The effect of acetyl-coenzyme A on phosphate-activated glutaminase from pig kidney and brain

Phosphate-activated glutaminase (EC 3.5.1.2; l-glutamine amidohydrolase) purified from pig kidney and brain is activated by CoA and short-chain acyl-CoA derivatives. Acetyl-CoA is the most powerful activator (K(A) about 0.2mm). Acetyl-CoA is maximally effective in the absence of other activating anions such as phosphate and citrate, and at low glutamine concentrations. The negative co-operative substrate activation observed at pH7 becomes more pronounced in the presence of acetyl-CoA. Similarly to phosphate, acetyl-CoA produces at high protein concentrations a different type of activation, which is time-dependent, depends on protein concentration and is accompanied by an increase in the sedimentation coefficient. Acetyl-CoA, phosphate and citrate appear to have binding sites in common. No significant difference was observed between kidney and brain phosphate-activated glutaminase.     

Feinstrukturanalyse der komplexen Geißeln von Rhizobium lupini H 13-3

Zusammenfassung Zellen von Rhizobium lupini H 13-3 besitzen 5–10 peritrich inserierte komplexe Geißeln, deren Feinstruktur durch Hochauflösungs-Elektronenmikroskopie und lichtoptische Diffraktion analysiert wurde. Das Geißelfilament hat einen Durchmesser von 160 Å und besteht aus einem zylindrischen Kern (Durchmesser ca. 110 Å), der fest von drei Bändern einer helikalen Scheide umgeben ist. Die Scheidenbänder sind 49 Å breit, durch 49 Å-Intervalle voneinander getrennt und haben eine Steigung von 31°. Die komplexen Geißelfilamente bestehen aus einem 43 000-Dalton-Protein, das den Kern und die helikale Scheide aufbaut. Beide gehen übergangslos aus dem proximalen Geißelhaken hervor, der einen Durchmesser von 150 Å und eine Länge von 600 bis 800 Å hat. Die Diffraktionsanalyse des Geißelhakens zeigte eine helikale Grundanordnung von globulären Untereinheiten, die ein Oberflächengitter von 5 parallelen Schrauben (Steigung 29° bzw. 33°) bilden, von denen jede fast 11 Untereinheiten pro Helixungang trägt. Die komplexen Geißeln von R. lupini H 13-3 und Pseudomonas rhodos [Schmitt et al.: J. Bact. 117, 844–857 (1974)] sind ein neuer Typ von Bakteriengeißeln. Sie zeigen deutliche Übereinstimmung in der Feinstruktur, der festen Verbindung von helikaler Scheide und Geißelhaken sowie in der Fragilität ihrer Filamente; sie unterscheiden sich deutlich im Molekulargewicht der Flagellinmonomeren (43 000 bzw. 55 000). Zellen von R. lupini H 13-3 führen schnelle, vibrierende Translationsbewegungen aus. Mögliche Mechanismen der Bewegung komplexer Geißeln werden diskutiert.

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Fine structure analysis of the complex flagella of Rhizobium lupini H 13-3

Cells of Rhizobium lupini H 13-3 possess 5 to 10 peritrichously inserted complex flagella, which were analyzed by high resolution electron microscopy and by optical diffraction. The flagellar filament has a diameter of 160 Å; it consists of a cylindrical core (diameter approximately 110 Å) surrounded by three close-fitting bands of a helical sheath. The helical bands are 49 Å wide, separated by axial intervals, 49 Å wide, and run at an angle of 31°. Complex filaments consist of a 43 000-dalton protein representing the core and the helical sheath. These originate from the proximal hook, which has a diameter of 150 Å and a length of 600 to 800 Å. The diffraction analysis of the hook showed a helical arrangement of globular subunits forming a surface of 5 parallel small-scale helices (pitch-angles 29° and 33°, respectively), each carrying almost 11 subunits per period. The complex flagella of R. lupini H 13-3 and Pseudomonas rhodos [Schmitt, et al.: J. Bact. 117, 844–857 (1974)] represent a novel type of bacterial flagella. There is agreement in their fine structures, in the intimate connection of the helical sheath and the core, and in the fragility of their filaments. Thery are clearly distinguished by the molecular weights of their flagellin monomers (43 000 and 55 000, respectively). Cells of R. lupini H 13-3 show fast, vibrating, translational motions. Possible mechanisms of complex flagellar motion are discussed.

Herrn Professor Wolfram Heumann zum 60. Geburtstag gewidmet.     

Transactions of the One Hundredth Annual Meeting of The Canadian Medical Association: Part II

The purpose of this retrospective study was to compare (1) regular methadone withdrawal treatment and (2) prolonged methadone withdrawal treatment in 105 and 71 voluntary patients respectively, who attended the Narcotic Addiction Foundation (N.A.F.) between 1959 and mid-1964. Treatment consisted of individual counselling and medical care for all, and only residential care and psychiatric assessment for selected cases. The number of treatment sessions and the details of drug therapy are described.One hundred and fifty-three of 176 patients (87%) were interviewed approximately one to five years after the first clinic contact. Forty-three per cent showed some overall improvement in their behaviour. Rehabilitation was defined as change in a specific area—drug use, work, criminal behaviour, community associations, friendship patterns and family relationships—rather than in terms of abstention from drugs only. Age affected comparative results.     

Metabolic inhibitors and mitosis: III. Effects of dinitrophenol on spindle disassembly inPinnularia

Summary At telophase in the diatomPinnularia, the two half spindles that comprise the central spindle, separate and then disassemble unidirectionally from the end formerly in the central overlap, back to the pole (Soranno andPickett-Heaps 1982). The metabolic inhibitors dinitrophenol plus deoxyglucose were applied to cells at telophase, depleting their ATP levels at the early stages of half-spindle disassembly; the cells were maintained in this state for 5 minutes, before the inhibitors were washed out. Disassembly of the half spindles, as judged from their birefringence, ceased in ATP-depleted conditions, and recommenced soon after the inhibitors were removed, going to completion quite rapidly.We conclude that disassembly of these MTsin vivo requires energy, probably in the form of ATP.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study

Background

Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia and goblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereas smoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smoking cessation.

Methods

114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids, current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV₁ 63 ± 9% predicted. Squamous cell metaplasia (%), goblet cell (PAS/Alcian Blue⁺) area (%), proliferating (Ki-67⁺) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies.

Results

Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towards reduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference in EGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short-term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs. 13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001).

Conclusion

Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-term smoking cessation (>3.5 years).

Trial registration

NCT00158847     

Epigenetic instability at imprinting control regions in a KrasG12D-induced T-cell neoplasm

Although aberrant DNA methylation within imprinted domains has been reported in a variety of neoplastic diseases, it remains largely uncharacterized in the context of carcinogenesis. In this study, we induced T-cell lymphoma in mice by employing a breeding scheme involving mouse strains, LSL-Kras^G12D and MMTV-Cre. We then systematically surveyed imprinted domains for DNA methylation changes during tumor progression using combined bisulfite restriction analysis and NGS-based bisulfite sequencing. We detected hyper- or hypo-methylation at the imprinting control regions (ICRs) of the Dlk1, Peg10, Peg3, Grb10, and Gnas domains. These DNA methylation changes at ICRs were more prevalent and consistent than those observed at the promoter regions of well-known tumor suppressors, such as Mgmt, Fhit, and Mlh1. Thus, the changes observed at these imprinted domains are the outcome of isolated incidents affecting DNA methylation settings. Within imprinted domains, DNA methylation changes tend to be restricted to ICRs as nearby somatic differentially methylated regions and promoter regions experience no change. Furthermore, detailed analyses revealed that small cis-regulatory elements within ICRs tend to be resistant to DNA methylation changes, suggesting potential protection by unknown trans-factors. Overall, this study demonstrates that DNA methylation changes at ICRs are dynamic during carcinogenesis and advocates that detection of aberrant DNA methylation at ICRs may serve as a biomarker to enhance diagnostic procedures.     

Congenital neosporosis in goats from the State of Minas Gerais, Brazil

Congenital Neospora caninum infection was diagnosed in two Saanen goat kids from two distinct herds with a history of abortion and weak newborn goat kids in the Southern region of the State of Minas Gerais, Brazil. The first kid was weak at birth, had difficulty to rise and was unable to nurse. Gross lesions of porencephaly and hydrocephalus ex vacuo were seen. Multifocal necrosis, gliosis and non-supurative encephalitis were observed in the brain. Several parasitic cysts with a thick wall that reacted strongly only with polyclonal antiserum to Neospora caninum were seen in the cerebral cortex, brain stem and cerebellum. The second kid was born from a Neospora caninum seropositive mother that aborted in the last pregnancy. It was born without clinical signs. The diagnosis of neosporosis was based on antibody titer of 1:800 to N. caninum by indirect fluorescence antibody test obtained from blood collected before the goat kid ingested the colostrum and Neospora caninum DNA was detected by polymerase chain reaction and sequenced from placenta. This is the first report of neosporosis in goats in the southeast region of Brazil.