των αδυναμιών του, ο ερευνητής σχεδίασε ένα εκπαιδευτικό σενάριο με βασικό αντικείμενο τη διδασκαλία ενός σχεδίου για τον προγραμματισμό κινούμενων σχημάτων στο ψηφιακό εργαλείο «Χελωνόσφαιρα», σε μαθητές της Στ’ τάξης του Δημοτικού. Πιο συγκεκριμένα διερευνήθηκε 1)Τι είδους νοήματα ανέπτυξαν οι μαθητές για τις προγραμματιστικές έννοιες με τις οποίες ήρθαν σε επαφή μέσα από τις δραστηριότητες που σχεδιάστηκαν για αυτούς, και 2) Πώς λειτούργησε ο εκπαιδευτικός σχεδιασμός του ερευνητή ως διδακτική πρόταση ως προς την κατάκτηση του προγραμματιστικού σχεδίου των κινούμενων σχημάτων στη χελωνόσφαιρα από τους μαθητές. Με βάση τη φύση του αντικειμένου και των ερευνητικών ερωτημάτων επιλέχθηκε ως μεθοδολογία η έρευνα σχεδιασμού. Στο τέλος της έρευνας παρατίθενται τα αποτελέσματα της έρευνας μέσα από την ποιοτική ανάλυση κρίσιμων συμβάντων στην πορεία εργασίας των μαθητών.The objective of this study is to present a research project on the way a didactic approach concerning the instruction of elementary programming concepts and a specific programming plan, functioned in educational practice. The researcher developed a theoretical didactic design, having as starting point the need of explicit instruction of programming strategies and plans, as it is pointed out by the respective research, in order to have novice programmers equipped with appropriate cognitive schemes that will enable them to comprehend and write code. In order for the efficiency and deficiencies of the theoretical didactic design to be assessed, an educational scenario was designed. The scenario’s main subject was the instruction of the programming plan of coding shapes that could move in a digital Logo-like environment, called MaLT, to senior students of a primary education school. More specifically, the researcher examined 1) what kinds of meaning were developed by the students on the elementary programming concepts that they encountered while working on the scenario’s activities, and 2) how the theoretical didactic design functioned regarding the acquisition of the “moving shapes” programming plan. Due to the nature of the main research objective and research questions, design-based research was chosen as research methodology. In the last part of the study, the results of the research are listed, through the qualitative analysis of critical incidents

Dermatofibrosarcoma protuberans of the vulva

Dermatofibrosarcoma protuberans occurs infrequently in the genital area, and a few cases have been reported in the literature. This type of sarcoma, although it has a relatively low metastatic potential, has a tendency to recur locally. The present study deals with a 76-year-old woman with dermatofibrosarcoma protuberans of the mons pubis, She underwent a wide local excision of the tumor. No recurrence of the disease was noted 2 years postoperatively, The above-mentioned surgical procedure seems to be a sufficient treatment for this rare sarcoma, given the biologic nature of the tumor

Structure of the regulatory domain of scallop myosin at 2.8 Ä resolution

The regulatory domain of scallop myosin is a three-chain protein complex that switches on this motor in response to Ca2+ binding. This domain has been crystallized and the structure solved to 2.8 Å resolution. Side-chain interactions link the two light chains in tandem to adjacent segments of the heavy chain bearing the IQ-sequence motif. The Ca2+-binding site is a novel EF-hand motif on the essential light chain and is stabilized by linkages involving the heavy chain and both light chains, accounting for the requirement of all three chains for Ca2+ binding and regulation in the intact myosin molecule

Localization of N-methyl-norsalsolinol within rodent and human brain

The isoquinoline derivative N-methyl-6,7-dihydroxytetrahydroisoquinoline (N-methyl-norsalsolinol) is present in normal human brain and has been identified in the cerebrospinal fluid of patients with Parkinson\u27s disease (PD). Endogenously, N-methyl-norsalsolinol may be derived from dopamine by condensation with aldehydes or α-ketoacids. In vitro experiments suggest that N-methyl-norsalsolinol is neurotoxic. In this study, high-performance liquid chromatography with electrochemical detection (HPLC-EC) was used to determine N-methyl-norsalsolinol concentrations in mouse, rat, normal human, and PD brain. In addition, a monoclonal antibody was generated against N-methyl-norsalsolinol and used to determine the cellular localization of N-methyl-norsalsolinol in brain. With HPLC-EC, N-methyl-norsalsolinol was detected in all regions of rodent and human brain subjected to analysis. In rodent brains, N-methyl-norsalsolinol tissue concentrations were similar among frontal cortex, ventral midbrain, striatum, hippocampus, and cerebellum. Conversely, in normal human control brains, N-methyl-norsalsolinol was concentrated in the substantia nigra and striatum. In comparison to normal human controls, N-methyl-norsalsolinol levels were significantly lower in the substantia nigra and caudate nuclei from PD patients, a finding possibly related to the death of nigrostriatal dopaminergic neurons. N-methyl-norsalsolinol immunoreactivity colocalized with a general neuronal marker (neuron-specific enolase) and a monoaminergic marker (tyrosine hydroxylase) but not with a glial marker (glial fibrillary acidic protein). The widespread neuronal localization of N-methyl-norsalsolinol in several mammalian species suggests that, in isolation, this compound is a weak neurotoxin. However, endogeneously derived N-methyl-norsalsolinol could contribute to the pathobiology of PD in genetically predisposed individuals after years of accumulation in dopaminergic neurons. © 2008 Wiley-Liss, Inc