Dislocation of the hip after normal sonographic screening examination: a case report and literature review.

We report the case of a five-month-old girl presenting with a subluxed left hip following normal neonatal clinical examination and serial ultrasound screening. Her only risk factor for developmental dysplasia of the hip (DDH) was breech presentation. She underwent closed reduction with successful concentric reduction. This case demonstrates that hip subluxation can occur after normal ultrasound screening, and has important clinical and medicolegal implications. Consideration should be given to further follow-up in children with overt risk factors for DDH, even after normal ultrasound examination.

Comparative outcomes following endoscopic ureteral detachment and formal bladder cuff excision in open nephroureterectomy for upper urinary tract transitional cell carcinoma.

PURPOSE: The introduction of laparoscopic nephroureterectomy highlights the need for the critical appraisal of approaches to the distal ureter at surgery for upper tract transitional cell carcinoma. We compared differences after endoscopic ureteral detachment and open bladder cuff excision in nephroureterectomy. MATERIALS AND METHODS: A total of 138 patients underwent open nephroureterectomy for upper urinary tract transitional cell carcinoma from 1982 to 2005 with a median followup of 43 months. Of these patients 90 underwent endoscopic ureteral detachment and 48 underwent bladder cuff excision. Demographic, perioperative and oncological outcome data were collected in all cases. Statistical analyses were performed using the Student t test, chi-square and log rank tests, and logistic and Cox regression. RESULTS: Mean operative duration was significantly lower in the endoscopic detachment group than in the bladder cuff group (p <0.01). There were 49 (54.4%) bladder recurrences in the endoscopic detachment group, of which 8 (16.3%) were muscle invasive and 3 (3.3%) developed at the resection site. There were 23 (47.9%) bladder recurrences in the bladder cuff group, of which 3 (13.0%) were muscle invasive and 2 (4.2%) developed at the resection site. All 5 resection site tumors occurred after excision of muscle invasive distal ureteral tumors and 4 of these had positive margins. There were no differences in recurrence-free survival or disease specific survival between the groups. Operation subtype did not predict oncological outcome on univariate or multivariate analysis. CONCLUSIONS: Endoscopic ureteral detachment reduces operative duration and is associated with equivalent oncological outcomes compared with open bladder cuff excision in nephroureterectomy. Caution should be exercised in patients with low ureteral tumors.

Does a dedicated hip fracture unit improve clinical outcomes? A five-year case series.

INTRODUCTION: The aim of the study was to establish whether a dedicated hip fracture unit, geographically separate from the local major trauma centre, could improve clinical outcomes for patients sustaining proximal femoral fragility fractures. MATERIALS AND METHODS: This study was a retrospective case series, using data collected from Brighton and Sussex University Hospitals NHS Trust's submissions to the National Hip Fracture Database between 1 April 2011 and 16 September 2016. The outcomes measured were mortality, length of hospital stay, time from admission to surgical intervention and return to premorbid residence. Patients were compared before and after reconfiguration of services into a separate dedicated hip fracture unit geographically distinct from the major trauma centre. RESULTS: A total of 2117 patients (2178 injuries) were managed before the existence of the hip fracture unit, while 660 patients (673 injuries) were treated within the hip fracture unit. During the five-year study period, the 30-day mortality rate (pre-hip fracture unit 5.47% vs hip fracture unit 3.13%, P = 0.014), variance in the length of hospital stay (P < 0.001), mean time to surgical intervention (P = 0.044) and return to premorbid residence were significantly improved. An immediate 12-month comparison demonstrated significantly improved variance in length of hospital stay (P = 0.020) and return to premorbid residence (P = 0.015). DISCUSSION: The reconfiguration of services significantly reduced variance in length of stay, enabling accurate resource planning in future. Multiple incremental improvements in service provision, in addition to the hip fracture unit, may explain the lower mortality observed. CONCLUSION: While further research is required, replication of the hip fracture unit service model may potentially afford significant clinical and financial gains.

Evidence that generation of inositol 1,4,5-trisphosphate and hydrolysis of phosphatidylinositol 4,5-bisphosphate are rapid responses following addition of fungal elicitor which induces phytoalexin synthesis in lucerne (Medicago sativa) suspension culture cells.

Treatment of lucerne suspension culture cells with glycoprotein elicitor from the phytopathogenic fungus Verticillium albo-atrum R & B triggers Ca(2+)-mediated induction of antimicrobial secondary metabolites termed phytoalexins. The present study investigated the possible role of polyphosphoinositide signal transduction in phytoalexin elicitation. Within 1 min of addition of elicitor to lucerne suspension culture cells we found a 100-160% (15-25 pmol/g fresh wt) increase in the level of compound with chromatographic and electrophoretic properties expected for an inositol trisphosphate (InsP3) and which was strongly bound by an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-specific binding protein; after 3 min the level of this compound had fallen below that observed prior to elicitor challenge. In 32P-prelabelled cells, the relative proportion of radioactivity which cochromatographed with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was found to have decreased by 48% 1 min after elicitor addition and that rapid depletion of membrane lipid radioactivity was specific to this lipid fraction. The rapid, transient increase in level of Ins(1,4,5)P3 and concomitant fall in PtdIns(4,5)P2 suggests that Ins(1,4,5)P3 generated by hydrolysis of PtdIns(4,5)P2 may provide a Ca(2+)-mobilizing signal in phytoalexin elicitation in lucerne.

Corticosterone response to the plus-maze: high correlation with risk assessment in rats and mice.

Exposure to the elevated plus-maze induces behavioural and physiological effects in rodents consistent with fear/anxiety. Maze-naive animals display high levels of risk assessment towards the open arms, and explore these areas less extensively than other parts of the maze while, immediately following the test, pain latencies, skin conductance levels, and plasma corticosterone titres (CORT) are significantly elevated. Although previous research has suggested a link between the plasma CORT response and open-arm exploration, significant elevations in CORT have also been found with restricted exposure to the closed arms. The present study employed ethological measures in an attempt to further characterise the relationship between behavioural and CORT responses to this widely used animal model of anxiety. Our results confirm that, relative to home-cage controls, 5-min exposure to the plus-maze significantly increases plasma CORT levels in test-naive male Wistar rats and male Swiss-Webster mice. Furthermore, in both species, the CORT response was found to be highly correlated with measures of risk assessment (mice: rs = +0.87; rats: rs = +0.58), but not with measures of open-arm activity (entries, time), general locomotor activity, rearing, or head dipping. Findings are discussed in relation to the functional significance of risk assessment in potentially dangerous situations and the potential involvement of glucocorticoids in this process. All rights reserved.

DNA demethylation and histone deacetylation inhibition co-operate to re-express estrogen receptor beta and induce apoptosis in prostate cancer cell-lines.

INTRODUCTION: Epigenetic silencing mechanisms are increasingly thought to play a major role in the development of human cancers, including prostate cancer. Promoter CpG island hypermethylation and histone hypoacetylation, catalyzed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC), respectively, are associated with transcriptional repression in a number of cancers. Evidence is accumulating the two mechanisms are dynamically linked, yet few studies have examined a potential interaction in prostate cancer. METHODS: LNCaP, DU-145, and PC-3 prostate cancer cells were co-treated with a DNMT inhibitor, 5'-aza-2'-deoxycytidine (5-AZAC), and an HDAC inhibitor, trichostatin A (TSA). Following treatment cells were processed for cell proliferation/apoptosis assays, or harvested for real-time RT-PCR. Assessed target genes were estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), androgen receptor (AR), progesterone receptor (PGR), and prostate specific antigen (PSA). RESULTS: In all cell-lines, co-treatment was associated with reduced cell proliferation compared with control groups (P<0.05). A reciprocal rise in caspase activation was identified, indicating apoptosis was the major mechanism of cell death. Most marked effects were seen in the androgen-dependent, AR-positive LNCaP cell-line. In all cell-lines, an additive re-expression of ERbeta was identified in the co-treatment group, a finding not seen for either AR or PSA. CONCLUSION: At concentrations associated with gene re-expression, the DNA demethylating agent 5-AZAC and the HDAC inhibitor TSA co-operate to induce apoptosis in prostate cancer cell-lines. Increased apoptosis in the co-treatment group was associated with marked re-expression of ERbeta, raising the possibility of further targeting of prostate cancer cells with ERbeta-selective agents.

Biosynthesis of Cutin: Enzymatic Conversion of omega-Hydroxy Fatty Acids to Dicarboxylic Acids by Cell-free Extracts of Vicia Faba Epidermis.

Long chain dicarboxylic acids are constituents of the protective biopolymers cutin and suberin of plants. Cell-free extracts from the excised epidermis of Vicia faba leaves catalyzed conversion of 16-hydroxy[G-(3)H]hexadecanoic acid to the corresponding dicarboxylic acid with nicotinamide-adenine dinucleotide phosphate as the preferred cofactor. This enzymatic activity, located largely in the 100,000g supernatant fraction, had a pH optimum near 8. This dehydrogenase showed an apparent Km of 1.25 x 10(-5)m and 3.6 x 10(-4)m for 16-hydroxyhexadecanoic acid and NADP, respectively. Modification of the substrate, either by esterification of the carboxyl group or by introduction of another hydroxyl group at C-10, resulted in a substantial (two-thirds) decrease in the rate of reaction, and hexadecanol was not a good substrate. The enzyme was inhibited by thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate. The aldehyde intermediate was trapped by the inclusion of dinitrophenyl hydrazine in the reaction mixture, and the 16-oxo compound was regenerated and identified. Furthermore, synthetic 16-oxo-[G-(3)H] hexadecanoic acid was readily converted to the dicarboxylic acid by the cell-free preparation. These results demonstrate that epidermis of Vicia faba contains an omega-hydroxyacid dehydrogenase and an omega-oxoacid dehydrogenase.

Systemic cholesterol crystal embolisation with pulmonary involvement: a fatal combination after coronary angiography.

Cholesterol crystal embolisation (CCE) is a rare but serious complication of invasive arterial procedures associated with a high mortality, and is a condition that medical staff undertaking invasive vascular procedures should be aware of. It is manifest as a multisystem disorder commonly involving the kidneys and peripheries, but rarely affecting the lungs. A case of fatal CCE with pulmonary involvement is reported, and similar published case reports are reviewed. The pathogenesis of lung involvement in CCE is unclear, but the combination is reported to be invariably fatal.

Irreversible effects of calcium ions on the plasma membrane calcium pump.

The calcium pump of human red cells can be irreversibly activated by preincubation of the membranes in the presence of calcium ions, with a pattern reminiscent of that produced by controlled trypsin attack. With 1 mM Ca2+, the activity of the basal enzyme increases three to fourfold over 30 to 60 min, to levels about half those obtained in the presence of calmodulin. On the whole, the effect occurs slowly, with a very low Ca2+ affinity at 37 degrees C and is unaffected by serine-protease inhibitors. The activation caused by 1 mM Ca2+ is little affected by leupeptin (a thiol-protease inhibitor) and that obtained at 10 microM Ca2+ is not inhibited. Preincubations at 0 degrees C also lead to activation, to a level up to half that seen at 37 degrees C, and the effect is not affected by leupeptin or antipain. No activation is observed by preincubating soluble purified Ca,Mg-ATPase in Ca(2+)-containing solutions at 37 degrees C. Instead, calcium ions protect the detergent-solubilized enzyme from thermal inactivation, the effect being half-maximal between 10 and 20 microM Ca2+. We conclude that the activation of the membrane-bound Ca,Mg-ATPase by Ca2+ should result from an irreversible conformational change in the enzyme and not from attack by a membrane-bound protease, and that this change presumably arises from the release of inhibitory particles existing in the original membrane preparations.

Characterisation of membrane phospholipids and glycolipids from a halophilic archaebacterium by high-performance liquid chromatography/electrospray mass spectrometry.

Combined high-performance liquid chromatography and electrospray mass spectrometry (LC/ES-MS) has been used for direct characterisation of the polar membrane lipids in total lipid extracts from Halobacterium salinarium, a species of halophilic archaebacterium. The principle phospholipids found were the diphytanyl archaeol phosphatidylglycerol and diphytanyl archaeol phosphatidylglycerolphosphate methyl ester. The application of LC/ES-MS revealed the additional presence of diphytanyl archaeol phosphatidylglycerol sulphate The extracts also contained an archaeol glycolipid, initially detected in preliminary offline ES-MS studies, which was further characterised by LC/ES-MS and by product ion tandem mass spectrometry (MS/MS) as a sulphate ester of diglycosyl-2,3-di-O-phytanyl-sn-glycerol. Whilst archaeol phospho- and glycolipids containing a (C(20)-C(20))-isopranyl glycerol ether core predominated, LC/ES-MS of the extracts from Halobacterium salinarium indicated the presence of an analogue containing one double bond in its isoprenyl ether core as a minor component of the phosphatidylglycerolphosphate methyl ester fraction, providing a further example of the previously recognised existence of isoprenologues of diphytanyl archaeols which occur as minor components of archaebacterial membrane lipids. The value of these techniques in compositional analysis of archaebacterial lipid extracts is discussed.

Quantitation by fast-atom bombardment mass spectrometry: assay of cytidine 3',5'-cyclic monophosphate-responsive protein kinase.

A protein kinase, stimulated by cytidine 3',5'-cyclic monophosphate, is conventionally assayed by monitoring the incorporation of radiolabelled phosphate from adenosine triphosphate into a histone substrate. Here the assay of the protein kinase is carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data so obtained show good agreement with data obtained by the conventional radiometric assay: the intrinsic advantage of the mass spectrometric assay is the capacity for multiple component monitoring; the ability of the kinase to bind competing cyclic nucleotides together with integral adenosine triphosphatase (ATPase) and phosphodiesterase activity can also be assessed.

Tandem mass spectrometry in vitamin E analysis.

Tandem mass spectrometry is applied to the tocopherols and representative tocotrienols of the vitamin E family. The collision-induced dissociation/mass analysed ion kinetic energy spectra generated from three ions in the electron impact ionization spectra of 5,7,8-trimethyltocol, 5,7,8-trimethyltocotrienol, 5,7-, 5,8- and 7,8-dimethyltocol and 7,8-dimethyltocotrienol are described. The technique allowed direct physical characterization of each class of tocochromanol, and in the case of monomethyltocols differentiation of 5-methyltocol from the 7- and 8-methyltocol isomers, and its value in analysis of biological tissue extracts is established.

Quantitation by fast-atom bombardment/mass-analysed ion kinetic energy spectrometry: kinetic analysis of cyclic nucleotide phosphodiesterase activity.

Quantitation of cyclic nucleotide phosphodiesterase activity by means of fast-atom bombardment (FAB) mass spectrometry with mass-analysed ion kinetic energy (MIKE) spectrum scanning is described. Characteristic peaks of the substrate, cyclic AMP, and product, AMP, were identified in positive-ion FAB mass spectra and MIKE scans of the protonated molecules. By spiking enzyme incubates with known quantities of cyclic AMP and AMP and measuring peak heights in the MIKE spectra of both spiked and unspiked samples, the concentrations of cyclic AMP and AMP in solution at the end of a series of enzyme incubations have been estimated. From the data obtained the Km and Vmax of the enzymes were calculated as 181 microM and 28.6 nmol/min respectively, showing excellent agreement with values of the Michaelis constant, Km = 205 microM and the maximum velocity Vmax = 33.2 nmol/min obtained by radioactive assay.

Analysis of cyclic nucleotide-related enzymes by continuous-flow fast-atom bombardment mass spectrometry.

Continuous-flow fast-atom bombardment mass spectrometry has been developed to directly monitor cyclic nucleotide (substrate) and its product levels from an on-going phosphodiesterase reaction. Analysis of cAMP and cCMP phosphodiesterase incubates have been performed where the temporal evolution of the enzymic reaction is monitored and the effect of enzyme concentration upon the rate of reaction determined. Quantitative data on the enzyme kinetics have been obtained, in the form of Lineweaver-Burke plots, that are shown to correlate well with well-established radiometric methods.

Assay of adenosine 3',5'-cyclic monophosphate-dependent protein kinase activity by quantitative fast atom bombardment mass spectrometry.

Cyclic AMP-dependent protein kinase is conventionally assayed by measuring the incorporation of radiolabeled phosphate into a histone substrate. Here the assay of the protein kinase is carried out by the positive-ion fast atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data obtained are in good agreement with those obtained from the conventional radiometric assay of the same kinase preparation. The inherent advantage of this mass spectrometric assay is the capacity for multiple component monitoring; in addition to the kinase activity, the ability of the enzyme to bind cyclic nucleotides, together with integral ATPase and phosphodiesterase activity, can also be estimated from the same spectra.

Kinetic analysis and multiple component monitoring of effectors of adenylyl cyclase activity by quantitative fast-atom bombardment mass spectrometry.

The enzyme adenylyl cyclase catalyses the conversion of adenosine 5'-triphosphate (ATP) to adenosine-3',5'-cyclic monophosphate (cyclic AMP), and is an important pharmaceutical target. Quantitation of this enzyme's activity has been carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The kinetic data obtained are in good agreement with those obtained by the conventional radiometric assay, and this mass spectrometry-based assay offers the facility to monitor the turnover of several components of the incubation simultaneously. This is utilized to study the relative efficiencies of two ATP-regenerating systems, three phosphodiesterase inhibitors and two modified substrates, and to monitor the uptake and conversion of two competing substrates, adenosine 5' triphosphate and 2'-deoxyadenosine-5-triphosphate, to cyclic AMP and to cyclic deoxyAMP, respectively.

Fast-atom bombardment tandem mass spectrometry of cyclic nucleotide analogues used as site-selective activators of cyclic nucleotide-dependent protein kinases.

The mass spectrometric behaviour of six cyclic nucleotide analogues which activate cyclic AMP-dependent protein kinase was studied by positive-ion fast-atom bombardment (FAB) and collision-induced dissociation (CID) mass-analysed ion kinetic energy (MIKE) spectrometry. The compounds studied were 1,N6-ethenoadenosine-3',5'-cyclic monophosphate, (epsilon-cyclic AMP) and 2'-aza-1,N6-ethenoadenosine-3',5'-cyclic monophosphate, which each activate both isoforms of cyclic AMP-dependent protein kinase and have similar affinity for both the 'fast' and the 'slow' regulatory site of each isoform, N6-phenyl-cyclic AMP, which is selective for the 'fast' regulatory site of each isoform, and 6-chloropurine riboside-3',5'-cyclic monophosphate, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphate and 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphate, which are each selective for the 'slow' regulatory site and preferentially activate isoform II. The FAB- and CID/MIKE spectra of the analogues are discussed in relation to their use in studies of the regulation of protein kinase activity by quantitative FAB mass spectrometry.