Lysine 269 is essential for cyclin D1 ubiquitylation by the SCF(Fbx4/alphaB-crystallin) ligase and subsequent proteasome-dependent degradation.

Protein ubiquitylation is a complex enzymatic process that results in the covalent attachment of ubiquitin, through Gly-76 of ubiquitin, to an varepsilonNH2 group of an internal lysine residue in a given substrate. Although E3 ligases frequently use lysines adjacent to the degron within the substrate, many substrates can be targeted to the proteasome through the polyubiquitylation of any lysine. We have assessed the role of lysine residues proximal to the cyclin D1 phosphodegron for ubiquitylation by the SCF(Fbx4/alphaB-crystallin) ubiquitin ligase and subsequent proteasome-dependent degradation of cyclin D1. The work described herein reveals a requisite role for Lys-269 (K269) for the rapid polyubiquitin-mediated degradation of cyclin D1. Mutation of Lys-269, which is proximal to the phosphodegron sequence surrounding Thr-286 in cyclin D1, not only stabilizes cyclin D1 but also triggers cyclin D1 accumulation within the nucleus, thereby promoting cell transformation. In addition, D1-K269R is resistant to genotoxic stress-induced degradation, similar to non-phosphorylatable D1-T286A, supporting the critical role for the post-translational regulation of cyclin D1 in response to DNA-damaging agents. Strikingly, although mutation of lysine 269 to arginine inhibits cyclin D1 degradation, it does not inhibit cyclin D1 ubiquitylation in vivo, showing that ubiquitylation of a specific lysine can influence substrate targeting to the 26S proteasome.

Perceptual equivalence between vision and touch is complexity dependent.

We experience the shape of objects in our world largely by way of our vision and touch but the availability and integration of information between the senses remains an open question. The research presented in this article examines the effect of stimulus complexity on visual, haptic and crossmodal discrimination. Using sculpted three-dimensional objects whose features vary systematically, we perform a series of three experiments to determine perceptual equivalence as a function of complexity. Two unimodal experiments--vision and touch-only, and one crossmodal experiment investigating the availability of information across the senses, were performed. We find that, for the class of stimuli used, subjects were able to visually discriminate them reliably across the entire range of complexity, while the experiments involving haptic information show a marked decrease in performance as the objects become more complex. Performance in the crossmodal condition appears to be constrained by the limits of the subjects' haptic representation, but the combination of the two sources of information is of some benefit over vision alone when comparing the simpler, low-frequency stimuli. This result shows that there is crossmodal transfer, and therefore perceptual equivalency, but that this transfer is limited by the object's complexity.

Outcome in elderly acute myeloid leukaemia.

We reviewed the reports of 79 newly diagnosed acute myeloid leukaemia patients admitted to our department between 1996 and 2005. Patients' characteristics and outcome were analysed. The acute myeloid leukaemia rate for all patients who received intensive chemotherapy, i.e. age > or = 60 years was 52.9%; in the age group (60-69) and > or = 70 years it was 62.5% and 44.4%, respectively. The acute myeloid leukaemia rate difference between the diagnosis periods (1996-2000) and (2001-2005) are statistically significant only in the age group (60-69) years (P = 0.018). For patients who received intensive chemotherapy and treatment other than intensive chemotherapy, the median overall survival was 9.73 and 3.6 months, respectively (P < 0.0001). There was no significant difference in overall survival between the age groups for the two diagnosis periods, except in patients aged > or = 80 years 3.0 and 0.63 month, respectively (P = 0.023). This study improved knowledge of characteristics and outcome of elderly acute myeloid leukaemia patients in our department.

Isolated limb infusion with cytotoxic agent for treatment of localized refractory cutaneous T-cell lymphoma.

We described a 57-yr-old male diagnosed with cutaneous T-cell lymphoma that had failed multiple treatment options, as his disease was mainly confined to one limb. We attempted a novel approach in this condition using a technique of intra-arterial limb infusion with cytotoxic agent Melphalan (ILI) which has been proven beneficial in management of localised malignant melanoma. This treatment approach was well tolerated with mild myelosuppression and moderate limb toxicity. However, a significant improvement has been noted in the affected limb. This case demonstrated the successful use of isolated limb infusion with Melphalan in the management of localised cutaneous T-cell lymphoma. However, this result needs to be confirmed and further study is recommended. We are unaware there have been similar cases reported in the literature.

Surfactant lavage with lidocaine improves pulmonary function in piglets after HCl-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality. The pathophysiology of ARDS includes abnormalities of surfactant function as well as pulmonary inflammation. Immunomodulating drugs, like Lidocaine, have shown some success in decreasing inflammation in ARDS. We attempted to combine surfactant lavage's ability to reverse the surfactant dysfunction, while acting as a vehicle to deliver Lidocaine. Gravity-driven surfactant (Infasurf) lavage (35 ml/kg) was administered alone or mixed with Lidocaine after severe HCl acid injury (0.3 N; 3 cc/kg) in neonatal piglets. Treatment groups included: control (C) ( n = 5), surfactant lavage (SL) (35 ml/kg-diluted Infasurf) ( n = 7) and SL mixed with Lidocaine (SL+L) ( n = 7). About 26-27% of the lavage was retained (phospholipid 73-74 mg/kg; Lidocaine 1.8 mg/kg). Oxygenation progressively increased in the SL and SL+L groups over the 4-hour period (at 240 min: C = 99 +/- 14; SL = 154 +/- 39; SL+L = 230 +/- 40 mmHg) ( p < 0.05). PaCO(2) increased in all groups from 43 +/- 0.3 to 55 +/- 0.7 mmHg. Only SL+L showed a reduction in PaCO(2) (at 240 min: C = 54 +/- 4; SL = 53 +/- 7; SL+L = 49 +/- 2 mmHg) ( p < 0.05). Finally, SL and SL + L had superior characteristics during the quasi-static pressure volume (PV) procedure as compared to Control ( p < 0.05). In our HCl ALI model, SL improved oxygenation and quasi-static lung compliance over C. The pulmonary function effects of SL were further enhanced by the addition of Lidocaine to the surfactant suspension. Combining therapeutic agents with surfactant lavage may be an effective strategy in ALI.

Primary care anticoagulant management using near patient testing.

BACKGROUND: Indications for anticoagulant treatment are increasing and new approaches to anticoagulant services require a shift from hospital to primary care. AIM: To pragmatically test the validity and effectiveness of primary care anticoagulant management using near patient testing. METHODS: Twelve CoaguCheck monitors were supplied to 16 rural practices that had previously provided supervision of anticoagulant therapy. Practices were required to record data for eligible patients from September 1998 to April 1999 and to forward one blood sample per week to the regional hospital laboratory for parallel testing. RESULTS: Nine practices returned data on 122 patients. Indications for anticoagulation Included atrial fibrillation (n = 56), valve replacement (n = 12) and deep venous thrombosis or pulmonary embolus (n=12). Regression of the mean of 185 paired readings against their difference confirmed the validity of the CoaguCheck monitor (r2 = 0.00 [95% CI -0.38 to 0.38]). There were 692 International Normalised Ratio (INR) tests performed representing an average of 5.7 tests per patient. The desired therapeutic range was provided for 609 (88%) of these tests; 294 (48.3%) were within the desired therapeutic range. Results differed significantly between practices. CONCLUSIONS: This study confirmed the validity of anticoagulant management using the CoaguCheck monitor in primary care.

The hypothermic effect of 5-CT in mice is mediated through the 5-HT7 receptor.

The 5-HT(7) receptor is a recent addition to the 5-HT receptor family and to date there is no clear idea as to its potential role in the CNS. The receptor has been mapped by in situ hybridization and 5-HT(7)-like immunoreactivity and has been detected in discrete areas of the brain including the hypothalamus (Oliver et al., 1999). This suggests the receptor may be involved in temperature regulation and have shown that a selective 5-HT(7) receptor antagonist reverses the hypothermic effect of 5-CT in guinea-pigs. The current study confirmed that the 5-HT(7) receptor antagonists, SB-269970 (1-30 mg/kg, i.p.) and SB-258719 (5-20 mg/kg, i.p.), but not the 5-HT(1A) receptor antagonist, WAY 100635(0.1-1 mg/kg, s.c.), or the 5-HT(1B/D) antagonist, GR127935 (1.25-5 mg/kg, i.p.), reversed the hypothermic effect of 5-CT in mice. In addition the effect of 5-CT on body temperature was examined on 5-HT(7) receptor null mutant mice. 5-CT (0.1-1 mg/kg, i.p.) significantly reduced rectal temperature in wildtype but not 5-HT(7) receptor knockout mice. This suggests that the hypothermic effects of 5-CT are mediated through the 5-HT(7) receptor. All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986).

Component-specific surface and physiological activity in bovine-derived lung surfactants.

Composition, surface activity and effects on pressure-volume (P-V) mechanics are examined for lavaged calf lung surfactant (LS) and the clinical exogenous surfactants Infasurf and Survanta. Lavaged LS and Infasurf had closely-matching compositions of phospholipids and neutral lipids. Survanta had higher levels of free fatty acids and triglycerides consistent with its content of added synthetic palmitic acid and tripalmitin. Infasurf and Survanta both contained less total protein than LS because of extraction with hydrophobic solvents, but the total protein content relative to phospholipid in Survanta was about 45% lower than in Infasurf. This difference was primarily due to surfactant protein (SP)-B, which was present by ELISA at a mean weight percent relative to phospholipid of 1.04% in LS, 0.90% in Infasurf, and 0.044% in Survanta. Studies on component fractions separated by gel permeation chromatography showed that SP-B was a major contributor to the adsorption, dynamic surface activity, and P-V mechanical effects of Infasurf, which approached whole LS in magnitude. Survanta had lower adsorption, higher minimum surface tension, and a smaller effect on surfactant-deficient P-V mechanics consistent with minimal contributions from SP-B. Addition of 0.05% by weight of purified bovine SP-B to Survanta did not improve surface or physiological activity, but added 0.7% SP-B improved adsorption, dynamic surface tension lowering, and P-V activity to levels similar to Infasurf. The SP-B content of lung surfactants appears to be a crucial factor in their surface activity and efficacy in improving surfactant-deficient pulmonary P-V mechanics.

High-versus low-threshold surfactant retreatment for neonatal respiratory distress syndrome.

UNLABELLED: Surfactant therapy has become an effective standard therapy for infants with respiratory distress syndrome (RDS). The first dose may be given either as prophylaxis immediately after delivery, or as rescue after an infant has developed RDS. Second and subsequent doses are currently recommended by the manufacturers to be administered at minimal levels of respiratory support. PURPOSE: This study compared the relative efficacy of administering second and subsequent doses of Infasurf surfactant at a low threshold (FIO(2) >30%, still requiring endotracheal intubation) versus a high threshold (FIO(2) >40%, mean airway pressure >7 cm H(2)O) of respiratory support. METHODS: A total of 2484 neonates received a first dose of surfactant; 1267 reached conventional retreatment criteria and were randomized to be retreated according to low- or high-threshold criteria. They were then retreated at a minimum of 6-hour intervals each time they reached their assigned threshold until receiving a maximum of 4 total doses. Subjects were stratified by whether they received their first dose by prophylaxis or rescue and by whether their lung disease was considered complicated (evidence of perinatal compromise or sepsis) or uncomplicated. RESULTS: Among the patients randomized, 33% of prophylaxis and 23% of rescue subjects met criteria for the complicated stratum. Although infants allocated to the high-threshold strategy were receiving slightly more oxygen at 72 hours, there was no difference in the number receiving mechanical ventilation at 72 hours or in the secondary respiratory outcomes (requirement for supplemental oxygen or mechanical ventilation at 28 days, supplemental oxygen at 36 weeks' postconceptional age, inspired oxygen concentration >60% at any time). However, there was a significantly higher mortality for infants with complicated RDS who had received retreatment according to the high-threshold strategy. CONCLUSIONS: We conclude that equal efficacy can be realized by delaying surfactant retreatment of infants with uncomplicated RDS until they have reached a higher level of respiratory support than is the current standard. We speculate that this would result in a substantial cost-saving from less utilization of drug. Conversely, we believe that infants with complicated RDS should continue to be treated by the low-threshold retreatment strategy, which is currently recommended by the manufacturers of the commercially available surfactants.

Combined subcutaneous recombinant alpha-interferon and interleukin-2 in metastatic renal cell cancer: results of the Multicentre All Ireland Immunotherapy Study Group.

OBJECTIVE: To analyse the toxicity and efficacy of combined interferon-alpha and interleukin-2, administered subcutaneously in a general multicentre setting, as treatment for metastatic renal cell carcinoma. METHODS: Thirty-three patients with metastatic renal cell carcinoma were scheduled to receive 2 cyclical doses of subcutaneous interferon-alpha (week 1: 5 MU x 3 days) and interleukin-2 (week 2: 36 MU x 2 days, 9 MU x 3 days; weeks 3-5: 9 MU daily). Karnofsky scores ranged from 80 to 100 (median 90). Metastases occurred in multiple organs (lung 63%, retroperitoneal 39%, liver 24%). Patients were categorised according to the risk of disease progression. Treatment toxicity, therapeutic response and actuarial survival were analysed. RESULTS: All patients received recommended doses of treatment, but 6 received less than 2 cycles. Most were treated as outpatients, although hospitalisation was usual during the 1st week of a cycle. All complained of mild flu-like symptoms. Severe side effects developed in 13 patients (39%), and treatment was discontinued in 3 of these patients. No deaths occurred as a result of treatment. The overall median survival was 10 months. The overall actuarial survival rate at 3 years was 22%. On statistical analysis, actuarial survival rates were not influenced by either response to treatment or risk group category. CONCLUSION: Subcutaneously administered, combined interferon-alpha and interleukin-2 therapy achieves durable survival rates in a minority of patients with renal cell carcinoma. Toxicity is remedial, and not fatal, when subcutaneous therapy is administered by multiple medical disciplines at a variety of centres.

An extraretinally expressed insect cryptochrome with similarity to the blue light photoreceptors of mammals and plants.

Photic entrainment of insect circadian rhythms can occur through either extraretinal (brain) or retinal photoreceptors, which mediate sensitivity to blue light or longer wavelengths, respectively. Although visual transduction processes are well understood in the insect retina, almost nothing is known about the extraretinal blue light photoreceptor of insects. We now have identified and characterized a candidate blue light photoreceptor gene in Drosophila (DCry) that is homologous to the cryptochrome (Cry) genes of mammals and plants. The DCry gene is located in region 91F of the third chromosome, an interval that does not contain other genes required for circadian rhythmicity. The protein encoded by DCry is approximately 50% identical to the CRY1 and CRY2 proteins recently discovered in mammalian species. As expected for an extraretinal photoreceptor mediating circadian entrainment, DCry mRNA is expressed within the adult brain and can be detected within body tissues. Indeed, tissue in situ hybridization demonstrates prominent expression in cells of the lateral brain, which are close to or coincident with the Drosophila clock neurons. Interestingly, DCry mRNA abundance oscillates in a circadian manner in Drosophila head RNA extracts, and the temporal phasing of the rhythm is similar to that documented for the mouse Cry1 mRNA, which is expressed in clock tissues. Finally, we show that changes in DCry gene dosage are associated predictably with alterations of the blue light resetting response for the circadian rhythm of adult locomotor activity.

A genetic linkage map for zebrafish: comparative analysis and localization of genes and expressed sequences.

Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes with essential functions. To facilitate the identification of candidate genes for these mutations, we have genetically mapped 104 genes and expressed sequence tags by scoring single-strand conformational polymorphisms in a panel of haploid siblings. To integrate this map with existing genetic maps, we also scored 275 previously mapped genes, microsatellites, and sequence-tagged sites in the same haploid panel. Systematic phylogenetic analysis defined likely mammalian orthologs of mapped zebrafish genes, and comparison of map positions in zebrafish and mammals identified significant conservation of synteny. This comparative analysis also identified pairs of zebrafish genes that appear to be orthologous to single mammalian genes, suggesting that these genes arose in a genome duplication that occurred in the teleost lineage after the divergence of fish and mammal ancestors. This comparative map analysis will be useful in predicting the locations of zebrafish genes from mammalian gene maps and in understanding the evolution of the vertebrate genome.

Instillation of calf lung surfactant extract (calfactant) is beneficial in pediatric acute hypoxemic respiratory failure. Members of the Mid-Atlantic Pediatric Critical Care Network.

OBJECTIVE: Prospective study of the efficacy of calf lung surfactant extract in pediatric respiratory failure. DESIGN: Multi-institutional, prospective, randomized, controlled, unblinded trial. SETTING: Eight pediatric intensive care units (ICU) of tertiary medical centers. PATIENTS: Forty-two children with acute hypoxemic respiratory failure characterized by diffuse, bilateral pulmonary infiltrates, need for ventilatory support, and an oxygenation index of >7. INTERVENTION: Instillation of intratracheal surfactant (80 mL/m2). MEASUREMENTS AND MAIN RESULTS: Ventilator parameters, arterial blood gases, and derived oxygenation and ventilation indices were recorded before and at intervals after surfactant administration. Complications and outcome measures, including mortality, duration of mechanical ventilation, and length of pediatric ICU and hospital stay, were also examined. Patients who received surfactant demonstrated rapid improvement in oxygenation and, on average, were extubated 4.2 days (32%) sooner and spent 5 fewer days (30%) in pediatric intensive care than control patients. There was no difference in mortality or overall hospital stay. Surfactant administration was associated with no serious adverse effects. CONCLUSIONS: Administration of calf lung surfactant extract, calfactant, appears to be safe and is associated with rapid improvement in oxygenation, earlier extubation, and decreased requirement for intensive care in children with acute hypoxemic respiratory failure. Further study is needed, however, before widespread use in pediatric respiratory failure can be recommended.

Increased survival in low birth weight neonates given prophylactic surfactant.

OBJECTIVE: To compare the effectiveness of a prophylactic surfactant treatment strategy (PRO) to the effectiveness of a rescue (RESC) surfactant treatment strategy in patients at high risk for developing hyaline membrane disease (HMD). STUDY DESIGN: We analyzed data from a retrospective cohort consisting of all patients admitted to the neonatal intensive care units at the centers participating in the recently completed Infasurf-Survanta Comparative Trial. To be in the cohort, a patient had to be admitted during the trial, be <48 hours of age on admission, have a gestational age of <30 weeks, have a birth weight of 501 to 1250 gm, and be free of congenital anomalies. Twelve centers participated in this study. They contributed 1097 patients of whom 381 were treated with a PRO strategy. RESULTS: Survival was significantly higher in the PRO-strategy patients (84% vs 72%, p < 0.05) as was survival without oxygen requirement at a postconceptional age of 36 weeks (60% vs 46%, p < 0.05). In addition, the patients with PRO had a lower prevalence of grade III and IV intraventricular hemorrhage (IVH, 9% vs 14%, p < 0.05). All analyses were controlled for birth weight and type of study center. CONCLUSION: These data support the conclusion that using a PRO treatment strategy results in improved survival in patients at risk for developing HMD. A PRO treatment strategy may also decrease the likelihood of developing a severe IVH.

Zebrafish organizer development and germ-layer formation require nodal-related signals.

The vertebrate body plan is established during gastrulation, when cells move inwards to form the mesodermal and endodermal germ layers. Signals from a region of dorsal mesoderm, which is termed the organizer, pattern the body axis by specifying the fates of neighbouring cells. The organizer is itself induced by earlier signals. Although members of the transforming growth factor-beta (TGF-beta) and Wnt families have been implicated in the formation of the organizer, no endogenous signalling molecule is known to be required for this process. Here we report that the zebrafish squint (sqt) and cyclops (cyc) genes have essential, although partly redundant, functions in organizer development and also in the formation of mesoderm and endoderm. We show that the sqt gene encodes a member of the TGF-beta superfamily that is related to mouse nodal. cyc encodes another nodal-related proteins, which is consistent with our genetic evidence that sqt and cyc have overlapping functions. The sqt gene is expressed in a dorsal region of the blastula that includes the extraembryonic yolk syncytial layer (YSL). The YSL has been implicated as a source of signals that induce organizer development and mesendoderm formation. Misexpression of sqt RNA within the embryo or specifically in the YSL induces expanded or ectopic dorsal mesoderm. These results establish an essential role for nodal-related signals in organizer development and mesendoderm formation.

Lavage administration of dilute surfactants after acute lung injury in neonatal piglets.

Exogenous surfactant therapy is not standard in the acute respiratory distress syndrome (ARDS) because of a lack of proven benefit. Nonuniform surfactant distribution after either bolus or aerosol administration may be an important factor limiting response. In a previous study of acute lung injury, we demonstrated that lavage administration of Exosurf (13.5 mg phospholipid/ml) was both effective and distributed uniformly in the lungs. Since the endogenous surfactant pool is much smaller than the typical dose of exogenous surfactant administered, we hypothesized that dilute surfactant preparations (4-4.5 mg phospholipid/ml) administered by lung lavage would be equally effective in reversing pulmonary dysfunction in a piglet model of acute lung injury. We compared three dilute surfactants: Infasurf (n = 5), KL4-Surfactant (n = 6), and Exosurf (n = 5) with controls (n = 6) and undiluted Exosurf (13. 5 mg phospholipid/ml; n = 6). All dilute surfactant preparations were effective in improving oxygenation and other parameters of pulmonary function. Surfactant administered by lavage resulted in uniform lung distribution. We conclude that dilute surfactants administered by lung lavage are effective in reversing pulmonary dysfunction after acute lung injury. We speculate that doses in the range of 20-40 mg phospholipid/kg may be adequate to improve lung function in ARDS when exogenously administered surfactant is uniformly distributed in the lung.

Mutant rescue by BAC clone injection in zebrafish.

Genes essential for vertebrate body plan specification, organ development, and organ function are likely to be shared between mammals and zebrafish, but only in zebrafish have large-scale, genome-wide mutagenesis screens been conducted to isolate embryonic lethal mutations. Discovering the roles played by these disrupted genes requires their molecular characterization, which would be facilitated by assaying large cloned genomic DNAs for their potential to rescue mutant phenotypes. Here we demonstrate that bacterial artificial chromosomes can rescue the phenotype of floating head (flh) mutants. Homozygous flh embryos lack a differentiated notochord and have a reduced, discontinuous floor plate. Mutant embryos injected with genomic clones containing the flh+ gene often had stretches of several to many notochord cells overlaid by a row of floor-plate cells. In contrast, control mutant embryos injected with artificial chromosomes lacking the flh+ gene failed to form notochord. We conclude that the injection of large-insert genomic clones will speed the isolation of zebrafish genes disrupted by mutation and hence the identification of gene functions necessary for development of vertebrate embryos.