New chemical entities, steroidal C-17 benzoazoles (5, 6, 9 and 10) and pyrazines (14 and 15) were rationally designed and synthesized. The key reaction for synthesis of the benzoazoles involved the nucleophilic vinylic "addition-elimination" substitution reaction of 3beta-acetoxy-17-chloro-16-formylandrosta-5,16-diene (2) and benzoazole nucleophiles, while that for synthesis of pyrazines involved palladium-catalyzed cross-coupling reaction of 17-iodoandrosta-5,16-dien-3beta-ol (13) with tributylstannyl diazines. Some of the compounds were shown to be potent inhibitors of human CYP17 enzyme as well as potent antagonist of both wild type and mutant androgen receptors (AR). The most potent CYP17 inhibitors were 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (5, code named VN/124-1), 3beta-hydroxy-17-(5(1)-pyrimidyl)androsta-5,16-diene (15) and 17-(1H-benzimidazole-1-yl)androsta-4,16-dien-3-one (6), with IC(50) values of 300, 500 and 915 nM, respectively. Compounds 5, 6, 14 and 15 were effective at preventing binding of (3)H-R1881 (methyltrienolone, a stable synthetic androgen) to both the mutant LNCaP AR and the wild-type AR, but with a 2.2- to 5-fold higher binding efficiency to the latter. Compounds 5 and 6 were also shown to be potent pure AR antagonists. The cell growth studies showed that 5 and 6 inhibit the growth of DHT-stimulated LNCaP and LAPC4 prostate cancer cells with IC(50) values in the low micromolar range (i.e., <10 microM). Their inhibitory potencies were comparable to that of casodex but remarkably superior to that of flutamide. The pharmacokinetics of compounds 5 and 6 in mice were investigated. Following s.c. administration of 50 mg/kg of 5 and 6, peak plasma levels of 16.82 and 5.15 ng/mL, respectively, occurred after 30 to 60 min, both compounds were cleared rapidly from plasma (terminal half-lives of 44.17 and 39.93 min, respectively), and neither was detectable at 8 h. Remarkably, compound 5 was rapidly converted into a metabolite tentatively identified as 17-(1H-benzimidazol-1-yl)androsta-3-one. When tested in vivo, 5 proved to be very effective at inhibiting the growth of androgen-dependent LAPC4 human prostate tumor xenograft, while 6 was ineffective. Compound 5 (50 mg/kg/twice daily) resulted in a 93.8% reduction (P = 0.00065) in the mean final tumor volume compared with controls, and it was also significantly more effective than castration. To our knowledge, this is the first example of an antihormonal agent (an inhibitor of androgen synthesis (CYP17 inhibitor)/antiandrogen) that is significantly more effective than castration in suppression of androgen-dependent prostate tumor growth. In view of these impressive anticancer properties, compound 5 is a strong candidate for development for the treatment of human prostate cancer.

Transgenic mice were generated by using the alpha-myosin heavy chain promoter coupled to the coding sequence of a constitutively active mutant alpha 1B-adrenergic receptor (AR). These transgenic animals demonstrated cardiac-specific expression of this alpha 1-AR with resultant activation of phospholipase C as shown by increased myocardial diacylglycerol content. A phenotype consistent with cardiac hypertrophy developed in adult transgenic mice with increased heart/body weight ratios, myocyte cross-sectional areas, and ventricular atrial natriuretic factor mRNA levels relative to nontransgenic controls. These transgenic animals may provide insight into the biochemical triggers that induce hypertrophy in cardiac disease and serve as a convenient experimental model for studies of this condition.

Adenosine is an autacoid that plays a critical role in regulating cardiac function, including heart rate, contractility, and coronary flow. In this chapter, current knowledge of the functions and mechanisms of action of coronary flow regulation and electrophysiology will be discussed. Currently, there are four known adenosine receptor (AR) subtypes, namely A(1), A(2A), A(2B), and A(3). All four subtypes are known to regulate coronary flow. In general, A(2A)AR is the predominant receptor subtype responsible for coronary blood flow regulation, which dilates coronary arteries in both an endothelial-dependent and -independent manner. The roles of other ARs and their mechanisms of action will also be discussed. The increasing popularity of gene-modified models with targeted deletion or overexpression of a single AR subtype has helped to elucidate the roles of each receptor subtype. Combining pharmacologic tools with targeted gene deletion of individual AR subtypes has proven invaluable for discriminating the vascular effects unique to the activation of each AR subtype. Adenosine exerts its cardiac electrophysiologic effects mainly through the activation of A(1)AR. This receptor mediates direct as well as indirect effects of adenosine (i.e., anti-beta-adrenergic effects). In supraventricular tissues (atrial myocytes, sinuatrial node and atriovetricular node), adenosine exerts both direct and indirect effects, while it exerts only indirect effects in the ventricle. Adenosine exerts a negative chronotropic effect by suppressing the automaticity of cardiac pacemakers, and a negative dromotropic effect through inhibition of AV-nodal conduction. These effects of adenosine constitute the rationale for its use as a diagnostic and therapeutic agent. In recent years, efforts have been made to develop A(1)R-selective agonists as drug candidates that do not induce vasodilation, which is considered an undesirable effect in the clinical setting.

Deletions involving the chromosomal band 5q21 are among the most frequent alterations in prostate cancer. Using single-nucleotide polymorphism (SNP) arrays, we mapped a 1.3 megabase minimally deleted region including only the repulsive guidance molecule B (RGMB) and chromodomain helicase DNA-binding protein 1 (CHD1) genes. Functional analyses showed that CHD1 is an essential tumor suppressor. FISH analysis of 2,093 prostate cancers revealed a strong association between CHD1 deletion, prostate-specific antigen (PSA) biochemical failure (P = 0.0038), and absence of ERG fusion (P < 0.0001). We found that inactivation of CHD1 in vitro prevents formation of ERG rearrangements due to impairment of androgen receptor (AR)-dependent transcription, a prerequisite for ERG translocation. CHD1 is required for efficient recruitment of AR to responsive promoters and regulates expression of known AR-responsive tumor suppressor genes, including NKX3-1, FOXO1, and PPARγ. Our study establishes CHD1 as the 5q21 tumor suppressor gene in prostate cancer and shows a key role of this chromatin remodeling factor in prostate cancer biology.

Classic work by Huggins and Hodges demonstrated that human prostate cancer regresses dramatically during antihormonal therapy but recurs frequently with androgen independence. Perturbations in the androgen receptor (AR) and PTEN-AKT signaling axes are significantly correlated with the progression of prostate cancer. Genetic alterations of the AR cause receptor hypersensitivity, promiscuity, and androgen-independent receptor transactivation. Prostate cancers maintain an elevated AKT activity through the loss of PTEN function or the establishment of autocrine signaling by growth factors and cytokines. We used an in vivo prostate regeneration system to investigate the biological potency of the potential crosstalk between these two signal transduction pathways. We demonstrate a direct synergy between AKT and AR signaling that is sufficient to initiate and progress naïve adult murine prostatic epithelium to frank carcinoma and override the effect of androgen ablation. Both genotropic and nongenotropic signals mediated by AR are essential for this synergistic effect. However, phosphorylation of AR by AKT at Ser-213 and Ser-791 is not critical for this synergy. These results suggest that more efficient therapeutics for advanced prostate cancer may need to target simultaneously AR signaling and AKT or the growth factor receptor tyrosine kinases that activate AKT.

OBJECTIVE

The autosomal-dominant form of the hyperimmunoglobulin E syndrome (AD-HIES) has been described as a multisystem disorder including immune, skeletal, and dental abnormalities. Variants of AD-HIES are known but not well defined.

METHODS

We evaluated 13 human immunodeficiency virus-seronegative patients from six consanguineous families with an autosomal-recessive form of hyperimmunoglobulin E syndrome (AR-HIES) and 68 of their relatives.

RESULTS

Persons affected with AR-HIES presented with the classical immunologic findings of hyperimmunoglobulin E syndrome, including recurrent staphylococcal infections of the skin and respiratory tract, eczema, elevated serum immunoglobulin E, and hypereosinophilia. In addition, severe recurrent fungal and viral infections with molluscum contagiosum, herpes zoster, and herpes simplex were noted. Autoimmunity was seen in two patients. Central nervous system sequelae, including hemiplegia, ischemic infarction, and subarachnoid hemorrhages, were common and contributed to high mortality. Notably, patients with AR-HIES did not have skeletal or dental abnormalities and did not develop pneumatoceles, as seen in AD-HIES. In lymphocyte proliferation assays, patients' cells responded poorly to mitogens and failed to proliferate in response to antigens, despite the presence of normal numbers of lymphocyte subpopulations.

CONCLUSIONS

The autosomal-recessive form of hyperimmunoglobulin E syndrome is a primary immunodeficiency with elevated immunoglobulin E, eosinophilia, vasculitis, autoimmunity, central nervous system symptoms, and high mortality. AR-HIES lacks several of the key findings of AD-HIES and therefore represents a different, previously unrecognized disease entity.

Mutations of the ubiquitin ligase parkin account for most autosomal recessive forms of juvenile Parkinson's disease (AR-JP). Several studies have suggested that parkin possesses DNA-binding and transcriptional activity. We report here that parkin is a p53 transcriptional repressor. First, parkin prevented 6-hydroxydopamine-induced caspase-3 activation in a p53-dependent manner. Concomitantly, parkin reduced p53 expression and activity, an effect abrogated by familial parkin mutations known to either abolish or preserve its ligase activity. ChIP experiments indicate that overexpressed and endogenous parkin interact physically with the p53 promoter and that pathogenic mutations abolish DNA binding to and promoter transactivation of p53. Parkin lowered p53 mRNA levels and repressed p53 promoter transactivation through its Ring1 domain. Conversely, parkin depletion enhanced p53 expression and mRNA levels in fibroblasts and mouse brains, and increased cellular p53 activity and promoter transactivation in cells. Finally, familial parkin missense and deletion mutations enhanced p53 expression in human brains affected by AR-JP. This study reveals a ubiquitin ligase-independent function of parkin in the control of transcription and a functional link between parkin and p53 that is altered by AR-JP mutations.

The adenosine receptors (ARs) in the nervous system act as a kind of "go-between" to regulate the release of neurotransmitters (this includes all known neurotransmitters) and the action of neuromodulators (e.g., neuropeptides, neurotrophic factors). Receptor-receptor interactions and AR-transporter interplay occur as part of the adenosine's attempt to control synaptic transmission. A(2A)ARs are more abundant in the striatum and A(1)ARs in the hippocampus, but both receptors interfere with the efficiency and plasticity-regulated synaptic transmission in most brain areas. The omnipresence of adenosine and A(2A) and A(1) ARs in all nervous system cells (neurons and glia), together with the intensive release of adenosine following insults, makes adenosine a kind of "maestro" of the tripartite synapse in the homeostatic coordination of the brain function. Under physiological conditions, both A(2A) and A(1) ARs play an important role in sleep and arousal, cognition, memory and learning, whereas under pathological conditions (e.g., Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, epilepsy, drug addiction, pain, schizophrenia, depression), ARs operate a time/circumstance window where in some circumstances A(1)AR agonists may predominate as early neuroprotectors, and in other circumstances A(2A)AR antagonists may alter the outcomes of some of the pathological deficiencies. In some circumstances, and depending on the therapeutic window, the use of A(2A)AR agonists may be initially beneficial; however, at later time points, the use of A(2A)AR antagonists proved beneficial in several pathologies. Since selective ligands for A(1) and A(2A) ARs are now entering clinical trials, the time has come to determine the role of these receptors in neurological and psychiatric diseases and identify therapies that will alter the outcomes of these diseases, therefore providing a hopeful future for the patients who suffer from these diseases.

BACKGROUND

Individual-based models can provide the most reliable estimates of the spread of infectious diseases. In the present study, we evaluated the diffusion of pandemic influenza in Italy and the impact of various control measures, coupling a global SEIR model for importation of cases with an individual based model (IBM) describing the Italian epidemic.

RESULTS

We co-located the Italian population (57 million inhabitants) to households, schools and workplaces and we assigned travel destinations to match the 2001 census data. We considered different R(0 )values (1.4; 1.7; 2), evaluating the impact of control measures (vaccination, antiviral prophylaxis -AVP-, international air travel restrictions and increased social distancing). The administration of two vaccine doses was considered, assuming that first dose would be administered 1-6 months after the first world case, and different values for vaccine effectiveness (VE). With no interventions, importation would occur 37-77 days after the first world case. Air travel restrictions would delay the importation of the pandemic by 7-37 days. With an R(0 )of 1.4 or 1.7, the use of combined measures would reduce clinical attack rates (AR) from 21-31% to 0.3-4%. Assuming an R(0) of 2, the AR would decrease from 38% to 8%, yet only if vaccination were started within 2 months of the first world case, in combination with a 90% reduction in international air traffic, closure of schools/workplaces for 4 weeks and AVP of household and school/work close contacts of clinical cases. Varying VE would not substantially affect the results.

CONCLUSIONS

This IBM, which is based on country-specific demographic data, could be suitable for the real-time evaluation of measures to be undertaken in the event of the emergence of a new pandemic influenza virus. All preventive measures considered should be implemented to mitigate the pandemic.

A low-temperature plasma (LTP) probe has been developed for ambient desorption ionization. An ac electric field is used to induce a dielectric barrier discharge through use of a specially designed electrode configuration. The low-temperature plasma is extracted from the probe where it interacts directly with the sample being analyzed, desorbing and ionizing surface molecules in the ambient environment. This allows experiments to be performed without damage to the sample or underlying substrate and, in the case of biological analysis on skin surfaces, without electrical shock or perceptible heating. Positive or negative ions are produced from a wide range of chemical compounds in the pure stateand as mixtures in the gaseous, solution, or condensed phases, using He, Ar, N2, or ambient air as the discharge gas. Limited fragmentation occurs, although it is greater in the cases of the molecular than the atomic discharge gases. The effectiveness of the LTP probe has been demonstrated by recording characteristic mass spectra and tandem mass spectra of samples containing hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) from poly(tetrafluoroethylene) (PTFE) surfaces where limits of detection are as low as 5 pg. Other performance characteristics, when using a commercial ion trap mass spectrometer, include 3-4 orders of magnitude linear dynamic range in favorable cases. Demonstration applications include direct analysis of cocaine from human skin, determination of active ingredients directly in drug tablets, and analysis of toxic and therapeutic compounds in complex biological samples. Ionization of chemicals directly from bulk aqueous solution has been demonstrated, where limits of detection are as low as 1 ppb. Large surface area sampling and control of fragmentation by a simple adjustment of the electrode configuration during operation are other demonstrated characteristics of the method.

Axenfeld-Rieger syndrome (ARS) is a rare autosomal dominant disorder, which encompasses a range of congential malformations affecting the anterior segment of the eye. ARS shows genetic heterogeneity and mutations of the two genes, PITX2 and FOXC1, are known to be associated with the pathogenesis. There are several excellent reviews dealing with the complexity of the phenotype and genotype of ARS. In this study, we will attempt to give a brief review of the clinical features and the relevant diagnostic approaches, together with a detailed review of published PITX2 and FOXC1 mutations.

Myocardial preconditioning with brief coronary artery occlusions before a sustained ischemic period is reported to reduce infarct size. To determine the number of occlusive episodes required to produce the preconditioning effect, we performed single or multiple occlusions of the left circumflex coronary artery (LCx) followed by a sustained occlusion (60 minutes) of the LCx. Anesthetized dogs underwent one (P1), six (P6), or 12 (P12) 5-minute occlusions of the LCx. Each occlusion period was followed by a 10-minute reperfusion period. A 60-minute occlusion of the LCx followed the preconditioning sequences. A control group received a 60-minute occlusion of the LCx without preconditioning. All groups were subjected to 6 hours of reperfusion after which the heart was removed for calculating infarct size (IS), area at risk (AR), and left ventricular mass (LV). The IS/AR ratio for the control group was 29.8 +/- 4.4% (n = 17), which was substantially greater (p less than 0.001) than that of the preconditioned groups: P1, 3.9 +/- 1.3% (n = 14); P6, 0.4 +/- 0.3% (n = 5); and P12, 2.9 +/- 2.8% (n = 5). There were no significant differences in the IS/AR ratio among the three preconditioned groups. The AR/LV ratio was comparable among all groups and did not differ statistically: control, 40.4 +/- 1.3%; P1, 36.2 +/- 1.7%; P6, 36.1 +/- 1.7%; and P12, 37.3 +/- 2.1%. Collateral blood flow to the inner two thirds of the risk region determined with radiolabeled microspheres during ischemia did not differ significantly between the control group (0.03 +/- 0.01 ml/min/g, n = 8) and single occlusion group (0.06 +/- 0.02 ml/min/g, n = 8), indicating that the marked disparity in infarct size could not be attributed to differences in collateral blood flow. The data indicate that preconditioning with one brief ischemic interval is as effective as preconditioning with multiple ischemic periods.

OBJECTIVE

To report the results from a randomized trial comparing accelerated radiotherapy (AR) with accelerated radiotherapy plus carbogen inhalation and nicotinamide (ARCON) in laryngeal cancer.

METHODS

Patients with cT2-4 squamous cell laryngeal cancer were randomly assigned to AR (68 Gy within 36 to 38 days) or ARCON. To limit the risk of laryngeal necrosis, ARCON patients received 64 Gy on the laryngeal cartilage. The primary end point was local control. Secondary end points were regional control, larynx preservation, toxicity, disease-free survival, and overall survival. In a translational side study, the hypoxia marker pimonidazole was used to assess the oxygenation status in tumor biopsies.

RESULTS

From April 2001 to February 2008, 345 patients were accrued. After a median follow-up of 44 months, local tumor control rate at 5 years was 78% for AR versus 79% for ARCON (P = .80), with larynx preservation rates of 84% and 87%, respectively (P = .48). The 5-year regional control was significantly better with ARCON (93%) compared with AR (86%, P = .04). The improvement in regional control was specifically observed in patients with hypoxic tumors and not in patients with well-oxygenated tumors (100% v 55%, respectively; P = .01). AR and ARCON produced equal levels of toxicity.

CONCLUSIONS

Despite lack of benefit in local tumor control for advanced laryngeal cancers, a significant gain in regional control rate, with equal levels of toxicity, was observed in favor of ARCON. The poor regional control of patients with hypoxic tumors is specifically countered by ARCON treatment.

The androgen receptor (AR) is transcriptionally activated by high affinity binding of testosterone (T) or its 5alpha-reduced metabolite, dihydrotestosterone (DHT), a more potent androgen required for male reproductive tract development. The molecular basis for the weaker activity of T was investigated by determining T-bound ligand binding domain crystal structures of wild-type AR and a prostate cancer somatic mutant complexed with the AR FXXLF or coactivator LXXLL peptide. Nearly identical interactions of T and DHT in the AR ligand binding pocket correlate with similar rates of dissociation from an AR fragment containing the ligand binding domain. However, T induces weaker AR FXXLF and coactivator LXXLL motif interactions at activation function 2 (AF2). Less effective FXXLF motif binding to AF2 accounts for faster T dissociation from full-length AR. T can nevertheless acquire DHT-like activity through an AR helix-10 H874Y prostate cancer mutation. The Tyr-874 mutant side chain mediates a new hydrogen bonding scheme from exterior helix-10 to backbone protein core helix-4 residue Tyr-739 to rescue T-induced AR activity by improving AF2 binding of FXXLF and LXXLL motifs. Greater AR AF2 activity by improved core helix interactions is supported by the effects of melanoma antigen gene protein-11, an AR coregulator that binds the AR FXXLF motif and targets AF2 for activation. We conclude that T is a weaker androgen than DHT because of less favorable T-dependent AR FXXLF and coactivator LXXLL motif interactions at AF2.

The mechanism(s) of load-induced muscle hypertrophy is as yet unclear, but increasing evidence suggests a role for locally expressed insulin-like growth factor I (IGF-I). We investigated the effects of concentric (CON) vs. eccentric (ECC) loading on muscle IGF-I mRNA concentration. We hypothesized a greater IGF-I response after ECC compared with CON. Ten healthy subjects (24.4 +/- 0.7 yr, 174.5 +/- 2.6 cm, 70.9 +/- 4.3 kg) completed eight sets of eight CON or ECC squats separated by 6-10 days. IGF-I, IGF binding protein-4 (IGFBP-4), and androgen receptor (AR) mRNA concentrations were determined in vastus lateralis muscle by RT-PCR before and 48 h after ECC and CON. Serum total testosterone (TT) and IGF-I were measured serially across 48 h, and serum creatine kinase activity (CK), isometric maximum voluntary contraction (MVC), and soreness were determined at 48 h. IGF-I mRNA concentration increased 62% and IGFBP-4 mRNA concentration decreased 57% after ECC (P < 0.05). Changes after CON were similar but not significant (P = 0.06-0.12). AR mRNA concentration increased (P < 0.05) after ECC (63%) and CON (102%). Serum TT and IGF-I showed little change. MVC fell 10% and CK rose 183% after ECC (P < 0.05). Perceived soreness was higher (P < 0.01) after ECC compared with CON. Results indicate that a single bout of mechanical loading in humans alters activity of the muscle IGF-I system, and the enhanced response to ECC suggests that IGF-I may somehow modulate tissue regeneration after mechanical damage.

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.

A glycoprotein, termed amphiregulin (AR), inhibits growth of several human carcinoma cells in culture and stimulates proliferation of human fibroblasts and certain other tumor cells. It has been purified to apparent homogeneity from serum-free conditioned medium of MCF-7 human breast carcinoma cells that had been treated with phorbol 12-myristate 13-acetate. AR is a single-chain extremely hydrophilic glycoprotein containing cysteines in disulfide linkage(s) that are essential for biological activity; it is stable between pH 2 and pH 12 and after heating for 30 min at 56 degrees C but unstable at 100 degrees C. The apparent molecular weights of AR and N-Glycanase-treated AR are 14,000 and 15,000, respectively, as assessed by gel chromatography, and approximately 22,500 and approximately 14,000, respectively, as determined by polyacrylamide gel electrophoresis. Treatment of AR with N-Glycanase, O-Glycanase, or neuraminidase does not affect its activity. The pI of AR is approximately 7.8. The amino-terminal amino acid sequence of AR has been determined, and no significant sequence homology between AR and other proteins was found. The molecule thus appears to be a distinct growth regulatory protein.

Rab GTPases are recognized as critical regulatory factors involved in vesicular membrane transport and endosomal fusion. For example, Rab5 directs the transport and fusion of endocytic vesicles to and with early endosomes, whereas Rab4 is thought to control protein trafficking from early endosomes back to the plasma membrane. In the present study, we investigated the role of Rab5 and Rab4 GTPases in regulating the endocytosis, intracellular sorting, and the plasma membrane recycling of the beta(2)AR. In cells expressing the dominant-negative Rab5-S34N mutant, beta(2)AR internalization was impaired, and beta(2)AR-bearing endocytic vesicles remained in either close juxtaposition or physically attached to the plasma membrane. In contrast, a constitutively active Rab5-Q79L mutant redirected internalized beta(2)AR to enlarged endosomes but did not prevent beta(2)AR dephosphorylation and recycling. The expression of either wild-type Rab4 or a Rab4-N121I mutant did not prevent beta(2)AR dephosphorylation. However, the dominant-negative Rab4-N121I mutant blocked beta(2)AR resensitization by blocking receptor recycling from endosomes back to the cell surface. Our data indicate that, in addition to regulating the intracellular trafficking and fusion of beta(2)AR-bearing endocytic vesicles, Rab5 also contributes to the formation and/or budding of clathrin-coated vesicles. Furthermore, beta(2)AR dephosphorylation occurs as the receptor transits between Rab5- and Rab4-positive compartments.

OBJECTIVE

The authors' aim was to determine survival and recurrence rates in patients undergoing resection of rectal cancer achieved by abdominoperineal resection (APR), coloanal anastomosis (CAA), and anterior resection (AR) without adjuvant therapy.

BACKGROUND

The surgery of rectal cancer is controversial; so, too, is its adjuvant management. Questions such as preoperative versus postoperative radiation versus no radiation are key. An approach in which the entire mesorectum is excised has been proposed as yielding low recurrence rates.

METHODS

Of 1423 patients with resected rectal cancers, 491 patients were excluded, leaving 932 with a primary adenocarcinoma of the rectum treated at Mayo. Eighty-six percent were resected for cure. Surgery plus adjuvant treatment was performed in 418, surgery alone in 514. These 514 patients are the subject of this review. Among the 514 patients who underwent surgery alone, APR was performed in 169, CAA in 19, AR in 272, and other procedures in 54. Eighty-seven percent of patients were operated on with curative intent. The mean follow-up was 5.6 years; follow-up was complete in 92%. APR and CAA were performed excising the envelope of rectal mesentery posteriorly and the supporting tissues laterally from the sacral promontory to the pelvic floor. AR was performed using an appropriately wide rectal mesentery resection technique if the tumor was high; if the tumor was in the middle or low rectum, all mesentery was resected. The mean distal margin achieved by AR was 3 +/- 2 cm.

RESULTS

Mortality was 2% (12 of 514). Anastomotic leaks after AR occurred in 5% (16 of 291) and overall transient urinary retention in 15%. Eleven percent of patients had a wound infection (abdominal and perineal wound, 30-day, purulence, or cellulitis). The local recurrence and 5-year disease-free survival rates were 7% and 78%, respectively, after AR; 6% and 83%, respectively, after CAA; and 4% and 80%, respectively, after APR. Patients with stage III disease, had a 60% disease-free survival rate.

CONCLUSIONS

Complete resection of the envelope of supporting tissues about the rectum during APR, CAA, and AR when tumors were low in the rectum is associated with low mortality, low morbidity, low local recurrence, and good 5-year survival rates. Appropriate "tumor-specific" mesorectal excision during AR when the tumor is high in the rectum is likewise consistent with a low rate of local recurrence and good long-term survival. However, the overall failure rate of 40% in stage III disease (which is independent of surgical technique) means that surgical approaches alone are not sufficient to achieve better long-term survival rates.

BACKGROUND

Influenza causes substantial morbidity and annual vaccination is the most important prevention strategy. Accurately measuring vaccine effectiveness (VE) is difficult. The clinical syndrome most closely associated with influenza virus infection, influenza-like illness (ILI), is not specific. In addition, laboratory confirmation is infrequently done, and available rapid diagnostic tests are imperfect. The objective of this study was to estimate the joint impact of rapid diagnostic test sensitivity and specificity on VE for three types of study designs: a cohort study, a traditional case-control study, and a case-control study that used as controls individuals with ILI who tested negative for influenza virus infection.

METHODS

We developed a mathematical model with five input parameters: true VE, attack rates (ARs) of influenza-ILI and non-influenza-ILI and the sensitivity and specificity of the diagnostic test.

RESULTS

With imperfect specificity, estimates from all three designs tended to underestimate true VE, but were similar except if fairly extreme inputs were used. Only if test specificity was 95% or more or if influenza attack rates doubled that of background illness did the case-control method slightly overestimate VE. The case-control method usually produced the highest and most accurate estimates, followed by the test-negative design. The bias toward underestimating true VE introduced by low test specificity increased as the AR of influenza- relative to non-influenza-ILI decreases and, to a lesser degree, with lower test sensitivity.

CONCLUSIONS

Demonstration of a high influenza VE using tests with imperfect sensitivity and specificity should provide reassurance that the program has been effective in reducing influenza illnesses, assuming adequate control of confounding factors.

Androgen receptor (AR) null male mice (AR(L-/Y)) revealed late-onset obesity, which was confirmed by computed tomography-based body composition analysis. AR(L-/Y) mice were euphagic compared with the wild-type male (AR(X/Y)) controls, but they were also less dynamic and consumed less oxygen. Transcript profiling indicated that AR(L-/Y) mice had lower transcripts for the thermogenetic uncoupling protein 1, which was subsequently found to be ligand-dependently activated by AR. We also found enhanced secretion of adiponectin, which is insulin sensitizing, from adipose tissue and a relatively lower expression of peroxisome proliferator-activated receptor-gamma in white adipose tissue in comparison to AR(X/Y) mice. Both factors might explain why the overall insulin sensitivity of AR(L-/Y) mice remained intact, despite their apparent obesity. The results revealed that AR plays important roles in male metabolism by affecting the energy balance, and it is negative to both adiposity and insulin sensitivity.

Ligand-dependent nuclear import is crucial for the function of the androgen receptor (AR) in both health and disease. The unliganded AR is retained in the cytoplasm but, on binding 5alpha-dihydrotestosterone, it translocates into the nucleus and alters transcription of its target genes. Nuclear import of AR is mediated by the nuclear import factor importin-alpha, which functions as a receptor that recognises and binds to specific nuclear localisation signal (NLS) motifs on cargo proteins. We show here that the AR binds to importin-alpha directly, albeit more weakly than the NLS of SV40 or nucleoplasmin. We describe the 2.6-angstroms-resolution crystal structure of the importin-alpha-AR-NLS complex, and show that the AR binds to the major NLS-binding site on importin-alpha in a manner different from most other NLSs. Finally, we have shown that pathological mutations within the NLS of AR that are associated with prostate cancer and androgen-insensitivity syndrome reduce the binding affinity to importin-alpha and, subsequently, retard nuclear import; surprisingly, however, the transcriptional activity of these mutants varies widely. Thus, in addition to its function in the nuclear import of AR, the NLS in the hinge region of AR has a separate, quite distinct role on transactivation, which becomes apparent once nuclear import has been achieved.

We have used the chromatin immunoprecipitation technique to analyze the formation of the androgen receptor (AR) transcription complex onto prostate-specific antigen (PSA) and kallikrein 2 promoters in LNCaP cells. Our results show that loading of holo-AR and recruitment of RNA polymerase II to the promoters occur transiently. The cyclic nature of AR transcription complex assembly is also illustrated by transient association of coactivators GRIP1 and CREB-binding protein and acetylated histone H3 with the PSA promoter. Treatment of cells with the pure antiandrogen bicalutamide also elicits occupancy of the promoter by AR. In contrast to the agonist-liganded AR, bicalutamide-bound receptor is not capable of recruiting polymerase II, GRIP1, or CREB-binding protein, indicating that the conformation of AR bound to anti-androgen is not competent to assemble transcription complexes. Proteasome is involved in the regulation of AR-dependent transcription, as a proteasome inhibitor, MG-132, prevents the release of the receptor from the PSA promoter, and it also blocks the androgen-induced PSA mRNA accumulation. Furthermore, occupancy of the PSA promoter by the 19 S proteasome subcomplex parallels that by AR. Collectively, formation of the AR transcription complex, encompassing AR, polymerase II, and coactivators, on a regulated promoter is a cyclic process involving proteasome function.