A bivariate radioimmunoassay for arginine vasopressin and the synthetic antidiuretic agent 1-deamino-8-D-arginine vasopressin (desmopressin)

Pairs of radioimmunoassays, each of which include a two-dimensional matrix of standards, have been previously employed to resolve specificity problems in steroid immunoassay. In this study the bivariate radioimmunoassay principle has been applied to simultaneous measurement of plasma antidiuretic hormone, arginine vasopressin, and the synthetic antidiuretic agent 1-deamino-8-D-arginine vasopressin (desmopressin), by utilizing two arginine vasopressin antisera which show significantly different cross-reactivities with the synthetic analog. Data processing consists of mathematical representation of two curved dose-response surfaces followed by solution of this pair of nonlinear simultaneous equations for the unknown arginine vasopressin and desmopressin concentrations. Details of numerical procedures are given in the Appendix. The assay appears entirely adequate in terms of sensitivity, accuracy, and precision for measurement of these antidiuretic agents in clinical samples. No evidence of significant covariance in estimated concentrations could be detected but precision of estimation is (not unexpectedly) a function of the concentration of both agents. The plasma disappearance half-time of desmopressin (probably the second of a biphasic disappearance) was estimated as 37 min in one normal subject, which is in good agreement with a previously reported value of 30 min.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Microcalorimetric assay of acetylcholinesterase

Comparative assays were made in a spectrophotometer and a microcalorimeter for the reaction between acetylcholinesterase (EC 3.1.1.7) and acetylthiocholine. The rate of light absorbance change and the rate of heat flow were measured from similar and simultaneous reactions in spectrophotometer and microcalorimeter, respectively. At the enzyme activity levels studied, i.e., 0.05–0.15 I.U. in calorimetry and 1–4 I.U. in spectrophotometry, the reaction rates were linear and showed first-order kinetics. A highly significant positive correlation was seen between the two methods (r = 0.997). More importantly, spectrophotometric assay with acetylthiocholine (which utilized a secondary reaction with chromagen, dithiobisnitrobenzoic acid) stood in highly significant positive correlation with calorimetric assays (which did not require a chromagen) either with the same substrate (r = 0.976) or with acetylcholine (r = 0.900). It appears that microcalorimetry can be used in preference to spectrophotometry for enzyme kinetic studies to overcome the complexity of reaction mixture and interference problems and with the advantage of using natural substrates.     

Climate change and seasonal reproduction in mammals

Seasonal reproduction is common among mammals at all latitudes, even in the deep tropics. This paper (i) discusses the neuroendocrine pathways via which foraging conditions and predictive cues such as photoperiod enforce seasonality, (ii) considers the kinds of seasonal challenges mammals actually face in natural habitats, and (iii) uses the information thus generated to suggest how seasonal reproduction might be influenced by global climate change. Food availability and ambient temperature determine energy balance, and variation in energy balance is the ultimate cause of seasonal breeding in all mammals and the proximate cause in many. Photoperiodic cueing is common among long-lived mammals from the highest latitudes down to the mid-tropics. It is much less common in shorter lived mammals at all latitudes. An unknown predictive cue triggers reproduction in some desert and dry grassland species when it rains. The available information suggests that as our climate changes the small rodents of the world may adapt rather easily but the longer lived mammals whose reproduction is regulated by photoperiod may not do so well. A major gap in our knowledge concerns the tropics; that is where most species live and where we have the least understanding of how reproduction is regulated by environmental factors.     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

Aminoadamantanes containing monoterpene-derived fragments as potent tyrosyl-DNA phosphodiesterase 1 inhibitors

The ability of a number of nitrogen-containing compounds that simultaneously carry the adamantane and monoterpene moieties to inhibit Tdp1, an important enzyme of the DNA repair system, is studied. Inhibition of this enzyme has the potential to overcome chemotherapeutic resistance of some tumor types. Compound (+)-3c synthesized from 1-aminoadamantane and (+)-myrtenal, and compound 4a produced from 2-aminoadamantane and citronellal were found to be most potent as they inhibited Tdp1 with IC₅₀ values of 6 and 3.5 µM, respectively. These compounds proved to have low cytotoxicity in colon HCT-116 and lung A-549 human tumor cell lines (CC₅₀ > 50 µM). It was demonstrated that compound 4a at 10 µM enhanced cytotoxicity of topotecan, a topoisomerase 1 poison in clinical use, against HCT-116 more than fivefold and to a lesser extent of 1.5 increase in potency for A-549.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.     

Bird sensitivity to disturbance as an indicator of forest patch conditions: An issue in environmental assessments

An Environmental Assessment (EA) is one of the steps within the Environmental Impact Assessment process. Birds are often used in EA to help decision makers evaluate potential human impacts from proposed development activities. A “sensitivity to human disturbance” index, created by Parker III et al. (1996) for all Neotropical species, is commonly considered an ecological indicator. However, this parameter was created subjectively and, for most species, there have been no rigorous field test to validate its effectiveness as such. Therefore, in this study, we aim to: (1) evaluate if, at the local scale, birds from forest patches in a human-modified landscape (HML) may differ in sensitivity from Parker's sensitivity classification; (2) evaluate the effectiveness of the species richness value at each sensitivity level as an ecological indicator; (3) gather information on how often and in which manner Parker's classification has been used in EA. To do so, bird sampling was performed in eight forest patches in a HML over one year. Then, we created a local sensitivity to disturbance using information about threat, endemism, spatial distribution and relative abundance of all species in the study area. We found that 37% of the forest birds showed different local sensitivity levels when compared with Parker's classification. Our results show that only the richness of high-sensitivity species from our local classification fitted the ecological indicator assumptions helping the environmental conditions evaluation of the studied patches. We conclude that species richness of each Parker's bird sensitivity levels do not necessarily perform as an ecological indicator at the local scale, and particularly in HML. Nevertheless, Parker's Neotropical bird sensitivity classification was used in 50% of EA we reviewed. In these, 76% assumed that it was an accurate ecological indicator of the local forest conditions for birds. The lack of clear criteria used in Parker's classification allows diverse interpretations by ornithologists, and there is no agreement about the ecological meaning of each sensitivity level and what environmental conditions each level may indicate of. Therefore, the use of Parker's classification in EA may jeopardize accurate interpretations of proposed anthropogenic impacts. Furthermore, because a bird species’ sensitivity often varies between locations, we argue that Parker's generalized classification of bird sensitivity should not be used as an indicator of forest environmental conditions in EA throughout HMLs in Neotropics. Rather, local bird ecological indices should be explored, otherwise, erroneous predictions of the anthropogenic impacts will continue to be common.     

Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

In vitro rooting of the apple rootstock M 26 in adult and juvenile growth phases and acclimatization of the plantlets

In order to obtain optimum conditions for in vitro propagation of the apple rootstock M 26 ( Malus pumila Mill.) in adult and juvenile growth phases, several rooting experiments were performed. Supraoptimal concentrations of indole-3-butyric acid (IBA) added to the rooting media resulted in profuse callus formation. Since extensive callus production is detrimental to the survival of the plantlets, modified culture conditions were established to reduce callus formation. A reduction of the time of exposure to IBA to 5 days and, thereafter, transfer to a hormone-free medium did not eliminate callus production. Exposure to darkness during the root initiation phase increased rooting. When the rooting medium was based on the Lepoivre formula instead of the Murashige and Skoog formula, callus formation was reduced. Optimum conditions for rooting were obtained at much lower concentration than earlier reported, being 1.25 μM for the juvenile and 0.5 μM for the adult growth phase in the range of IBA concentrations tested. Anatomical studies revealed that root initials are formed after 5 days of IBA-treatment. Therefore, we transferred shoots directly to non-sterile conditions after the root-inducing phase. This resulted in a 90% survival of the plantlets. Subculture on hormone-free medium can thus be eliminated when the optimum auxin concentration is known.