"Quantal" Ca2+ release at the cytoplasmic aspect of the Ins(1,4,5)P3R channel in smooth muscle.

Smooth muscle responds to activation of the inositol (1,4,5)-trisphosphate receptor [Ins(1,4,5)P(3)R] with a graded concentration-dependent ("quantal") Ca2+ release from the sarcoplasmic reticulum (SR) store. Graded release seems incompatible both with the finite capacity of the store and the Ca2+-induced Ca2+ release (CICR)-like facility, at Ins(1,4,5)P3Rs, that, once activated, should release the entire content of SR Ca2+. The structural organization of the SR and the regulation of Ins(1,4,5)P3R activity by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and Ca2+ have each been proposed to explain ;quantal' Ca2+ release.

Low pressure air injury to the hand.

We present a case of accidental low-pressure inflation injury to the hand sustained while inflating a football with a foot-operated bellows pump when the nozzle slipped and punctured the first web space. The patient made a full recovery after ecacuation of a haematoma, elevation, antibiotics, and physiotherapy.

Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells.

An ideal technology for direct imaging of post-translationally modified proteins would be one in which the appearance of a fluorescent signal is linked to a modification dependent protein-activation event. Herein, we utilize the protein semisynthesis technique, expressed protein ligation (EPL), to prepare caged analogues of the signaling protein Smad2; the function and fluorescence of the analogues were then photocontrolled in a correlated fashion. We show that this strategy permits titration of the cellular levels of active phosphorylated Smad2 in its biologically relevant, full-length form.

Osmotic and metabolic responses to dehydration and urea-loading in a dormant, terrestrially hibernating frog.

Physiological responses to dehydration in amphibians are reasonably well documented, although little work has addressed this problem in hibernating animals. We investigated osmotic and metabolic responses to experimental manipulation of hydration state in the wood frog (Rana sylvatica), a terrestrial hibernator that encounters low environmental water potential during autumn and winter. In winter-conditioned frogs, plasma osmolality varied inversely with body water content (range 69-79%, fresh mass) primarily due to increases in sodium and chloride concentrations, as well as accumulation of glucose and urea.

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis.

Expressed protein ligation (EPL) is a protein semisynthesis technique that allows the site-specific introduction of unnatural amino acids and biophysical probes into proteins. In the present study, we illustrate the utility of the approach through the generation of two semisynthetic proteins bearing spectroscopic probes. Dihydrofolate reductase containing a single (13)C probe in an active site loop was generated through the ligation of a synthetic peptide-alpha-thioester to a recombinantly generated fragment containing an N-terminal Cys.

Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt.

Controlling protein function through posttranslational manipulations has emerged as an attractive complementary technology to existing genetic systems. Often these methods involve developing pharmacological agents to probe protein function without the need to generate a unique compound for each protein family. One common strategy uses small molecules that act as chemical inducers of dimerization by mediating the interaction of two proteins.

Rectocele repair: a randomized trial of three surgical techniques including graft augmentation.

Objective: This study was undertaken to compare outcomes of 3 different rectocele repair techniques.

Study Design: One hundred six women with stage II or greater posterior vaginal wall prolapse were randomly assigned to either posterior colporrhaphy (n = 37), site-specific rectocele repair (n = 37), or site-specific rectocele repair augmented with a porcine small intestinal submucosa graft (Fortagen, Organogenesis, Inc, Canton, MA; n = 32). Subjects underwent a physical examination and completed 3 validated pelvic floor instruments at baseline and 6 months, 1 year, and 2 years after surgery.

Post-translational enzyme activation in an animal via optimized conditional protein splicing.

Control over the timing, location and level of protein activity in vivo is crucial to understanding biological function. Living systems are able to respond to external and internal stimuli rapidly and in a graded fashion by maintaining a pool of proteins whose activities are altered through post-translational modifications. Here we show that the process of protein trans-splicing can be used to modulate enzymatic activity both in cultured cells and in Drosophila melanogaster.

Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation.

Potassium channels are K+-selective protein pores in cell membrane. The selectivity filter is the functional unit that allows K+ channels to distinguish potassium (K+) and sodium (Na+) ions. The filter's structure depends on whether K+ or Na+ ions are bound inside it.

Ca2+ microdomains in smooth muscle.

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store.

Structural and functional consequences of an amide-to-ester substitution in the selectivity filter of a potassium channel.

The selectivity filter of K(+) channels comprises four contiguous ion binding sites, S1 through S4. Structural and functional data indicate that the filter contains on average two K(+) ions at any given time and that these ions reside primarily in two configurations, namely to sites S1 and S3 or to sites S2 and S4. Maximum ion flux through the channel is expected to occur when the energy difference between these two binding configurations is zero.

Semisynthetic proteins in mechanistic studies: using chemistry to go where nature can't.

The manipulation of the chemical structure of proteins beyond what is feasible with standard genetics, offers a powerful strategy to investigate protein mechanisms. By allowing the incorporation of biophysical probes, unnatural amino acids, and post-translational modifications in proteins, chemistry-driven approaches have greatly contributed to the understanding of the molecular basis of protein function.

Solution structure and folding characteristics of the C-terminal SH3 domain of c-Crk-II.

Crk-II is a signaling adaptor protein that is involved in many cellular processes including apoptosis, proliferation, and differentiation. It has a modular domain architecture consisting of an Src homology 2 domain (SH2) followed by two Src homology 3 (SH3) domains. The structures and ligand-binding properties of the SH2 and the middle SH3 domains are well-characterized.

Intermittent urinary retention secondary to a uterine leiomyoma.

Urinary retention rarely affects reproductive-age women. In obstructive retention, the source must be determined and then removed to allow the patient to void normally. The most common causes of obstructive retention are gynecologic surgery and pelvic masses.

Protein ligation: an enabling technology for the biophysical analysis of proteins.

Biophysical techniques such as fluorescence spectroscopy and nuclear magnetic resonance (NMR) spectroscopy provide a window into the inner workings of proteins. These approaches make use of probes that can either be naturally present within the protein or introduced through a labeling procedure. In general, the more control one has over the type, location and number of probes in a protein, then the more information one can extract from a given biophysical analysis.

Role of the basic helix-loop-helix protein ITF2 in the hormonal regulation of Sertoli cell differentiation.

Sertoli cells are a post-mitotic terminally differentiated cell population that forms the seminiferous tubules in the adult testis and provides the microenvironment and structural support for developing germ cells. During pubertal development, Sertoli cells are responsive to follicle-stimulating hormone (FSH) to promote the expression of differentiated gene products. The basic helix-loop-helix (bHLH) and inhibitors of differentiation (Id) transcription factors are involved in the differentiation of a variety of cell lineages during development.

In smooth muscle, FK506-binding protein modulates IP3 receptor-evoked Ca2+ release by mTOR and calcineurin.

Ca2+ release from the sarcoplasmic reticulum (SR) by the IP3 receptors (IP3Rs) crucially regulates diverse cell signalling processes from reproduction to apoptosis. Release from the IP3R may be modulated by endogenous proteins associated with the receptor, such as the 12 kDa FK506-binding protein (FKBP12), either directly or indirectly by inhibition of the phosphatase calcineurin. Here, we report that, in addition to calcineurin, FKPBs modulate release through the mammalian target of rapamycin (mTOR), a kinase that potentiates Ca2+ release from the IP3R in smooth muscle.

IP3-mediated Ca2+ increases do not involve the ryanodine receptor, but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in guinea-pig colonic smooth muscle cells.

Smooth muscle responds to IP3-generating (sarcolemma acting) neurotransmitters and hormones by releasing Ca2+ from the sarcoplasmic reticulum (SR) via IP3 receptors (IP3Rs). This release may propagate as Ca2+ waves. The Ca2+ signal emanating from IP3 generation may be amplified by its activating further Ca2+ release from ryanodine receptors (RyRs) in the process of Ca2+-induced Ca2+ release (CICR).

Development of a tandem protein trans-splicing system based on native and engineered split inteins.

Protein trans-splicing involving naturally or artificially split inteins results in two polypeptides being linked together by a peptide bond. While this phenomenon has found a variety of applications in chemical biology and biotechnology, precious little is known about the molecular recognition events governing the initial fragment association step. In this study, fluorescence approaches have been used to measure the dissociation constant for the Ssp DnaE split intein interaction and to determine the on and off rates of fragment association.