Comparison of ultrasound versus fluoroscopic guided rotator cuff interval approach for MR arthrography.

Our purpose is to compare ultrasound and fluoroscopic-guided approaches to the glenohumeral joint at the rotator cuff interval for magnetic resonance (MR) arthrography of the shoulder. Forty consecutive patients were prospectively studied. 20 patients underwent ultrasound-guided and 20 patients underwent fluoroscopic guided injection. Successful rate of ultrasound guidance is 95%. No significant difference in pain score and duration of injection between two methods (P>.05). Ultrasound-guided rotator cuff interval injection of the glenohumeral joint for MR arthrography is comparable with fluoroscopic-guided injection. Ultrasound is the preferred method as there is no ionizing radiation.

Mercury profiles in sediments of the Pearl River Estuary and the surrounding coastal area of South China.

The spatial and temporal variations of mercury (Hg) in sediments of the Pearl River Estuary (PRE) and the surrounding coastal area (South China Sea) were studied. In surface sediments, the concentrations of Hg ranged from 1.5 to 201ng/g, with an average of 54.4ng/g, displaying a decreasing trend with the distance from the estuary to the open sea. This pattern indicates that the anthropogenic emissions from the Pearl River Delta (PRD) region are probably the main sources of Hg in this coastal region. Using the (210)Pb dating technique, the historical changes in the concentrations and influxes of Hg in the last 100 years were also investigated. The variations in Hg influxes in sediment cores obviously correlate with the economic development and urbanization that has occurred the PRD region, especially in the last three decades.

Trace metal distribution in sediments of the Pearl River Estuary and the surrounding coastal area, South China.

Surface sediments and sediment cores collected at the Pearl River Estuary (PRE) and its surrounding coastal area were analysed for total metal concentrations, chemical partitioning, and Pb isotopic compositions. The distribution of Cu, Cr, Pb, and Zn demonstrated a typical diffusion pattern from the land to the direction of the sea. Two hotspots of trace metal contamination were located at the mixed zone between freshwater and marine waters. The enrichment of metals in the sediments could be attributed to the deposition of the dissolved and particulate trace metals in the water column at the estuarine area. The similar Pb isotopic signatures of the sediments at the PRE and its surrounding coastal area offered strong evidence that the PRE was a major source of trace metals to the adjacent coastal area. Slightly lower (206)Pb/(207)Pb ratios in the coastal sediments may indicate other inputs of Pb in addition to the PRE sources, including the inputs from Hong Kong and other parts of the region.

Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1).

Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).

Dimerization and the effectiveness of ICAM-1 in mediating LFA-1-dependent adhesion.

Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single "fully competent" LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an alpha-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/DeltaD1-2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/DeltaD1-2 and with an approximately 2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/DeltaD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/DeltaD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, "fully competent" LFA-1-binding surface.

Multicenter trial of sentinel node biopsy for breast cancer using both technetium sulfur colloid and isosulfan blue dye.

OBJECTIVE: To determine the factors associated with false-negative results on sentinel node biopsy and sentinel node localization (identification rate) in patients with breast cancer enrolled in a multicenter trial using a combination technique of isosulfan blue with technetium sulfur colloid (Tc99). SUMMARY BACKGROUND DATA: Sentinel node biopsy is a diagnostic test used to detect breast cancer metastases. To test the reliability of this method, a complete lymph node dissection must be performed to determine the false-negative rate. Single-institution series have reported excellent results, although one multicenter trial reported a false-negative rate as high as 29% using radioisotope alone. A multicenter trial was initiated to test combined use of Tc99 and isosulfan blue. METHODS: Investigators (both private-practice and academic surgeons) were recruited after attending a course on the technique of sentinel node biopsy. No investigator participated in a learning trial before entering patients. Tc99 and isosulfan blue were injected into the peritumoral region. RESULTS: Five hundred twenty-nine patients underwent 535 sentinel node biopsy procedures for an overall identification rate in finding a sentinel node of 87% and a false-negative rate of 13%. The identification rate increased and the false-negative rate decreased to 90% and 4.3%, respectively, after investigators had performed more than 30 cases. Univariate analysis of tumor showed the poorest success rate with older patients and inexperienced surgeons. Multivariate analysis identified both age and experience as independent predictors of failure. However, with older patients, inexperienced surgeons, and patients with five or more metastatic axillary nodes, the false-negative rate was consistently greater. CONCLUSIONS: This multicenter trial, from both private practice and academic institutions, is an excellent indicator of the general utility of sentinel node biopsy. It establishes the factors that play an important role (patient age, surgical experience, tumor location) and those that are irrelevant (prior surgery, tumor size, Tc99 timing). This widens the applicability of the technique and identifies factors that require further investigation.

Treadmill injuries to the upper extremity in pediatric patients.

Injury is the leading cause of preventable death and disability in childhood and early adulthood. Approximately 25,000 children are injured on exercise equipment each year. Although hand injuries sustained on stationary bicycles in the home have been well documented, little has been reported on home treadmill injuries. Between September 1996 and March 2000, the authors treated 12 children at The Children's Hospital of Philadelphia for injuries to the upper extremity sustained on a treadmill. The age at the time of injury ranged from 14 months to 7 years, with a median age of 2.4 years. Average length of follow-up was 11.4 months. Ten of the 12 children sustained partial- or full-thickness burns to the volar aspect of the hand and digits. Overall, 6 patients required surgical repair. A total of 25% of the volar flexion creases involved required surgery. All patients had good functional results. The authors discuss the mechanism of injury, management, and measures to prevent treadmill injuries to children. To their knowledge, this is the first report of such injuries resulting from the use of a treadmill.

Mutational analysis of Gbetagamma and phospholipid interaction with G protein-coupled receptor kinase 2.

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2.

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.

Regulation of G protein-coupled receptor kinases by caveolin.

G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, we tested the direct binding of purified full-length GRK2 to various glutathione S-transferase-caveolin-1 fusion proteins, and we discovered a specific interaction of GRK2 with the caveolin scaffolding domain. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function.

Structure-function analysis of G protein-coupled receptor kinase-5. Role of the carboxyl terminus in kinase regulation.

Many G protein-coupled receptors are phosphorylated and regulated by a distinct family of G protein-coupled receptor kinases (GRKs) that specifically target the activated form of the receptor. Recent studies have revealed that the GRKs are also subject to post-translational regulation. For example, GRK5 activity is strongly inhibited by protein kinase C phosphorylation and by Ca2+-calmodulin binding. Ca2+-calmodulin binding also promotes GRK5 autophosphorylation, which further contributes to kinase inhibition. In this study we identify two important structural domains in GRK5, a phospholipid binding domain (residues 552-562) and an autoinhibitory domain (residues 563-590), that significantly contribute to GRK5 localization and function. We demonstrate that the C-terminal region of GRK5 (residues 563-590) contains residues autophosphorylated in the presence of calmodulin as well as the residues phosphorylated by protein kinase C. Deletion of this domain increases the apparent affinity of GRK5 for receptor substrates 3-4-fold but has no effect on nonreceptor substrates. These findings define residues 563-590 of GRK5 as an autoinhibitory domain with efficacy that is regulated by phosphorylation. Another C-terminal domain in GRK5 that appears to be functionally important is found between residues 552 and 562. Deletion of this region significantly inhibits kinase phosphorylation of membrane-bound receptor substrates but has no effect on soluble substrates. Additional studies reveal that this domain is critical for GRK5 interaction with phospholipids and for the intracellular localization of the kinase. Interestingly, similar regions in GRK4 and GRK6 appear to be palmitoylated (and involved in membrane interaction), suggesting evolutionary conservation of the function of this domain.

Binding and phosphorylation of tubulin by G protein-coupled receptor kinases.

Although the beta-adrenergic receptor kinase (betaARK) mediates agonist-dependent phosphorylation and desensitization of G protein-coupled receptors, recent studies suggest additional cellular functions. During our attempts to identify novel betaARK interacting proteins, we found that the cytoskeletal protein tubulin could specifically bind to a betaARK-coupled affinity column. In vitro analysis demonstrated that betaARK and G protein-coupled receptor kinase-5 (GRK5) were able to stoichiometrically phosphorylate purified tubulin dimers with a preference for beta-tubulin and, under certain conditions, the betaIII-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of betaARK contains the primary tubulin binding determinants. In vivo interaction of GRK and tubulin was suggested by the following: (i) co-purification of betaARK with tubulin from brain tissue; (ii) co-immunoprecipitation of betaARK and tubulin from COS-1 cells; and (iii) co-localization of betaARK and GRK5 with microtubule structures in COS-1 cells. In addition, GRK-phosphorylated tubulin was found preferentially associated with the microtubule fraction during in vitro assembly assays suggesting potential functional significance. These results suggest a novel link between the cytoskeleton and GRKs that may be important for regulating GRK and/or tubulin function.

G-protein-coupled receptors: turn-ons and turn-offs.

Advances in the study of G-protein-coupled receptor regulation have provided novel insights into the role of G-protein-coupled receptor kinases and arrestins in this process. Of particular interest are recent studies that have dramatically expanded the known cellular functions of these molecules to include roles in receptor endocytosis and activation of MAP kinase signalling pathways.

Cancer of the male breast.

A retrospective review of 28 male breast cancer patients at Eastern Virginia Medical School and affiliated hospitals was performed to learn about patient characteristics, treatment, and outcome. The mean age of the patients was 64 years, and 60 per cent of patients were obese. The most common presenting symptoms were mass in 79 per cent and nipple discharge in 29 per cent. The median duration of symptoms was 3.3 months. All patients except two underwent mastectomy, and most tumors were early stage (0, I, or II). Many patients, particularly those with advanced disease, were also treated with chemotherapy, radiation therapy, and/or endocrine therapy. At a median follow-up of 29 months, the actuarial 5-year survival was 43 per cent, somewhat worse than the survival of female breast cancer patients in the literature. Male breast cancer patients are treated in a similar fashion to female patients. Aggressive systemic treatment should be considered for patients with poor prognosis disease. The high frequency of estrogen receptor positivity suggests tamoxifen may prove to be particularly useful in these patients.

Body composition and spontaneous growth hormone secretion in normal short stature children.

This study was designed to compare the relationship of measured and estimated indices of adiposity with the spontaneous GH secretion (SGHS) in 37 normal short stature children. Fifteen of the 37 patients (10 males and 5 females) were pubertal, and 22 (17 males and 5 females) were prepubertal. All patients underwent a review of their medical history, a physical exam, laboratory tests, and a nutritional assessment that included anthropometry and evaluation of body composition by bioelectric impedance. The percentage of body fat and body fat mass index were used as measured indices of adiposity. The weight for height ratio, body mass index, and body mass index z-scores were calculated and used as estimated indices of adiposity. Our results showed that SGHS is greatly influenced by variations in adiposity in normal short stature children, and measured indices of adiposity demonstrated the strongest negative correlation with SGHS. Gender differences were apparent in the degree of adiposity that modified SGHS, and it appears that adiposity altered the amplitude of GH pulses in pubertal patients and the number of pulses in prepubertal children. These results suggest that interpretation of SGHS must take into account body composition and gender in addition to pubertal status.

Hypertension secondary to renin-secreting juxtaglomerular cell tumor: case report and review of 38 cases.

A 15-year-old girl with severe high renin hypertension caused by a juxtaglomerular cell tumor (JCT) was successfully treated with the calcium channel blocker nifedipine until surgical removal effected a permanent cure. This case was incorporated into a review of the 37 cases previously published. Comparison of the children and adolescents with the adult population showed that the features of JCT were similar in the two groups except for the average duration of symptoms prior to diagnosis (pediatric group 2.6 years vs. 6.0 years for the adult group). Analysis of all 38 cases demonstrated the following: 1. Teenagers constituted the largest single population with JCT (39%) and approximately two-thirds of the entire population were female. 2. Many patients failed to show persistent hypokalemia despite high plasma renin activity and secondary hyperaldosteronism. 3. Renal angiography was initially negative in more than half the cases. 4. Renal vein renin failed to show lateralization to the affected kidney in 52% of the cases. 5. Computerized tomography demonstrated a renal mass in all of the cases in which it was performed, even when other imaging studies were negative. 6. Calcium channel blockers may evolve as the preferred treatment for the high renin hypertension of JCT.

Developing and maintaining of hemophilia programs in developing countries.

There are elements key to the success of developing and maintaining hemophilia programs in developing countries. Health care providers who are dedicated champions of hemophilia care are essential. Their training through the WFH International Hemophilia Training Centers brings them in contact with modern comprehensive care as well as establishes collegial linkages with treaters in developed centers. Affordable, safe, viral free coagulation products are essential for an effective hemophilia program. Developing essential for an effective hemophilia program. Developing countries may have to use intermediate purity products because of economic considerations, but technologies must be used which reduce the risk of viral contamination. Successful programs also result from linking to hemophilia programs with national health care initiatives. Hemophilia health care must be recognized as a priority within the developing country's health care system.