The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.     

Mytilid bivalveLithophaga in Upper Triassic coralPamiroseris from Zlambach beds compared with cretaceousLithophaga Alpina

Summary The mytilid genusLithophaga is confirmed for the Upper Triassic. The Rhaetian specimen, boring the dead part of coral, is compared with the SenonianL. alpina, which is associated with live coral.     

Diet-induced obesity increases NF-κB signaling in reporter mice

The nuclear factor (NF)-κB is a primary regulator of inflammatory responses and may be linked to pathology associated with obesity. We investigated the progression of NF-κB activity during a 12-week feeding period on a high-fat diet (HFD) or a low-fat diet (LFD) using NF-κB luciferase reporter mice. In vivo imaging of luciferase activity showed that NF-κB activity was higher in the HFD mice compared with LFD-fed mice. Thorax region of HFD females displayed fourfold higher activity compared with LFD females, while no such increase was evident in males. In male HFD mice, abdominal NF-κB activity was increased twofold compared with the LFD males, while females had unchanged NF-κB activity in the abdomen by HFD. HFD males, but not females, exhibited evident glucose intolerance during the study. In conclusion, HFD increased NF-κB activity in both female and male mice. However, HFD differentially increased activity in males and females. The moderate increase in abdomen of male mice may be linked to glucose intolerance.     

Real-time ultrasound imaging for predicting ovine fetal age

Fetal head width and thoracic depth were observed via real-time ultrasound imaging in 12 single-, 12 twin- and 4 triplet-bearing BooroolaxSouth Australian Merino ewes at weekly intervals during mid pregnancy. Fetal age varied by a maximum of 24 h. Relationships between fetal age and the linear measures (head width, thoracic depth) were determined within litter size categories, using both linear and quadratic terms. All relationships tested for linearity were statistically significant (P<0.05). A curvilinear relationship (P<0.05) was observed for fetal age and thoracic depth of twin fetuses. Significant (P<0.05) curvilinear relationships were also observed for fetal age and head width, and fetal age and thoracic depth when litter size data were pooled. We conclude that head width and thoracic depth are acceptable linear measurements for estimating fetal age in the ovine species. Implications of these results for research and for the commercial field are discussed.     

Metal binding to angiotensin converting enzyme: implications for the metal binding site

Angiotensin converting enzyme interacts with the chelator, 1,10-phenanthroline (OP) to form an OP-Zn-ACE ternary complex, which subsequently dissociates to OP-Zn and apoenzyme. The association and dissociation rate constants for the reaction OP + Zn-ACE in equilibrium OP-Zn-ACE have been determined and compared with those of known OP-metal complexes. Such constants were also used to calculate the rate constant for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissociation of zinc from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concentration, at different pH values, and with different buffers. The stability constant for the binding of zinc to apoACE log Kc = 8.2, determined by equilibrium dialysis using atomic absorption spectroscopy to assess metal concentration, is much smaller than that for Zn-carboxypeptidase A. Zn-thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the zinc dissociation rate constant of ACE, 7.5 X 10(-3) sec-1 at pH 7.0, which is much greater than that of the other zinc metalloenzymes. These results lead to inferences regarding the metal binding site of ACE.     

Within‐plant distribution of 1,4‐benzoxazin‐3‐ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore‐foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within‐plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4‐benzoxazin‐3‐one derivatives (BXDs), affects the foraging behaviour of two leaf‐chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well‐adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD‐free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non‐adapted herbivores and thereby likely contribute to feeding niche differentiation.     

氨氯地平联合依那普利治疗原发性高血压的临床疗效及对患者左心室肥厚的影响

摘要目的：探讨氨氯地平联合依那普利治疗原发性高血压的临床效果,观察联合用药对左心室肥厚的影响。方法：选择本院收治的原发性高血压患者92例,随机分为观察组和对照组,各46例,对照组给予苯磺酸左旋氨氯地平5mg,1次／d,口服;观察组在对照组基础上加用马来酸依那普利10mg,2次／d,口服,疗程均为24周。观察两组治疗前后血压变化,应用超声心动图测量两组左心室厚度变化。结果：治疗后,观察组总有效率为91_3％;对照组总有效率为73．9％,观察组总有效率高于对照组（P〈0．05）。治疗前两组心率、血压比较无统计学差异（P〉0．05）,治疗后两组血压均明显降低,观察组收缩压、舒张压明显低于对照组（P〈O．05）;观察组心率明显低于对照组（P〈0．01）。治疗前两组左心室舒张末期室间隔厚度（Leaventricularend—diastolicventricularseptalthickness,IVST）、左心室后壁厚度（1eftventricularposteriorwallthickness,U,PwT）和左室射血分数（Leftventricularejectionfxaction,LVEF）比较无统计学差异（P〉0．05）;治疗后观察组IVST、L、,PwT明显低于对照组,LVEF明显高于对照组（P〈0．05）。结论：氨氯地平联合依那普利治疗原发性高血压能有效扭转左心室肥厚,降压效果较单独应用氨氯地平更佳。     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.