Fgfr2 is integral for bladder mesenchyme patterning and function.

While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor 2 (Fgfr2) is necessary for kidney and ureter mesenchymal development. Our objective was to determine the role of Fgfr2 in bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in bladder mesenchyme (Fgfr2BM-/-). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblotting, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with controls, embryonic (E) day 16.5 (E16.5) Fgfr2BM-/- bladders have thin muscle layers with reduced α-smooth muscle actin levels and thickened lamina propria with increased collagen expression that intrudes into muscle. From postnatal (P) day 1 (P1) to P30, Fgfr2BM-/- bladders demonstrate progressive muscle loss and increased collagen expression. Postnatal Fgfr2BM-/- bladder sheets exhibit decreased contractility and increased passive stretch tension compared with controls. In vivo cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2BM-/- bladders compared with controls. Mechanistically, while Shh expression appears normal, mRNA and protein readouts of hedgehog activity are increased in E16.5 Fgfr2BM-/- bladders compared with controls. Moreover, E16.5Fgfr2BM-/- bladders exhibit higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, than controls. Fgfr2 is critical for bladder mesenchyme patterning by virtue of its role in modulation of hedgehog signaling.

Fgfr2 is integral for bladder mesenchyme patterning and function.

While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor (Fgfr)2 is necessary for kidney and ureter mesenchymal development. The objective of the present study was to determine the role of Fgfr2 in the bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in the bladder mesenchyme (Fgfr2(BM-/-)). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblot analysis, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with control bladders, embryonic day 16.5 (E16.5) Fgfr2(BM-/-) bladders had thin muscle layers with less α-smooth muscle actin and thickened lamina propria with increased collagen type Ia and IIIa that intruded into the muscle. The reciprocal changes in mutant layer thicknesses appeared partly due to a cell fate switch. From postnatal days 1 to 30, Fgfr2(BM-/-) bladders demonstrated progressive muscle loss and increased collagen expression. Postnatal Fgfr2(BM-/-) bladder sheets exhibited decreased agonist-mediated contractility and increased passive stretch tension versus control bladder sheets. Cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2(BM-/-) versus control bladders. Mechanistically, whereas Shh expression appeared normal, mRNA and protein readouts of hedgehog activity were increased in E16.5 Fgfr2(BM-/-) versus control bladders. Moreover, E16.5 Fgfr2(BM-/-) bladders exhibited higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, compared with control bladders. In conclusion, loss of Fgfr2 in the bladder mesenchyme leads to abnormal bladder morphology and decreased compliance and contractility.

A nine country study of the burden of non-severe nocturnal hypoglycaemic events on diabetes management and daily function.

AIMS: The purpose of this study was to explore the burden and impact of non-severe nocturnal hypoglycaemic events (NSNHEs) on diabetes management, patient functioning and well-being in order to better understand the role that NSNHEs play in caring for persons with diabetes and facilitate optimal diabetes treatment management strategies. METHODS: A 20-min survey assessing the impact of NSNHEs was administered to patients with self-reported diabetes age 18 or older via the Internet in nine countries (USA, UK, Germany, Canada, France, Italy, Spain, The Netherlands and Sweden) who experienced an NSNHE in the last month. Questions captured reasons for and length of the event, and impacts on diabetes management, daily function, sleep and well-being. RESULTS: A total of 20 212 persons with Type 1 diabetes mellitus (T1DM) and Type 2 diabetes mellitus (T2DM) were screened of which 2108 respondents were eligible. Respondents initiated, on average, an additional 3.6 glucose monitoring tests, and did not resume usual functioning for an average of 3.4 hours after the NSNHE. Of the respondents using insulin, 15.8% decreased their insulin dose over an average of 3.6 days. NSNHEs also impacted sleep, with 10.4% not returning to sleep that night. Next day functioning was affected with 60.3% (n = 1273) feeling the need to take a nap and/or rest (with 65.5% of those actually taking a nap/rest) and 40.2% (n = 848) wanting to go to bed earlier than usual. A total of 21.4% were restricted in their driving the next day. These events also resulted in decreased well-being with 39.6% of respondents feeling 'emotional low' the following day. CONCLUSIONS: NSNHEs have serious consequences for patients. Greater attention to patient and physician education regarding the burden of NSNHEs and incorporation of corrective actions in treatment plans is needed to facilitate patients reaching optimal glycaemic control.

Comparison of 7-day and repeated 24-h recall of type 2 diabetes.

PURPOSE: Patient reporting of type 2 diabetes symptoms in a questionnaire with a 7-day recall period was expected to be different from symptom reports using a 7-day diary with repeated 24-h recall based on cognitive theory of memory processes and prior literature. This study compared these two types of recall in patients diagnosed with type 2 diabetes (T2D). METHODS: One hundred and forty adults with T2D completed a daily diary for 7 days containing 9 T2D-related symptom and impact items. On day 7, patients completed the same items with a 7-day recall period. We examined the concordance of 7-day recall with summary descriptors of the daily reports and compared the scores and the discriminant ability of 7-day recall and mean of daily reports. RESULTS: Seven-day recall was most concordant with the mean of daily reports. The average difference in scores was small (range 0.22-0.77 on 11-point scale) and less than 0.5 standard deviations. For some items, the difference was positively associated with the variation in daily reports. The discriminant ability was comparable. CONCLUSIONS: In this study population, a questionnaire with 7-day recall provided information consistent with a daily diary measure of the average week-long experience of T2D symptoms and impacts.

Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.

Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.

The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.

Architecture of RNA polymerase II and implications for the transcription mechanism.

A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

Selenomethionine incorporation in Saccharomyces cerevisiae RNA polymerase II.

A protocol for the incorporation of SeMet into yeast proteins is described. Incorporation at a level of about 50% suffices for the location of Se sites in an anomalous difference Fourier map of the 0.5 MDa yeast RNA polymerase II. This shows the utility of the approach as an aid in the model-building of large protein complexes.

The C-terminal domain revealed in the structure of RNA polymerase II.

The location of the CTD in the structure of RNA polymerase II has been determined by electron crystallography at 16 A resolution. Difference maps between wild-type enzyme and that lacking the CTD, or with an antibody fragment bound in place of the CTD, disclose the site of attachment of the CTD to the polymerase. Appropriate display of the polymerase structure reveals the CTD as an element projecting from this site of attachment into solution. A low relative density and large volume of this element identify the CTD as a conformationally mobile region.

Repeated tertiary fold of RNA polymerase II and implications for DNA binding.

X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein.

A Purified Zinc Protease of Pea Chloroplasts, EP1, Degrades the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

A previously reported endopeptidase (EP1) from pea chloroplasts was purified over 11,000-fold using a four-step protocol involving ultrafiltration, sucrose gradient centrifugation, isoelectric focusing, and high performance liquid chromatography gel filtration. The enzyme was determined to be a metalloprotease requiring bound Zn2+ and added Mg2+ or Ca2+ for proper activity. Its localization in the stroma of pea chloroplasts was confirmed by demonstrating its insensitivity to thermolysin when the envelope was intact. A contaminating serine protease that attacks EP1 was found. The contaminating protease was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not by o-phenanthroline, whereas EP1 sensitivities were the reverse. EP1 is able to hydrolyze the large subunit of native ribulose-1,5-bisphosphate carboxylase/oxygenase under physiological conditions.

Clinical outcome of patients with a 'low probability' of pulmonary embolism on ventilation-perfusion lung scan.

Lung ventilation and perfusion (V/Q) scintigraphy is usually indicated when pulmonary embolism (PE) is a suspected diagnosis. Typically, V/Q scintigraphic interpretation is reported as a "normal," "low," "intermediate," or "high probability" of PE. Although a "low probability" interpretation does not exclude the diagnosis of PE, it significantly reduces the likelihood. We retrospectively analyzed up to one year of follow-up in 90 patients who were clinically suspected of having PE, but in whom V/Q scintigraphy implied a low probability of PE. None of the 90 patients demonstrated clinical evidence of PE subsequent to the V/Q scan. Our findings suggest that significant pulmonary embolism is uncommon and that the clinical course appears to be predictable in patients with a low probability V/Q scan.

The influence of drag on human locomotion in water.

Propulsion in water requires a propulsive force to overcome drag. Male subjects were measured for cycle frequency, energy cost and drag (D) as a function of velocity (V), up to maximal V, for fin and front crawl swimming, kayaking and rowing. The locomotion with the largest propulsive arms and longest hulls traveled the greatest distance per cycle (d/c) and reached higher maximal V. D while locomotoring increased as a function of V, with lower levels for kayaking and rowing at lower Vs. For Vs below 1 m/s, pressure D dominated, while friction D dominated up to 3 m/s, after which wave D dominated total D. Sport training reduced the D, increased d/c, and thus lowered C and increased maximal V. Maximal powers and responses to training were similar in all types of locomotion. To minimize C or maximize V, D has to be minimized by tailoring D type (friction, pressure or wave) to the form of locomotion and velocity.

Two years of intensive glycemic control and left ventricular function in the Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus (VA CSDM).

OBJECTIVE: The Veterans Affairs Cooperative Study in Type 2 Diabetes Mellitus (VA CSDM) was a multicenter randomized prospective study of 153 male type 2 diabetic patients to assess the ability to sustain clinically significant glycemic separation between intensive and standard treatment arms. A trend toward an excess of combined cardiovascular events in the intensive treatment arm of this trial was reported earlier. The present analysis was done to evaluate the effect of 2 years of intensive glycemic control on the left ventricular (LV) function. RESEARCH DESIGN AND METHODS: The patients were randomized to intensive step treatment with insulin alone or with sulfonylurea (intensive treatment arm [INT], n = 75) or to standard once-daily insulin injection (standard treatment arm [STD], n = 78) treatment. A total of 136 patients (standard treatment arm [STD], n = 70; INT, n = 66) had radionuclide ventriculography at entry and at 24 months for the assessment of LV function. RESULTS: There was no difference in the mean LV ejection fraction (at entry: STD 57.1+/-9.51%; INT 58.1+/-8.7%; at 24 months: STD 57.3+/-10.8%, INT 59.5+/-10.7%), peak filling rate (at entry: STD 2.6+/-0.7 end diastolic volume per second, INT 2.4+/-0.8 end diastolic volume per second; at 24 months: STD 2.7+/-1.0 end diastolic volume per second, INT 2.5+/-0.7 end diastolic volume per second), or time to peak filling rate (at entry: STD 195.3+/-69.5 ms, INT 185.6 +/-62.4 ms; at 24 months: STD 182.6+/-64.8 ms, INT 179.2+/-61.2 ms) between the 2 treatment arms. A subgroup analysis of 104 patients (STD, n = 53; INT, n = 51) that omitted individuals with intervening cardiac events/revascularization or a change in cardioactive medications also showed no difference in the LV function at entry and at 24 months between the 2 groups. Abnormal LV ejection fraction at baseline predicted cardiac events (interval between cardiac beats [RR] = 2.5). CONCLUSIONS: Two years of intensive glycemic control does not affect the LV systolic or diastolic function in patients with type 2 diabetes.

Prediction of language and neurologic recovery after cerebral infarction with SPECT imaging using N-isopropyl-p-(I 123) iodoamphetamine.

Fourteen patients (10 with left-sided and 4 with right-sided cerebral infarction) were prospectively studied with single-photon emission computed tomography (SPECT) using N-isopropyl-p-(I 123) iodoamphetamine (IMP, SPECTamine) to determine its usefulness in predicting neurologic/language recovery after cerebral infarction. All neuro-SPECT imaging was performed within 30 days after infarction. Detailed assessment of neurologic and/or language recovery (after 3 months) was carried out prospectively in each patient. Patients with smaller volume IMP defects in the region of infarction demonstrated significantly better neurologic and language recovery than patients with large IMP defects. Analysis of the IMP "redistribution" phenomenon failed to demonstrate definitively a relationship with clinical recovery. It was concluded that the volume of the IMP defect can aid in predicting recovery potential after cerebral infarction.

Cortical blindness and residual vision: is the "second" visual system in humans capable of more than rudimentary visual perception?

We studied 12 patients with static cortical blindness to evaluate residual vision after destruction of area 17 and to assess the visual capacity of the subcortical "second" visual system in humans. In each case, the cause was bilateral infarction of the occipital lobes. Five patients had total blindness, and four had residual rudimentary vision (RRV), characterized by homonymous areas of light perception in the peripheral field and ability to detect moving objects. Only three patients had the ability to read; two of these had spared macular vision, and the other had spared left homonymous hemimaculae and spared temporal crescent. Neuroimaging and visual evoked potentials (VEPs) correlated with the extent of the visual dysfunction. Total destruction of area 17 bilaterally was associated with total permanent visual loss. The larger the amount of spared visual cortex, the better the vision. Positron emission tomography (PET) or single photon emission computed tomography (SPECT) demonstrated retained metabolic activity in islands of preserved area 17 in patients with some residual vision. VEPs were present in totally blind individuals. We conclude that, in humans, useful visual function is preserved only when a critical amount of area 17 is spared. The subcortical second system may participate in the generation of VEPs, but is incapable of conscious visual perception.

Alteration in hematopoietic stem cell seeding and proliferation by both high and low dose rate irradiation of bone marrow stromal cells in vitro.

The mechanism of physiologic alteration by high (HDR) or low dose rate (LDR) (5 or 120 cGy/min) irradiation of plateau-phase bone marrow stromal cell cultures was investigated using a technique of in vitro bone marrow transplantation. Purified stromal cell cultures from C57BL/6J, C3H/HeJ, or (C57BL/6J X DBA2/J)F1 (B6D2F1) mouse marrow were irradiated to doses of 2.5 to 10 Gy at LDR or 10-100 Gy at HDR and were then engrafted in vitro with nonadherent hematopoietic cells from murine continuous bone marrow cultures. Parameters of engraftment quantitated included: (1) numbers of adherent proliferating hematopoietic cell colonies, "cobblestone islands" (2) cumulative production of nonadherent hematopoietic cells over 8 weeks after engraftment, (3) M-CSF, GM-CSF and multi-CSF (IL-3) dependent hematopoietic progenitor cells forming greater than or equal to 50 cell colonies in semisolid medium, (4) cumulative production of CFUs, and (5) number of adherent stromal cells positive for detectable extracellular laminin or collagen type IV (markers of endothelial cells, reticular adventitial cells, or sinus lining cells). There was a decrease in cobblestone island formation between 5 and 10 Gy and this parameter possibly increased at doses of 50 and 100 Gy. There was no difference between HDR and LDR irradiation to 10 Gy. Irradiation to doses above 10 Gy decreased support of engrafted cells forming CFU-GM and CFU-GEMM. Measures of CFUs after 10 Gy were variable but indicated a possible increase with HDR and no effect of LDR at 1 week and a decrease in both HDR and LDR groups at 3 weeks after engraftment. Thus, LDR and HDR irradiation in vitro alter several specific parameters of marrow stromal cell support for engrafted hematopoietic stem cells.

Synthesis and antitumor activity of novel 4-demethoxyanthracyclines.

A versatile and efficient synthetic route to 4-demethoxyanthracyclinones has been utilized in the preparation of a number of aglycons having 9-alkyl, 9-(hydroxylalkyl), or 9-carbamoyl substituents. Silver trifluoromethanesulfonate catalyzed coupling of these aglycons with various daunosamine derivatives has yielded a series of novel anthracyclines which have been evaluated as antitumor agents. 9-Alkylanthracyclines 22, 23, 33, and 34 have higher efficacy vs L-1210 leukemia than the parent 4-demethoxydaunorubicin (21), or the natural anthracyclines daunorubicin (1) and doxorubicin (2). 9-(Hydroxyalkyl) derivatives have in most cases high efficacy but are slightly less potent than 21. 9-Methyl analogue 22 has higher efficacy vs P388 leukemia than other anthracyclines tested, while 9-(hydroxymethyl) derivative 37 retains similar efficacy to anthracyclines 1, 2, and 21 but is considerably more potent. The N-substituted 9-carbamoylanthracyclines are devoid of antitumor activity.