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The central structural element of the title compound, C(24)H(29)NO(2), is a carbazole unit substituted with two acetyl residues and an octyl chain. The acetyl residues are nearly coplanar [dihedral angles = 5.37 (14) and 1.0 (3)°] with the carbazole unit which is essentially planar (r.m.s. deviation for all non-H atoms = 0.025 Å). The octyl chain adopts an all-trans conformation. The crystal packing is stabilized by C-H⋯O hydrogen bonds.

Six patients with chronic acquired demyelinating neuropathy (CADP) were treated with autologous peripheral blood stem cell transplantation (PBSCT). Two with polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome improved-improvement was sustained in one but relapsed and required repeat transplant in the other. Two of the three with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and one with an IgM paraprotein and antibodies to nerve improved--of the responders, one relapsed after 18 months and the other was in remission after 6 months. Four developed neutropenic septicemia and pneumonia. The role of PBSCT in CADP refractory to other treatment deserves further investigation but the serious adverse events and lack of sustained response in some patients emphasize the need for caution.

Disease relapse following an allogeneic transplant remains a major cause of treatment failure, often with a poor outcome. Second allogeneic transplant procedures have been associated with high TRM, especially with myeloablative conditioning. We hypothesized that the use of reduced-intensity conditioning (RIC) would decrease the TRM. We performed a retrospective national multicentre analysis of 71 patients receiving a second allogeneic transplant using RIC after disease relapse following an initial allogeneic transplant. The majority of patients had leukaemia/myelodysplasia (MDS) (N=57), nine had lymphoproliferative disorders, two had myeloma and three had myeloproliferative diseases. A total of 25% of patients had unrelated donors. The median follow-up was 906 days from the second allograft. The predicted overall survival (OS) and TRM at 2 years were 28 and 27%, respectively. TRM was significantly lower in those who relapsed late (>11 months) following the first transplant (2 years: 17 vs 38% in early relapses; P=0.03). Two factors were significantly associated with a better survival: late relapse (P=0.014) and chronic GVHD following the second transplant (P=0.014). These data support our hypothesis that the second RIC allograft results in a lower TRM than using MA. A proportion of patients achieved a sustained remission even when relapsing after a previous MA transplant.

Lymphoplasmacytic lymphoma (LL) is incurable by standard therapy (median survival: 60 months). UK transplant registry data 1984-2003 identified 18 cases of histologically verified LL (median age: 50 years, range: 38-58 years). Nine patients received high dose chemotherapy [plus total body irradiation (TBI) in 1/9] and autologous peripheral blood stem cells (PBSC). Disease status at transplant was complete remission (2), partial remission (5), primary refractory (1) or relapse (1). Transplant related mortality (TRM) at 12 months was 0%. Median follow-up is 44 months with 4 year disease free survival 43% and overall survival 73%. Karnofsky performance status (KPS) is 80-100%. The nine allografted patients (median age: 49 years, range: 39-56 years) were conditioned with standard TBI (2), BEAM (2) or FLU-MEL (5) and received PBSC from HLA-matched sibling (8) or unrelated (1) donors. Disease status at transplant was partial remission (7) or primary refractory (2). TRM at 12 months was 44%. Complications included graft failure (2), grades I-II acute graft versus host disease (aGVHD) (2), grades III-IV aGVHD (3) and chronic GVHD (4). Median follow-up is 32 months with 4 year disease free survival 44% and overall survival 56%. KPS is 70-100%.

We previously reported that daily dose pomalidomide (CC-4047), a thalidomide analogue, has excellent anti-myeloma activity but is associated with myelosuppression and deep vein thrombosis. We report here a phase 1 study to determine the maximum tolerated dose (MTD) of pomalidomide at 1 mg, 2 mg, 5 mg and 10 mg on alternate days (ad). Twenty patients with relapsed myeloma were treated. Grade 4 neutropenia occurred in all patients receiving 10 mg and the MTD was defined as 5 mg ad. No thrombotic events were observed. Pomalidomide was continued following the 4-week MTD study in 17/20 patients for a median of 14 months. 10% of patients had a complete response and >50% reduction in paraprotein was achieved in 50% of subjects. Progression-free survival was 10.5 months and median overall survival was 33 months. A significant rise was observed in the proportion of CD8(+) cells. Alternate day pomalidomide was associated with a marked reduction in the incidence of thrombosis whilst maintaining excellent anti-myeloma activity. This trial provides further in vivo evidence that pomalidomide modulates the immune system in myeloma patients. Phase 2 studies to further assess the optimal schedule of administration and anti-myeloma activity of this agent are planned.

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

Patients who relapse after High dose therapy and autologous stem cell transplant (ASCT) for Diffuse large B cell Lymphoma (DLBCL) have a poor prognosis with a median survival of only 3-6 month.1-2 This case demonstrates the ability of thalidomide at low doses to induce durable response in a patient with DLBCL who relapsed after full intensity allogeneic transplantation.

Objective: Assess the perception of stress amongst medical students and their coping strategies. METHODOLOGY/STUDY DESIGN: A cross sectional study using a semi-structured self administered questionnaire was carried out over four weeks, using a small sample of students of all categories and classes of a medical college., Results: A total of 264 students out of 300 (88%) filled in the questionnaire. Inability to cope, helplessness, increased psychological pressure, mental tension and too much workload are 'stress factors' for students. A considerable majority (> 90%) think that they have been stressed at one time or another. Ninety-four per cent of males have experienced stress. The senior students of the fourth and final year feel more stressed (95% and 98% respectively). Low moods, inability to concentrate, loss of temper are most common symptoms. Females report more symptoms. Academics and exams are the most powerful stressors. Sports, music, hanging out with friends, sleeping or going into isolation are various coping mechanisms. Stress can affect the academic performance. If needed, students prefer to talk to a peer. They demand more recreational activities on campus, revised schedule of academics and exams, better counselling facilities and improvement in student-teacher relationship., Conclusion: The prevalence of perceived stress seems to be high among medical students, which tends to affect not only their academic performances but also all aspects of health. Review of academics and exam schedules, more leisure time activities, better interaction with the faculty and proper guidance, advisory services and peer counselling at the campus could do a lot to reduce the stress.

Objective: Carotid artery stenting (CAS) for treatment of carotid stenosis has not received wide acceptance because of the availability of carotid endarterectomy (CEA) with its excellent results and because of the risk of embolic stroke associated with CAS. The feasibility and efficacy of cerebral protection devices that may prevent such embolic complications have yet to be shown. We report our initial results with CAS performed with cerebral protection., Methods: For a period of 28 months, 31 patients with carotid artery stenosis, most of whom were considered at high risk for CEA (87%), underwent treatment with CAS in conjunction with either the PercuSurge GuardWire (n = 19; Medtronic, Minneapolis, Minn), the Cordis Angioguard filter (n = 7; Cordis, Warren, NJ), or the ArteriA Parodi Anti-embolization catheter (n = 4; ArteriA, San Francisco, Calif) with US Food and Drug Administration-approved investigational device exemptions. Factors that made CEA high risk included restenosis after CEA (n = 6), hostile neck (n = 6), high or low lesions (n = 4), and severe comorbid medical conditions (n = 11). Preoperative neurologic symptoms were present in 58%, and the mean stenosis was 85% +/- 12%. Data were prospectively recorded and analyzed on an intent-to-treat basis. Neurologic evaluation was performed before and after CAS by a protocol neurologist., Results: CAS was performed with local anesthesia with the Wallstent (n = 23; Boston Scientific Corp, Natick, Mass) or the PRECISE carotid stent (n = 7; Cordis) in conjunction with one of the protection devices in an operating room with a mobile C-arm. Each patient received dual antiplatelet therapy before surgery. The overall technical success rate was 97% (30/31). In one patient, the lesion could not be crossed with a guidewire because of a severely stenosed and tortuous lesion. This patient was not a candidate for CEA and was treated conservatively. In the remaining 30 cases, CAS had a good angiographic result (residual stenosis, <10%). All patients tolerated the protection device well, and no intraprocedural neurologic complications occurred. Macroscopic embolic particles were recovered from each case. One patient (3%) with a severely tortuous vessel had a major stroke immediately after CAS, and no deaths occurred. The combined 30 day stroke/death rate was 3%. During a mean follow-up period of 17 months, one subacute occlusion of the stent occurred but did not result in a stroke. Three other patients had duplex scan-proven in-stent restenosis, and two underwent treatment with repeat percutaneous transluminal angioplasty with a good result. No patient had a stroke during the follow-up period., Conclusion: CAS with cerebral protection devices can be performed safely with a high technical success rate. Although many patients who underwent treatment with CAS were at high risk, the neurologic complication rate was low and CAS appears to be an acceptable treatment option for select patients at high risk for CEA. Tight lesions and tortuous anatomy may make the use of distal protection devices difficult. Further study is warranted.

High dose chemoradiotherapy with autologous peripheral blood progenitor cell transplantation (PBPCT) may improve outcome in myeloma. Melphalan is an effective drug in the treatment of myeloma, but is potentially toxic to progenitor cells. We studied 8 patients receiving intermittent intravenous melphalan (25 mg/m2) as induction therapy before PBPCT to assess engraftment characteristics post-transplantation. Comparison was made with an age-matched control group of patients with non-Hodgkins lymphoma who had not received melphalan during induction therapy. There was correlation (P=0.037) between the dose of melphalan per kg body weight given, premobilization, and days to neutrophil engraftment, but no significant difference between the two groups in neutrophil recovery. The study group had delayed platelet recovery (P=0.01) and required more platelet support post-transplantation (P=0.05). 3-4 weekly melphalan (25 mg/m2) up to 6 courses was delivered to patients who went on to PBPCT without significantly influencing neutrophil recovery but with a negative impact on platelet recovery.

G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.

Berberis aristata is an edible plant employed in South Asian traditional medicine; in particular, its fruit is used as a tonic remedy for liver and heart. In isolated cardiac tissues, Berberis aristata fruit extract exhibits a positive inotropic action. Activity-directed fractionation using organic solvents revealed that the cardiotonic activity is concentrated in the n-butanolic fraction (BF). The cardiac action of BF was investigated in spontaneously beating right atria and in electrically driven right ventricular strips and left atria obtained from reserpinized guinea pigs. The results show that this fraction produces a dose-dependent positive inotropic action with little effect on heart rate. To study its possible mode of action, guinea pig atria were pretreated with propranolol, a beta-adrenoceptor blocking agent. This treatment abolished the cardiotonic effect of isoprenaline, whereas the cardiotonic effect of BF remained unaltered, suggesting that this effect does not involve stimulation of beta-adrenoceptors. On the other hand, application of carbachol reverses only part of the BF-induced increase in ventricular force of contraction, indicating that besides a cyclic AMP (cAMP)-dependent mechanism, a cAMP-independent mechanism underlies the inotropic action of BF. This is in line with the observation that the dynamics of isometric twitch contractions are not significantly altered by BF. Investigations in skinned myocardial preparations showed that BF modulates the calcium-dependent interaction of actin and myosin, apparently by reducing the cooperativity of the calcium-dependent binding of myosin to actin, i.e., there is enhanced calcium activation at low to physiological intracellular calcium, and reduced calcium activation at high intracellular calcium concentrations as present, for example, in ischemic calcium overload. These data indicate that the edible plant, Berberis aristata, contains active principle(s) that cause(s) a selective inotropic effect, involving-in the form of the modulatory effect on actin myosin cooperativity-a novel mechanism of action. Further phytochemical and pharmacological studies may lead to isolation and structural identification of an attractive, new cardiotonic agent from Berberis aristata fruit.

Purpose: This study was designed to test whether palmitoylation and glycosylation are required for the formation of the green opsin visual pigment., Methods: Stable cell lines were established by transfecting EBNA-293 cells with a pMEP4ss recombinant plasmid containing wild-type bovine rhodopsin or wild-type or mutant (N32S) green opsin cDNA molecules that included a tag for the eight amino acid residues located at the C-terminus of rhodopsin. The opsins were induced by addition of CdCl2 into the medium and then reconstituted with 11-cis-retinal. The reconstituted opsins were purified by immunoaffinity chromatography, then analyzed by difference spectra, and by binding 35S-GTP in the presence of bovine transducin. Non-reconstituted opsins were analyzed by Western blotting and by pulse-labeling with 3H-palmitic acid followed by immunoprecipitation., Results: Elimination of glycosylation by mutagenesis of the N-linked glycosylation site did not impair the ability of the resulting cone opsin to absorb light at the appropriate wavelength nor to activate transducin. Furthermore, as judged by pulse-labeling with 3H-palmitic acid and immunoprecipitation and by gas chromatography-mass spectroscopy, the wild type green opsin differs from rhodopsin by not being palmitoylated., Conclusions: Glycosylation and palmitoylation are not required for the formation of cone opsin visual pigments. For the previously described green opsin C203R mutation, disruption of folding and transport, rather than altered glycosylation is sufficient to explain the associated color vision deficiency.

The variable absorption of melphalan from the gastrointestinal tract results in response rates between 40 and 60%. High dose melphalan increases response rates but at the cost of increased morbidity and mortality. We have investigated intravenous intermediate dose melphalan and dexamethasone in the treatment of patients presenting with de novo multiple myeloma with the object of reducing toxicity while preserving an improved response rate compared to oral melphalan and prednisolone. The results show that this treatment can be delivered safely on an outpatient basis in patients up to the age of 78 yr; 82% of patients achieved an objective response and 30% a complete haematological and clinical remission. Median overall survival for the whole group is 37 months.

The distinction between a specific factor inactivator and a non-specific inhibitor is important when confronted by a patient with a history of bleeding and abnormal in-vitro coagulation tests. We report on two patients who presented with bleeding and a prolonged activated partial thromboplastin time. Initial factor assays suggested combined deficiency of factors VIII and IX as a result of the presence of inactivators. The use of dilution studies, chromogenic assays, a novel in-house enzyme-linked-immunosorbent-assay-based technique and phospholipid neutralization, demonstrated that Case 1 had a genuine factor VIII inactivator resulting in factor VIII levels of less than 1 IU/dl but no factor IX deficiency. Case 2 had normal levels of factor VIII on further testing and no specific inactivator to either factor VIII or IX but a potent antiphospholipid antibody which had interfered with the phospholipid-dependent in-vitro assays. Care must be taken in the interpretation of laboratory assays in the presence of antiphospholipid antibodies to ensure that the correct diagnosis is made and inappropriate treatment avoided.

Purpose: To test the effects of disruption of a conserved proline in the green cone opsin molecule on light-activated isomerization, transducin activation, protein accumulation, glycosylation, and transport., Methods: Stable cell lines were established by transfecting EBNA-293 cells with a plasmid containing wild-type or mutant (P307L) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of CdCl2 and analyzed by spectra, transducin activation, Western blotting, and immunocytochemistry., Results: The P307L mutation diminished ability of the visual pigment to absorb light at the appropriate wavelength and to activate transducin. Protein glycosylation and transport to the cell membrane were unaffected. Although there was some diminution in the accumulation of the opsin, this was insufficient to account for the observed effect., Conclusions: Like rhodopsin, the formation of the cone opsins visual pigments is dependent on the binding of retinal into a hydrophobic pocket that is formed by the second and fourth transmembranous loops. Disruption of a conserved proline near the retinal binding site represents a cause of color vision deficiency that is unrelated to spectral shifts of the photopigment.

Purpose: To test the effects of disruption of a conserved cysteine in the green cone opsin molecule on light-activated isomerization, transducin activation, folding, transport, and protein half-life., Methods: Stable cell lines were established by transfecting 293-EBNA cells with a plasmid containing wild-type or mutant (C203R, C203S, C126S, C126S/C203S) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of cadmium chloride and analyzed by spectra, transducin activation, Western blotting, pulse-labeling with immunoprecipitation, and immunocytochemistry., Results: The C203R mutation disrupts the folding and half-life of the green opsin molecule and its abilities to absorb light at the appropriate wavelength and to activate transducin. Similar disruption of folding, half-life, and light activation occurs when Cys203 or its presumed partner for formation of a disulfide bond (Cys126) is replaced by serine residues., Conclusions: Like rhodopsin, the folding of the cone opsins appears to be dependent on the formation of a disulfide bond between the third transmembrane helix and the second extracellular loop. Disruption of this disulfide bond represents a cause of color vision deficiencies that is unrelated to spectral shifts of the photopigment.

A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.

The healing wound offers a clear example of the sequence of events in chronic inflammation leading to repair. Although angiogenesis has an obvious and essential role in this process, it has been little studied. For an angiogenic factor to seem relevant, it would have to be shown to precede the peak of increased vascularity. To define this peak, the vessel content of simple, incised mouse wounds was estimated using morphometry of histological sections, and found to rise to a maximum at days 5 and 6. Total angiogenic activity of aqueous extracts was found to reach a peak at day 3. The detection of such activity on the chick chorioallantoic membrane is very dependent on the preparation technique and the choice of proteinase inhibitors. Previous in vitro work by us using purified material has shown fibrin degradation products to be effective in stimulating angiogenesis. Fibrin degradation products are prominent on immunoblotting from day 3, when macrophages are plentiful, with a similar band pattern to human granulation tissue.

We have addressed the problem of distinguishing angiogenesis induced in the chick chorioallantoic membrane by injury and inflammation from angiogenesis induced by primary stimulation. Focal, slow-release application of trypsin stimulated a localized spoke-wheel pattern of vascularity. In comparison, a range of doses up to a sublethal amount of trypsin applied generally, in liquid form, resulted in no change in DNA synthesis or vessel content, despite a transient influx of inflammatory cells. This contrasts with previous work with fibrin degradation products, histamine and heparin which each produce characteristic patterns of increased DNA synthesis leading to angiogenesis in the entire 'dropped' area of the chorioallantoic membrane. Such general application, therefore, avoids the danger of misinterpretation of focal, toxic effects.