Kinetic studies of human immunodeficiency virus type 1 protease and its active-site hydrogen bond mutant A28S.

Human immunodeficiency virus type 1 (HIV-1) protease optimally catalyzes in the pH range of 4-6 in contrast to nearly all of the other eukaryotic aspartic proteases, which catalyze best in the pH range of 2-4. A possible structural reason for the higher optimal pH of HIV-1 protease is the absence of a hydrogen bond to the carboxyl group of active-site Asp25, which is nearly universally present in others. To investigate this hypothesis, we have mutated residue 28 in HIV-1 protease from alanine to serine. Both the wild-type and the mutant A28S enzymes have been overexpressed in Escherichia coli using a chemically synthesized gene and purified for a comparative study in enzyme kinetics. The kcat and Km values were determined by a radiometric assay for the wild-type enzyme from pH 3.2 to 7.0, and for the mutant enzyme from pH 3.2 to 6.0. The low pK values of the active site of the free enzyme, pKe1, are 3.3 and 3.4 for the wild-type and mutant enzymes, respectively. The low pK values of the active site of the enzyme bound to substrate, pKes1, are 5.1 and 4.3 for the wild-type and mutant enzymes, respectively. The high pK values of the free enzyme, pKe2, are 6.8 and 5.6, and the corresponding ones for the substrate-bound enzyme, pKes2, are 6.9 and 6.0 for the wild-type and mutant enzymes, respectively. The lowering of pK values in mutant HIV-1 protease indicates that the hydroxyl group of Ser28 forms a new hydrogen bond to active-site Asp25 to increase its acidity.     

Amino acid sequence of alpha-subunit in hen egg white ovomucin deduced from cloned cDNA.

The primary amino acid sequence of alpha-subunit in ovomucin (OVM) from hen thick egg white was determined. The 2087 amino acid residues with a relative molecular mass of 230.9 kDa along the full length of the alpha-subunit were represented. The alpha-subunit contains domains, arranged from the N- to C-terminals in the following order: D1-D2-D'-D3-R (central region)-D4-C1-CK (Cystine-knot), in a manner similar to the arrangement of D, C and CK domains in human pre-pro-von Willebrand factor (hpp-vWF) and hMUC2. The alpha-subunit showed identities on amino acid sequences with hpp-vWF and hMUC2 at 33 and 41% in the N-terminal region and 30 and 38% in the C-terminal region, respectively. The numbers and positions of cysteine residues were highly conserved among alpha-subunit, hpp-vWF and hMUC2. However, R showed no virtual sequence homology with the corresponding regions in two proteins. It was estimated that alpha-subunit was not part of a large peptide of OVM, but was independently synthesized from beta-subunit.     

Impact on fruit removal and seed predation of a secondary metabolite, emodin, in Rhamnus alaternus fruit pulp

There are two contradictory approaches to explaining the presence of secondary metabolites in ripe fruits. One holds that they evolved toward enhancing dispersal success (adaptive approach); the other claims that they evolved primarily to deter herbivores from eating leaves and seeds and that their presence in ripe fruits is a byproduct of that function (non‐adaptive approach). We tested the validity of three hypotheses of the adaptive approach that explain the presence of secondary metabolites in ripe fruits. We explored the current function of a secondary metabolite, emodin, in Mediterranean buckthorn (Rhamnus alaternus, Rhamnaceae) fruits by relating intraspecific variation and seasonal patterns of concentration to fruit removal and seed damage and by conducting feeding trials with captive birds presented artificial fruits that varied in emodin concentration. The concentration of emodin in the pulp of 10–13 Rhamnus plants from the same population was determined by HPLC every month during two fruiting seasons. Fruit removal by birds and seed predation by invertebrates and microbes were determined for the same plants. Emodin concentration rose during the first stages of ripening, reaching a peak before the fruits were ripe, and then decreased to a minimum when the fruits were ripe. No significant correlation between emodin concentration and ripe fruit removal rate among trees was observed in the first year, whereas in the second year the correlation was positive and significant. Thus, the impact of emodin on fruit selection varied between years, suggesting that emodin concentration does not solely govern fruit selection. A significant negative correlation was found in the first year between emodin concentration and seed predation during the first fruiting month. The yellow‐vented bulbul (Pycnonotus xanthopygos), a seed dispersing bird, distinguished between artificial foods that differed in emodin concentration (control, 0.001% and 0.002%), always preferring the lower concentration. In contrast, house sparrows, (Passer domesticus), a seed predator, did not detect such differences in emodin concentration but did distinguish between control foods and food with 0.005% and 0.001% emodin. We suggest that emodin has an ecological role, preventing seed predation by invertebrates and microbes without decreasing fruit removal by avian dispersers.     

Mitogenic activation and proliferation of mouse thymocytes : Comparison between isotope incorporation and flow-microfluorometry

Staining with acridine orange and flow microfluorometry was used to measure the distribution over the cell cycle of Concanavalin A (ConA)-stimulated mouse thymocytes. These data were compared with data on isotope incorporation. Mitogen-induced [¹⁴C]thymidine incorporation into DNA increased 10–20-fold upon addition of 2-mercaptoethanol or spleen-derived glass adherent cells. However, the proportion of proliferating cells as measured by flow microfluorometry increased only by a factor of two. Removal of nylonwool adherent cells nearly abolished ConA-induced thymidine incorporation; this was restored by the addition of glass-adherent cells. The fraction of mitogen-activated cells as measured by flow microfluorometry was hardly affected by nylonwool filtration and addition of mercaptoethanol or glass adherent cells enhanced the proportion of activated cells that can enter DNA synthesis. From these and similar experiments it is concluded that DNA synthesis measured as thymidine incorporation is only proportional to the number of mitogen-activated cells under restricted conditions. Differences in thymidine incorporation in different lymphocyte populations or measured under different conditions are difficult to interpret in terms of number of proliferating cells. The cytofluorographic method is a simple and rapid way to solve these problems quantitatively and rapidly.     

Differential expression of genes coding for EGF-like factors and ADAMTS1 following gonadotropin stimulation in normal and transformed human granulosa cells

We have demonstrated previously that the synthesis of epiregulin and amphiregulin, of the EGF-like growth factor family, is stimulated by luteinizing hormone in human follicular (granulosa) cells obtained from in vitro fertilization program. In the present work, we demonstrate that H89, a PKA inhibitor, attenuated the expression of these growth factors both in the mRNA and the protein levels, suggesting PKA involvement in this signaling pathway. SV40-transformed human granulosa cells showed higher basal levels of epiregulin and amphiregulin than normal cells, which were still elevated following cAMP stimulation by Forskolin. Cleavage by a disintegrin and metalloproteinases (ADAMs) is essential for activation of these growth factors, allowing their interaction with EGF receptor. Expression of ADAMTS1 and ADAM12 was downregulated by cAMP in normal, but not in SV40-transformed cells, suggesting that in normal cells epiregulin and amphiregulin activity is downregulated by a feedback mechanism that may be lost in SV40-transformed cells and their loss of downregulation may be involved in the development of ovarian tumors.     

Food preferences of captive gray short-tailed opossums (Monodelphis domestica)

The gray short-tailed opossum has been a subject in behavioral and biomedical studies for the last quarter century, but researchers know little about its preferred diet. The authors describe a study designed to determine food preferences of this species for the purpose of identifying suitable rewards to be used in behavioral studies.     

Brain trauma induces X-box protein 1 processing indicative of activation of the endoplasmic reticulum unfolded protein response

Brain trauma was induced in mice using a closed head injury (CHI) model. At 1, 6 or 24 h after trauma, brains were dissected into the cortex, striatum and hippocampus. Changes in levels of processed X-box protein 1 (xbp1), glucose-regulated protein 78 (grp78), growth arrest and DNA damage-inducible gene 153 (gadd153) and heat-shock protein 70 (hsp70) mRNA, indicating impaired endoplasmic reticulum (ER) and cytoplasmic functioning, were evaluated by quantitative PCR. In the cortex, processed xbp1 mRNA levels rose to 2000% of control 1 h after CHI, and stayed high throughout the experiments. In the hippocampus and striatum, processed xbp1 mRNA levels rose in a delayed fashion, peaking at 6 h (1000% of control) and 24 h after CHI (1500% of control) respectively. Levels of grp78 mRNA were only slightly increased in the cortex 24 h after CHI (150% of control), and were unchanged or transiently decreased in the hippocampus and striatum. Levels of gadd153 mRNA did not change significantly after trauma. A transient rise in hsp70 mRNA levels was observed only in the cortex, peaking at 1 h after CHI (600% of control). Processing of xbp1 mRNA is a sign of activation of the unfolded protein response indicative of ER dysfunction. The results suggest that brain trauma induces ER dysfunction, which spreads from the ipsilateral cortex to the hippocampus and striatum. These observations may have clinical implications and should therefore be considered for future investigations on therapeutic intervention of brain injury caused by contusion-induced neurotrauma.     

Equilibrium and balanced growth of a vegetative crop

MODEL: A previously developed dynamic model, NICOLET, designed to predict growth and nitrate content of a lettuce crop, is subjected to (virtual) constant environmental conditions. For every combination of shoot and root environment, the cell sap, here assumed to reside in the "vacuole" compartment, equilibrates at a certain nitrate concentration level. This, in turn, defines the composition of the crop in terms of carbon and nitrogen content in each of the three compartments of the model. Growth under constant environmental conditions is defined as "equilibrium" growth (EG). If, in addition, the source strengths of carbon and nitrogen balance each other, as well as the sink strength of the growing crop, the growth is said to be "balanced" (BG). RESULTS: It is shown that the range of BG approximately coincides with the range of "mild" nitrogen stress, where reduction in nitrogen availability results in a mild reduction of relative growth rate (RGR). Beyond a certain low nitrate concentration in the cell sap, the N-stress becomes "severe" and the loss of growth increases considerably. CONCLUSIONS: The model is able to mimic the five central observations of many constant-environment growth-chamber experiments, namely (1) the initial exponential growth and later decline of the RGR, (2) the constant chemical composition, (3) the equality of the RGR and the relative nutrient supply rate (RNR), (4) the proportionality between the N : C ratio and the RNR, and (5) the proportionality between the water content and the reduced N content. Guidelines for the optimal combination of the shoot and root environments are suggested.