Nonthermoregulatory control of cutaneous vascular conductance and sweating during recovery from dynamic exercise in women.

The purpose of the study was to examine the effect of 1) active (loadless pedaling), 2) passive (assisted pedaling), and 3) inactive (motionless) recovery modes on mean arterial pressure (MAP), cutaneous vascular conductance (CVC), and sweat rate during recovery after 15 min of dynamic exercise in women. It was hypothesized that an active recovery mode would be most effective in attenuating the fall in MAP, CVC, and sweating during exercise recovery. Ten female subjects performed 15 min of cycle ergometer exercise at 70% of their predetermined peak oxygen consumption followed by 20 min of 1) active, 2) passive, or 3) inactive recovery. Mean skin temperature (Tsk), esophageal temperature (Tes), skin blood flow, sweating, cardiac output (CO), stroke volume (SV), heart rate (HR), total peripheral resistance (TPR), and MAP were recorded at baseline, end exercise, and 2, 5, 8, 12, 15, and 20 min postexercise. Cutaneous vascular conductance (CVC) was calculated as the ratio of laser-Doppler blood flow to MAP. In the active recovery mode, CVC, sweat rate, MAP, CO, and SV remained elevated over inactive values (P < 0.05). The passive mode was equally as effective as the active mode in maintaining MAP. Sweat rate was different among all modes after 12 min of recovery (P < 0.05). TPR during active recovery remained significantly lower than during recovery in the inactive mode (P < 0.05). No differences in either Tes or Tsk were observed among conditions. The results indicate that CVC can be modulated by central command and possibly cardiopulmonary baroreceptors in women. However, differences in sweat rate may be influenced by factors such as central command, mechanoreceptor stimulation, or cardiopulmonary baroreceptors.     

Control of cutaneous vascular conductance and sweating during recovery from dynamic exercise in humans.

The purpose of the study was to examine the effect of 1) passive (assisted pedaling), 2) active (loadless pedaling), and 3) inactive (motionless) recovery modes on mean arterial pressure (MAP), skin blood flow (SkBF), and sweating during recovery after 15 min of dynamic exercise. It was hypothesized that an active recovery mode would be most effective in attenuating the fall in MAP, SkBF, and sweating during exercise recovery. Six male subjects performed 15 min of cycle ergometer exercise at 70% of their predetermined peak oxygen consumption followed by 15 min of 1) active, 2) passive, or 3) inactive recovery. Mean skin temperature (T(sk)), esophageal temperature (T(es)), SkBF, sweating, cardiac output (CO), stroke volume (SV), heart rate (HR), total peripheral resistance (TPR), and MAP were recorded at baseline, end exercise, and 2, 5, 8, 12, and 15 min postexercise. Cutaneous vascular conductance (CVC) was calculated as the ratio of laser-Doppler blood flow to MAP. In the active and passive recovery modes, CVC, sweat rate, MAP, CO, and SV remained elevated over inactive values (P < 0.05). The passive mode was equally as effective as the active mode in maintaining CO, SV, MAP, CVC, and sweat rate above inactive recovery. Sweat rate was different among all modes after 8 min of recovery (P < 0.05). TPR during active recovery remained significantly lower than during recovery in the passive and inactive modes (P < 0.05). No differences in either T(es) or T(sk) were observed among conditions. Given that MAP was higher during passive and active recovery modes than during inactive recovery suggests differences in CVC may be due to differences in baroreceptor unloading and not factors attributed to central command. However, differences in sweat rate may be influenced by factors such as central command and mechanoreceptor stimulation.     

Modeling and alanine scanning mutagenesis studies of recombinant pokeweed antiviral protein

The Phytolacca americana-derived naturally occurring ribosome inhibitory protein pokeweed antiviral protein (PAP) is an N-glycosidase that catalytically removes a specific adenine residue from the stem loop of ribosomal RNA. We have employed molecular modeling studies using a novel model of PAP-RNA complexes and site-directed mutagenesis combined with bioassays to evaluate the importance of the residues at the catalytic site and a putative RNA binding active center cleft between the catalytic site and C-terminal domain for the enzymatic deadenylation of ribosomal RNA by PAP. As anticipated, alanine substitutions by site-directed mutagenesis of the PAP active site residues Tyr(72), Tyr(123), Glu(176), and Arg(179) that directly participate in the catalytic deadenylation of RNA resulted in greater than 3 logs of loss in depurinating and ribosome inhibitory activity. Similarly, alanine substitution of the conserved active site residue Trp(208), which results in the loss of stabilizing hydrophobic interactions with the ribose as well as a hydrogen bond to the phosphate backbone of the RNA substrate, caused greater than 3 logs of loss in enzymatic activity. By comparison, alanine substitutions of residues (28)KD(29), (80)FE(81), (111)SR(112), (166)FL(167) that are distant from the active site did not significantly reduce the enzymatic activity of PAP. Our modeling studies predicted that the residues of the active center cleft could via electrostatic interactions contribute to both the correct orientation and stable binding of the substrate RNA molecule in the active site pocket. Notably, alanine substitutions of the highly conserved, charged, and polar residues of the active site cleft including (48)KY(49), (67)RR(68), (69)NN(70), and (90)FND(92) substantially reduced the depurinating and ribosome inhibitory activity of PAP. These results provide unprecedented evidence that besides the active site residues of PAP, the conserved, charged, and polar side chains located at its active center cleft also play a critical role in the PAP-mediated depurination of ribosomal RNA.     

Isolation of Five Sterols from the Liverwort,Scapania parvitexta Steph.

Castasterone (1), teasterone (2), and 6-deoxocastasterone (3), as well as monoolein, monolinolein, and monopalmitin, have been identified in immature seeds of rice (Oryza sativa) as active principles in the rice lamina inclination bioassay. Castasterone, as well as monoolein and monopalmitin, were likewise identified in immature seeds of Perilla frutescens, while monopalmitin was identified as a major active principle in cultured cells of Nicotiana tabacum. It seems that the occurrence of monoglycerides in higher plants as biologically active principles might be a general feature.     

A simple general method to determine the proportion of active ribosomes in eukaryotic cells

The resistance of 80S ribosomes derived from active polyribosomes to dissociation in high KCl cone, can conveniently be used to assay the proportion of active ribosomes in eukaryotic cells. The method described has been useful in studies of organisms as diverse as fungi (yeast), protozoa (Tetrahymena), higher plants (barley), echinoderms (sea urchin), insects (Musca), birds (chick) and mammals (mouse, rat). The technique is relatively insensitive to the use of harsh mechanical treatments for cell disruption or the presence of endogenous ribonuclease activity.     

Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

The glycoprotein hormones, luteinizing hormone (LH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH), play important roles in overall physiology and reproduction. These hormones are heterodimeric molecules consisting of an identical alpha subunit non-covalently associated with the hormone-specific beta subunit. The inherent structural intricacies possessed by these hormones make them very interesting model systems for structure-function relationship studies of complex dimeric glycoproteins. The structural studies, as well as, the therapeutic applications require large quantities of biologically active hormones free of any contaminants. In this study, we report hyperexpression and purification of biologically active recombinant hLH and hCG expressed using Pichia pastoris expression system. A combination of hydrophobic interaction chromatography and ion exchange chromatography has been used to purify these recombinant hormones to homogeneity. Using a number of biochemical and immunological criteria, the recombinant hormones have been shown to be similar to the natural hormones and were equally biologically active. The preliminary data also suggested that P. pastoris cells express a low molecular weight isoform of hCG that appeared to be less glycosylated. This isoform exhibited lesser affinity for the receptor as compared to hCG, but was found to be fully biologically active.     

Kinetics and mechanism of refolding of bovine carbonic anhydrase. A probe study of the formation of the active site.

In kinetic studies of the folding of bovine carbonic anhydrase from disorganized to native structure, an azosulfonamide, 2-(4-sulfomylphenylazo)-7-acetamido-1-hydroxynaphthalene-3,6-disulfonate (I), has been used as a probe to follow the dynamics of formation of the active site region. The probe is a specific inhibitor of the native enzyme that binds in the active site crevice. The experiments, with previous data (Yazgan, A., and Henkens, R. W. (1972), Biochemistry 11, 1314), show that a tight binding site for I forms at an intermediate stage in the folding process. A subsequent conformational change perturbs the visible absorption and circular dichroism of bound I and could result in even tighter binding. The subsequent change completes formation of the active site. This is shown by results from separate experiments on the kinetics of recovery of activity (p-nitrophenyl acetate as substrate). Similar probe methods could be used with other proteins and enzymes to study the kinetics and mechanism of regeneration of specific sites--for example, the active site.     

May active solute flux control the cell volume in the steady state?

The dynamics of a bioreactor with a variable volume and an active solute flux based on the thermodynamics of irreversible processes and stability analysis was studied. The active solute flux may control both the bioreactor volume and the hydrostatic pressure as well as the concentration of the solute inside the cell in the steady state. The range of the active solute flux is limited by amplitudes (j1(0), J2(0] of the active transport depending on the membrane transport parameters. The dynamic system is stable for j0 greater than jth0.     

Lipid A, the active part of bacterial endotoxins in inducing serum colony stimulating activity and proliferation of splenic granulocyte/macrophage progenitor cells.

An analysis of which component of lipopolysaccharides (LPS), the lipid or the polysaccharide (PS), is active in stimulating the murine granulopoietic system has been performed. LPS with different structures, isolated from different mutant strains of Salmonella and chemical degradation products of lipopolysaccharides have been used. Lipid A obtained by acid hydrolysys of the LPS and complexed to bovine serum albumin (BSA) (lipid A-BSA) was shown to be active in generating serum colony stimulating factor (CSF) and in increasing the splenic colony forming cells (CFC) levels, although it was less active than the parent LPS. The polysaccharide (PS) showed no significant activity at the concentrations used. LPS (glycolipids) from R mutants of Salmonella minnesota were active to the same extent as the LPS. The fact that even the most defective LPS from the R mutant R595 which contains lipid A and KDO only is a potent endotoxin, points unequivocally, to lipid A, as the active principle in stimulating the granulopoietic system.     

The key residues of active sites on the catalytic fragment for paclitaxel interacting with poly (ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (PARP) is regarded as a target protein for paclitaxel (PTX) to bind. An important issue is to identify the key residues as active sites for PTX interacting with PARP, which will help to understand the potential drug activity of PTX against cancer cells. Using docking method and MD simulation, we have constructed a refined structure of PTX docked on the catalytic function domain of PARP (PDB code: 1A26). The residues Glu327(988), Tyr246(907), Lys242(903), His165(826), Asp105(766), Gln102(763) and Gln98(759) in PARP are identified as potential sites involved in interaction with PTX according to binding energy (E(b)) between PTX and single residue calculated with B3LYP/6-31G(d,p). These residues form an active binding pocket located on the surface of the catalytic fragment, possibly interacting with the required groups of PTX leading to its activity against cancer cells. It is noted that most of the active sites make conatct with the "southern hemisphere" of PTX except for one residue, Tyr246(907), which interacts with the "northern hemisphere" of PTX. The conformation of PTX in complex with the catalytic fragment is observed as being T-shaped, similar to that complexed with β-tubulin. The total Eb of -269.9 kJ/mol represents the potent interaction between PTX and the catalytic fragment, implying that PTX can readily bind to the active pocket. The tight association of PTX with the catalytic fragment would inhibit PARP activation, suggesting a potential application of PTX as an effective antineoplastic agent.     

肠道菌群分析及粪便炎性标志物检测在炎症性肠病活动度评估中的作用

目的 探讨肠道菌群和粪便炎性标志物在炎症性肠病（IBD）活动度评估中的临床价值。方法 共纳入120例IBD患者为研究组,其中溃疡性结肠炎（UC）患者68例,克罗恩病（CD）患者52例。选择30例经结肠镜检查正常的健康体检者为对照组。采集全部研究对象的新鲜粪便标本进行粪便细菌培养及炎性标志物检测,比较不同疾病活动度IBD患者的肠道菌群及粪便钙卫蛋白（FC）、乳铁蛋白（LF）、基质金属蛋白酶-9（MMP-9）、髓过氧化酶（MPO）水平的变化。结果 与对照组比,UC和CD患者肠道中肠杆菌、肠球菌、拟杆菌、消化球菌及酵母菌数量均明显增加（P＜0.05）,双歧杆菌、乳杆菌及真杆菌数量明显减少（P＜0.05）。UC患者梭菌数量较对照组增加（P＜0.05）,CD患者梭菌数量较对照组减少（P＜0.05）。UC、CD活动期患者肠杆菌、肠球菌、拟杆菌、消化球菌及酵母菌数量明显多于缓解期患者（P＜0.05）,双歧杆菌、乳杆菌及真杆菌数量明显少于缓解期患者（P＜0.05）,且重度活动期患者肠道菌群改变较轻、中度活动期改变更明显（P＜0.05）。UC活动期患者梭菌数量明显多于缓解期（P＜0.05）,CD活动期患者梭菌数量明显少于缓解期（P＜0.05）。UC和CD患者粪便中FC、LF、MMP-9及MPO水平均显著高于对照组（P＜0.05）。UC、CD活动期患者FC、LF、MMP-9及MPO水平显著高于缓解期患者（P＜0.05）,且重度活动期患者高于轻、中度活动期患者（P＜0.05）。结论 肠道菌群变化和粪便中FC、LF、MMP-9及MPO水平可作为IBD患者疾病活动性评估的辅助指标。     

14-Dehydro-19-nortestosterone and its 7 -methyl derivative

14-Dehydro-19-nortestosterone and its 7α-methyl derivative were synthesized. The former was found to be approximately 100 and the latter 1000 times as active as testosterone in chick comb (local application) assays. In rat assays (subcutaneous), 14-dehydro-19-nortestosterone was approximately one-half as active as, or equal to testosterone in the ventral prostate or levator ani assays respectively, whereas its 7α-methyl derivative still retained its high potency (100 times as active as testosterone) in either type of assay.     

Crystal structure of the "cab"-type beta class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum

The structure of the "cab"-type beta class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) has been determined to 2.1-A resolution using the multiwavelength anomalous diffraction phasing technique. Cab exists as a dimer with a subunit fold similar to that observed in "plant"-type beta class carbonic anhydrases. The active site zinc is coordinated by protein ligands Cys(32), His(87), and Cys(90), with the tetrahedral coordination completed by a water molecule. The major difference between plant- and cab-type beta class carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes buffer molecule bound 8 A away from the active site zinc, which suggests a possible proton transfer pathway from the active site to the solvent.     

A search for an 'ouabain-like' substance from the electric organ of Electrophorus electricus which led to arachidonic acid and related fatty acids

The electric organ of Electrophorus electricus contains substances which inhibit (Na+ + K+)-ATPase activity, the specific binding of [3H]ouabain to purified (Na+ + K+)-ATPase and 86Rb+ uptake by chick cardiac cells in culture. The active organic material was extracted from microsomal membranes. Its purification was carried out by chromatography on Sep-Pak C-18 and thin-layer chromatography. Reverse-phase liquid chromatography and mass spectrometry identified the active material as a mixture of unsaturated fatty acids. Linoleic (18:2), arachidonic (20:4), linolenic (18:3) and docosahexaenoic acids (22:6) contributed to about 60% of the total activity of the active material. The other active substances could be arachidonic analogs, since they have both a lipophilic and carboxylic character. Pure unsaturated fatty acids have been shown to be active in the different biological assays used to analyze the endogenous 'ouabain-like' activity. Linolenic, arachidonic and docosahexaenoic acids were the most active, whereas saturated fatty acids and glyceryl esters or methyl esters of unsaturated fatty acids were inactive. It is possible that in pathological situations in which the level of unsaturated fatty acids increases, these molecules may then act as physiological inhibitors of the sodium pump.     

The isolation and partial characterization of catalase and a peroxidase active fraction from human white adipose tissue.

1. A procedure is described for the purification of catalase and a peroxidase active fraction from human white adipose tissue. 2. Gel electrophoresis on SDS-PAGE revealed relative molecular masses of 202,900 and 208,600 for the active catalase and peroxidase molecules respectively (nonreducing conditions), as compared to 56,800 and 49,800 for the monomers under reducing conditions, thus indicating the likelihood of tetramers in the intact state. 3. The two purified enzymes differ with regard to pH optima (5-9 for catalase and 3 for peroxidase), temperature stability (up to 50 degrees C for catalase and 70 degrees C for peroxidase) and Km values towards H2O2 (38.9 mM for catalase and 7.69 mM for peroxidase, which was also active in oxidizing a number of o-dihydricphenols as second substrates). 4. The catalase enzyme showed uncompetitive inhibition by the irreversible inhibitor 3-amino-1,2,4-triazole (AT), Ki = 5.4 mM.     

Cyclic AMP modulation of ion transport across frog retinal pigment epithelium. Measurements in the short-circuit state

In the frog retinal pigment epithelium (RPE), the cellular levels of cyclic AMP (cAMP) were measured in control conditions and after treatment with substances that are known to inhibit phosphodiesterase (PDE) activity (isobutyl-1-methylxanthine, SQ65442) or stimulate adenylate cyclase activity (forskolin). The cAMP levels were elevated by a factor of 5-7 compared with the controls in PDE-treated tissues and by a factor of 18 in forskolin-treated tissues. The exogenous application of cAMP (1 mM), PDE inhibitors (0.5 mM), or forskolin (0.1 mM) all produced similar changes in epithelial electrical parameters, such as transepithelial potential (TEP) and resistance (Rt), as well as changes in active ion transport. Adding 1 mM cAMP to the solution bathing the apical membrane transiently increased the short-circuit current (SCC) and the TEP (apical side positive) and decreased Rt. Microelectrode experiments showed that the elevation in TEP is due mainly to a depolarization of the basal membrane followed by, and perhaps also accompanied by, a smaller hyperpolarization of the apical membrane. The ratio of the apical to the basolateral membrane resistance increased in the presence of cAMP, and this increase, coupled with the decrease in Rt and the basolateral membrane depolarization, is consistent with a conductance increase at the basolateral membrane. Radioactive tracer experiments showed that cAMP increased the active secretion of Na (choroid to retina) and the active absorption of K (retina to choroid). Cyclic AMP also abolished the active absorption of Cl across the RPE. In sum, elevated cellular levels of cAMP affect active and passive transport mechanisms at the apical and basolateral membranes of the bullfrog RPE.     

Synthesis of active site-directed organometallic irreversible protease inhibitors.

p-Antimonybenzenesulfonyl fluoride and p-mercurybenzenesulfonyl fluoride irreversibly inhibit chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), and chromosomal protease, and these inhibitors appear to be as active as phenylmethanesulfonyl fluoride. The pretreatment of the proteases interferes with the phosphorylation of the active-site serine by diisopropylfluorophosphate suggesting that the organometallic inhibitors may also interact with the active site serine. The organometallic inhibitors may be used for localization of proteases in different parts of the cell by electron microscopy and p-mercurybenzenesulfonyl fluoride could also be used for isolation of proteases by sulfhydryl affinity chromatography.     

Identifying a surrogate metric for monitoring the population status of a secretive habitat specialist,the heath skink Liopholis multiscutata,in south‐eastern Australia

Threatened species that exist in small isolated populations are vulnerable to extinction processes, so effectively monitoring the trajectory of such populations will help determine the most appropriate management actions to combat extinction threats. In this study, we aimed to track the population status of the fossorial heath skink Liopholis multiscutata that is listed as threatened in Victoria, south‐eastern Australia, and exists there as a few small and highly disjunct populations, by using an appropriate surrogate population monitoring metric. This secretive lizard is a habitat specialist, is highly localised in Victoria and lives in warrens in semi‐arid heathland or mallee on large dunes. Survey data, which included every warren and their constituent burrows, as well as an assessment of whether each burrow was ‘active’, were collected for the four known Victorian populations in 2007 and annually during 2014–2018 inclusive. We compared five population indices per monitoring site: number of active warrens (NAW), number of active burrows (NAB), population area for 80% of active warrens (PA80), percentage of warrens that were active (PAW) and average number of active burrows per active warren. The heath skink currently occurs in small populations (8–46 active warrens) and these populations have typically declined over recent years. NAW was the most robust metric; NAB and PA80 did not reveal strong temporal trends. PAW indicated that inactive warrens and burrows persist less than a year and hence may provide information about recent (within months) population changes. It is imperative to establish a material link between the effective monitoring of small, vulnerable populations and the implementation of management actions that benefit such populations. Here, NAW could be used as a long‐term monitoring tool to provide an estimate of the minimum population size of the heath skink at a site. Its use would also ensure continuity in monitoring approaches for the Victorian populations.     

Correlation between the compact regions predicted by the Average Distance Map (ADM) and the biologically active sites in atrial natriuretic peptide

The prediction of the short-range compact regions of human atrial natriuretic peptide (a-hANP), one of the biologically active peptides, has been made by means of the Average Distance Map(ADM). We found out that the location of the predicted short-range compact regions is consistent with the structural units determined by the NMR analysis (Kobayashiet al., 1988). Furthermore, the short-range compact regions correspond well to the biologically active areas of atriopeptin (103–125)-amide (which is homologous peptide toa-hANP), detected by the glycine substitution technique (Konishiet al., 1987). The results suggest that a predicted short-range compact region can be regarded as a possible active site in a biologically active peptide.