Non-redox-active small-molecules can accelerate oxidative protein folding by novel mechanisms

Multi-disulfide-bond-containing proteins acquire their native structures through an oxidative folding reaction which involves formation of native disulfide bonds through thiol-disulfide exchange reactions between cysteines and disulfides coupled to a conformational folding event. Oxidative folding rates of the four-disulfide-bond-containing protein bovine pancreatic ribonuclease A (RNase A) in the presence of the synthetic redox-active molecule, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), and in combination with non-redox-active trimethylamine-N-oxide (TMAO), and trifluorethanol were determined by HPLC analysis. The data indicate that regeneration of RNase A is enhanced 2-fold by BMC (50 microM) and 3-fold upon addition of TMAO (0.2 M) and TFE (3% v/v) relative to control experiments performed in the absence of small-molecules. Examination of the native tendency of the fully-reduced polypeptide and the stability of key folding intermediates suggests that the increased oxidative folding rate can be attributed to native-like elements induced within the fully-reduced polypeptide and the stabilization of native-like species by added non-redox-active molecules.     

Nitrogen fixation ability explains leaf chemistry and arbuscular mycorrhizal responses to fertilization

Atmospheric nitrogen (N) and phosphorus (P) deposition rates are predicted to drastically increase in the coming decades. The ecosystem level consequences of these increases will depend on how plant tissue nutrient concentrations, stoichiometry and investment in nutrient uptake mechanisms such as arbuscular mycorrhizal fungi (AMF) change in response to increased nutrient availability, and how responses differ between plant functional types. Using a factorial nutrient addition experiment with seedlings of multiple N-fixing and non-N-fixing tree species, we examined whether leaf chemistry and AMF responses differ between these dominant woody plant functional groups of tropical savanna and dry forest ecosystems. We found that N-fixers have remarkably stable foliar chemistry that stays constant with external input of nutrients. Non-N-fixers responded to N and N + P addition by increasing both concentrations and total amounts of foliar N, but showed a corresponding decrease in P concentrations while total amounts of foliar P stayed constant, suggesting a ‘dilution’ of tissue P with increased N availability. Non-N-fixers also showed an increase in N:P ratios with N and N + P addition, probably driven by both an increase in N and a decrease in P concentrations. AMF colonization decreased with N + P addition in non-N-fixers and increased with N and N + P addition in N-fixers, suggesting differences in their nutrient acquisition roles in the two plant functional groups. Our results suggest that N-fixers and non-N-fixers can differ significantly in their responses to N and P deposition, with potential consequences for future nutrient and carbon cycling in savanna and dry forest ecosystems.     

Diterpenes derived from clerodanes from Pulicaria angustifolia

The investigation of Pulicaria angustifolia afforded, in addition to known triterpenes and caryophyllene epoxide, four methyl esters of diterpenes, all derived from clerodane. Three of them are derivatives of seco-nidoresedic acid. The structures were elucidated by spectroscopic methods.     

Sodium-gradient-driven, high-affinity, uphill transport of succinate in human placental brush-border membrane vesicles.

1. A high-density-lipoprotein (HDL) conversion factor was partially purified from human plasma by precipitation with (NH4)2SO4, ultracentrifugation, cation-exchange chromatography, anion-exchange chromatography and chromatography on a column of hydroxyapatite. 2. This factor modulates the particle size of HDL by converting a homogeneous population into new populations of particles, some of which are smaller and others larger than those in the original population. 3. The isolated HDL conversion factor appeared as one major band and at least three minor bands on SDS/polyacrylamide-gel electrophoresis; attempts to purify this factor further resulted in loss of conversion activity. 4. Preparations of the HDL conversion factor were stable after heating to 58 degrees C for 1 h, and were shown not to possess proteolytic activity. 5. The conversion factor was distinct from the known apolipoproteins, none of which had HDL conversion activity. 6. Addition of apolipoprotein A-IV had a dose-dependent potentiating effect on the process promoted by the HDL conversion factor.     

Selective release of follicle-stimulating hormone and luteinizing hormone by 5 alpha-dihydroprogesterone and 3 alpha, 5 alpha-tetrahydroprogesterone in pregnant mare's serum gonadotropin-primed immature rats exposed to constant light

The effects of 5 alpha-dihydroprogesterone (5 alpha-DHP) and 3 alpha, 5 alpha-tetrahydroprogesterone (3 alpha, 5 alpha-THP) on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release were examined in the pregnant mare's serum gonadotropin (PMSG)-primed immature female rat (8 IU PMSG at 28 days of age) maintained in constant light. Control rats kept in 14L:10D conditions exhibited proestrous-like surges of LH and FSH release with peak levels attained at 1800 h on the second day after PMSG treatment. In rats exposed to constant light, the PMSG-induced surges of LH and FSH were not only delayed until 1000 h on the third day after PMSG, resulting in a delay in ovulation, but were also significantly attenuated when compared to the gonadotropin surges that occurred on Day 2 in rats kept under normal light-dark conditions. The administration of 5 alpha-DHP significantly enhanced the release of FSH at 1000 h on Day 3 when compared to constant light-exposed controls, but had no effect on LH. Treatment with 3 alpha, 5 alpha-THP selectively potentiated the release of LH at 1000 h on Day 3 and had an attenuating effect on FSH release on Days 2 and 3. These observations confirm earlier findings in the immature ovariectomized estrogen-primed rat and suggest that 5 alpha-DHP and 3 alpha, 5 alpha-THP may have significant roles in the regulation of FSH and LH secretion.     

New β-phospholactam as a carbapenem transition state analog: Synthesis of a broad-spectrum inhibitor of metallo-β-lactamases

In an effort to test whether a transition state analog is an inhibitor of the metallo-β-lactamases, a phospholactam analog of carbapenem has been synthesized and characterized. The phospholactam 1 proved to be a weak, time-dependent inhibitor of IMP-1 (70%), CcrA (70%), L1 (70%), NDM-1 (53%), and Bla2 (94%) at an inhibitor concentration of 100 μM. The phospholactam 1 activated ImiS and BcII at the same concentration. Docking studies were used to explain binding and to offer suggestions for modifications to the phospholactam scaffold to improve binding affinities.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Stem cells: cross-talk and developmental programs

The thesis advanced in this essay is that stem cells-particularly those in the nervous system-are components in a series of inborn 'programs' that not only ensure normal development, but persist throughout life so as to maintain homeostasis in the face of perturbations-both small and great. These programs encode what has come to be called 'plasticity'. The stem cell is one of the repositories of this plasticity. This review examines the evidence that interaction between the neural stem cell (as a prototypical somatic stem cell) and the developing or injured brain is a dynamic, complex, ongoing reciprocal set of interactions where both entities are constantly in flux. We suggest that this interaction can be viewed almost from a 'systems biology' vantage point. We further advance the notion that clones of exogenous stem cells in transplantation paradigms may not only be viewed for their therapeutic potential, but also as biological tools for 'interrogating' the normal or abnormal central nervous system environment, indicating what salient cues (among the many present) are actually guiding the expression of these 'programs'; in other words, using the stem cell as a 'reporter cell'. Based on this type of analysis, we suggest some of the relevant molecular pathways responsible for this 'cross-talk' which, in turn, lead to proliferation, migration, cell genesis, trophic support, protection, guidance, detoxification, rescue, etc. This type of developmental insight, we propose, is required for the development of therapeutic strategies for neurodegenerative disease and other nervous system afflictions in humans. Understanding the relevant molecular pathways of stem cell repair phenotype should be a priority, in our view, for the entire stem cell field.     

Evidence for an imipramine-sensitive serotonin transporter in human placental brush-border membranes

Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+ is replaced by Li+, K+, Rb+, Cs+ or choline. The presence of Cl- seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+ (20-40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50 values are 70 nM and 20 microM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50 greater than 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt = 51 +/- 2 nM; Vmax = 6.4 +/- 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50 = 32 nM) and desipramine (IC50 = 160 nM) but relatively insensitive to reserpine and hydralazine.