Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.

Key Points: Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin.

Clinical activity of carfilzomib correlates with inhibition of multiple proteasome subunits: application of a novel pharmacodynamic assay.

While proteasome inhibition is a validated therapeutic approach for multiple myeloma (MM), inhibition of individual constitutive proteasome (c20S) and immunoproteasome (i20S) subunits has not been fully explored owing to a lack of effective tools. We utilized the novel proteasome constitutive/immunoproteasome subunit enzyme-linked immunosorbent (ProCISE) assay to quantify proteasome subunit occupancy in samples from five phase I/II and II trials before and after treatment with the proteasome inhibitor carfilzomib. Following the first carfilzomib dose (15-56 mg/m(2) ), dose-dependent inhibition of c20S and i20S chymotrypsin-like active sites was observed [whole blood: ≥67%; peripheral blood mononuclear cells (PBMCs): ≥75%].

Cellular properties and chemosensory responses of the human carotid body.

The carotid body (CB) is the major peripheral arterial chemoreceptor in mammals that mediates the acute hyperventilatory response to hypoxia. The CB grows in response to sustained hypoxia and also participates in acclimatisation to chronic hypoxaemia. Knowledge of CB physiology at the cellular level has increased considerably in recent times thanks to studies performed on lower mammals, and rodents in particular.

Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections.

X-gal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. However, this procedure does not permit the direct confocal acquisition of images, thus preventing the identification of labelled cells on the depth (Z) axis of tissue sections and leading sometimes to erroneous conclusions in co-localization and gene expression studies. Here we report a technique, based on X-gal fluorescence emission and mathematically-based optical correction, to obtain high quality fluorescence confocal images.

T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal.

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na(+) currents as well as transient Ca(2+) currents abolished by the external application of Ni(2+).

Carotid body chemosensory responses in mice deficient of TASK channels.

Background K(+) channels of the TASK family are believed to participate in sensory transduction by chemoreceptor (glomus) cells of the carotid body (CB). However, studies on the systemic CB-mediated ventilatory response to hypoxia and hypercapnia in TASK1- and/or TASK3-deficient mice have yielded conflicting results. We have characterized the glomus cell phenotype of TASK-null mice and studied the responses of individual cells to hypoxia and other chemical stimuli.

PKA-mediated Golgi remodeling during cAMP signal transmission.

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) is part of the set of signaling proteins that are stably associated to the cytosolic surface of Golgi membranes in mammalian cells. In principle, Golgi-associated PKA could participate in either signal transduction events and/or the coordination of Golgi transport activities. Here, we show data indicating that although Golgi-associated PKA is activated fast and efficiently during cell stimulation by an extracellular ligand it does not contribute significantly to cAMP signal transmission to the nucleus.

Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome.

Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits beta5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138+ tumor cells.

Developmental change of T-type Ca2+ channel expression and its role in rat chromaffin cell responsiveness to acute hypoxia.

Neonatal chromaffin cells of the adrenal medulla (AM) are intrinsic chemoreceptors that secrete catecholamines in response to hypoxia, thus contributing to fetal adaptation to extrauterine life. In most mammals studied, oxygen sensitivity of AM cells disappears a few days after birth, possibly due to innervation of the adrenal gland by the cholinergic fibres of the splanchnic nerve (approximately postnatal day 7 in the rat). The mechanisms underlying these homeostatic changes in chromaffin cells are unknown.

Development and validation of an improved inducer-regulator protein complex in the pBRES-regulated expression system.

Widespread adaptation of small molecule-regulated expression systems requires the development of selective inducer molecules that do not have any significant side effects on the endogenous receptors from which the regulated expression system is derived. Here we report the identification and in vitro validation of a novel inducer-receptor pair for the single-plasmid regulated expression system termed pBRES, which contains the ligand-binding domain from the human progesterone receptor (hPR). A small molecule inducer, BLX-913, has been identified as having a 30-fold lower IC(50) for the human progesterone receptor than mifepristone (MFP), the previously best characterized inducer for pBRES.

Decreased expression of maxi-K+ channel beta1-subunit and altered vasoregulation in hypoxia.

Background: Hypertension, a major cause of cardiovascular morbidity and mortality, can result from chronic hypoxia; however, the pathogenesis of this disorder is unknown. We hypothesized that downregulation of the maxi-K+ channel beta1-subunit by hypoxia decreases the ability of these channels to hyperpolarize arterial smooth muscle cells, thus favoring vasoconstriction and hypertension.

Methods And Results: Lowering O2 tension produced a decrease of maxi-K+ beta1-subunit mRNA levels in rat (aortic and basilar) and human (mammary) arterial myocytes.

Exo-mechanism proximity-accelerated alkylations: investigations of linkers, electrophiles and surface mutations in engineered cyclophilin-cyclosporin systems.

Investigations on the scope and utility of exo-mechanism proximity-accelerated reactions in engineered receptor-ligand systems are reported. We synthesized a series of electrophilic cyclosporin (CsA) derivatives by varying electrophiles and linker lengths, prepared a series of nucleophilic cysteine mutations on the surface of cyclophilin A (Cyp), and examined their reactivity and specificity in proximity-accelerated reactions. Acrylamide and epoxide electrophiles afforded useful reactivity and high specificity for alkylation of engineered receptors in Jurkat cell extracts.

Regulation of oxygen sensing by ion channels.

O(2) sensing is of critical importance for cell survival and adaptation of living organisms to changing environments or physiological conditions. O(2)-sensitive ion channels are major effectors of the cellular responses to hypoxia. These channels are preferentially found in excitable neurosecretory cells (glomus cells of the carotid body, cells in the neuroepithelial bodies of the lung, and neonatal adrenal chromaffin cells), which mediate fast cardiorespiratory adjustments to hypoxia.

Induction of T-type calcium channel gene expression by chronic hypoxia.

Cellular responses to hypoxia can be acute or chronic. Acute responses mainly depend on oxygen-sensitive ion channels, whereas chronic responses rely on the hypoxia-inducible transcription factors (HIFs), which up-regulate the expression of enzymes, transporters, and growth factors. It is unknown whether the expression of genes coding for ion channels is also influenced by hypoxia.

Selective inhibition of engineered receptors via proximity-accelerated alkylation.

A new approach for creating allele-specific inhibitors is demonstrated. In this approach, a receptor and ligand are engineered to contain complementary reactive groups that form a covalent bond via a proximity-accelerated reaction upon formation of the receptor-ligand complex, irreversibly modulating the biological function of the receptor. This approach is demonstrated in the cyclophilin-cyclosporin receptor-ligand system by introducing thiol and acrylamide functional groups in the receptor and ligand, respectively.

Osteoid osteoma of the calcaneus: an unusual cause of hindfoot pain in an adolescent athlete.

Osteoid osteomas are distinctive, benign tumors of bone that cause localized pain. This problem, although rare in athletes, must be resolved before pain-free competition can resume. We present a case of a 14-year-old white, male football player who complained of heel pain.

Digital nerves of the foot: anatomic variations and implications regarding the pathogenesis of interdigital neuroma.

Seventy-one cadaveric feet were dissected, with attention to communicating branches of the digital nerves, the diameters of the digital nerves, the distance between the metatarsal heads, and the presence or absence of interdigital neuromas. A communicating branch was absent in 52 feet (73.2%) and present in 19 specimens (26.

Total knee arthroplasty without patellar resurfacing. Clinical outcomes and long-term follow-up evaluation.

Total knee arthroplasty (TKA) without resurfacing of the patella by a single surgeon was reviewed retrospectively in 125 patients: 66 patients (79 knees) were included in the final study group, 32 patients were deceased, 13 were without current address and not able to be contacted, and 14 patients were unable to complete a questionnaire because of severe illness or language barrier. All patients had a diagnosis of osteoarthrosis. The decision to leave the patella unresurfaced was based on the presence of satisfactory patellar articular cartilage, absence of eburnated bone, congruent patellofemoral tracking, normal anatomic patellar shape, and no evidence of crystalline disease or an inflammatory synovitis.

Arterial and ischemic aspects of total knee arthroplasty.

Prospective and retrospective analyses of 1,182 consecutive patients undergoing primary total knee arthroplasty (TKA) were performed to determine (1) the incidence of chronic lower extremity ischemia (CLEI); (2) the effect of tourniquet occlusion; and (3) guidelines that will allow TKA to be performed safely. Despite the appropriately advanced age of our patients, the incidence of CLEI was only 2%. All ischemic complications occurred in six patients with CLEI (25%), but none resulted in death or amputation.

Methods for assessing condition-specific and generic functional status outcomes after total knee replacement.

Many assume that, relative to generic measures, condition-specific health measures are both more sensitive to the condition's severity and more specific because they are less affected by other conditions. We analyzed the sensitivity and specificity of the generic SF-36, condition-specific scales based on the SF-36, and condition-specific measures based on the Knee Society's Clinical Rating System in a study of osteoarthritis patients following knee replacement. As hypothesized, knee-specific role function and pain measures were more specific than generic measures among patients with other comorbid conditions, and less so among patients with only knee problems.

Evaluation of the painful prosthetic joint. Relative value of bone scan, sedimentation rate, and joint aspiration.

Seventy-two joint arthroplasties undergoing total hip or total knee surgery were studied prospectively with plain radiographs, three-phase bone imaging (3PBI), erythrocyte sedimentation rate (ESR), aspiration of the joint for culture, and multiple intraoperative cultures at the time of revision. Intraoperative cultures and the operative appearance were used to form a diagnosis of definite infection (unequivocal microbiology and gross sepsis), possible infection (positive microbiology or gross sepsis), or no infection (neither positive microbiology nor gross sepsis). For the preoperative diagnosis of infection, as opposed to aseptic loosening, 3PBI alone had a sensitivity of 33% and a specificity of 86%.

Immunoaffinity chromatography of a cellular tumor antigen from mouse neuroblastoma cells.

The cellular tumor antigen p53 was isolated from mouse neuroblastoma cells and was found in a form not complexed to another protein. The p53 in these cells was stable, turning over about every 10 h. Its methionine-labelled tryptic peptides were very similar to those of the p53 isolated from SV40-transformed mouse cells.