Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes

A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.     

Structural energetics of the molten globule state

Certain partly ordered protein conformations, commonly called “moltenglobule states,” are widely believed to represent protein folding intermediates. Recentstructural studies of molten globule states ofdifferent proteins have revealed features whichappear to be general in scope. The emergingconsensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define ageneral state of protein folding called “the molten globule state,” which is structurally andthermodynamically distinct from both the native state and the denatured state. Despite exaatensive knowledge of structural features of afew molten globule states, a cogent thermodynamic argument for their stability has not yetbeen advanced. The prevailing opinion of thelast decade was that there is little or no enthalpy difference or heat capacity differencebetween the molten globule state and the unfolded state. This view, however, appears to beat variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of α-lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other protential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states. © 1993 Wiley-Liss, Inc.     

Search for substrate-based inhibitors fitting the S2' space of malarial aspartic protease plasmepsin II.

Plasmepsin (Plm) has been identified as an important target for the development of new antimalarial drugs, since its inhibition leads to the starvation of Plasmodium falciparum. A series of substrate-based dipeptide-type Plm II inhibitors containing the hydroxymethylcarbonyl isostere as a transition-state mimic were synthesized. The general design principle was provision of a conformationally restrained hydroxyl group (corresponding to the set residue at the P2' position in native substrates) and a bulky unit to fit the S2' pocket.     

Research into the Rhizobium/Leguminosae symbiosis in Latin America

More than 60 institutions and 100 researchers were involved in Rhizobium research in 1978 in Latin America. Half of these researchers were located in Argentina and Brazil. Research activity and the application of research findings vary widely among countries. Problems that plague research include 1) inadequate training of research personnel and insufficient attention paid to the Rhizobium/Legume symbiosis at agriculture schools; 2) poorly-established research priorities that do not sufficiently weigh the immediate needs for the farmers such as the identification of limiting environmental factors (e.g. nutritional deficiencies), techniques for smallscale inoculant production, and quality control of available inoculants; 3) isolation of the researchers and a lack of adequate library support; 4) poorly integrated research teams (e.g. in many institutes researchers are either microbiologists with no agricultural background or agronomists lacking microbiological training); and 5) insufficient dissemination of research findings. Problems with inoculant production and control include 1) a local dependence on national or imported inoculants rather than on locally-selected strains, 2) poor inoculant quality control which results in low inoculation success rates and subsequent discredit to the inoculation practice, and 3) high prices for inoculants. Extension problems include 1) lacking or deficient legume-promotion programs by government agencies, 2) poor contact between research and extension workers, and 3) administrators, leaders, extension workers and agronomists working in the field that lack adequate knowledge of the Rhizobium/Legume symbiosis. Immediate measures to foster extension and legume promotion programs and informal and/or official quality control are needed in Argentina, Uruguay, Brazil, Mexico, and probably Colombia. Countries where combined efforts should primarily be directed toward stimulating research and extension include Peru, Venezuela, Costa Rica, and Chile. In Ecuador, Paraguay, Bolivia, Nicaragua, Honduras, Guatemala, the Dominican Republic and Panama, priority should be given to research. Colombia should also be included in this group as national research institutions need to be strengthened. Table 2 lists these priorities more fully.     

Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.     

Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin

The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbants in order to assess the magnitude of the interactions within the B-subunits. For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbants, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion. At pH 7.5 and in the absence of any additional perturbants, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacity function centered at 77 degrees C and characterized by a delta Hcal of 328 kcal/mol of B-subunit pentamer and delta Hvh/delta Hcal of 0.3. Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van't Hoff enthalpy. The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3. Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 degrees C. Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer. Similar behavior is obtained with GuHCl. In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three triterpenes and other terpenoids from Catha cassinoides

The investigation of stems and leaves of Catha cassinoides afforded, in addition to sitosterol, β-amyrin, ursolic acid, lup-20(29)-en-3β,30-diol and friedelin, three new pentacyclic triterpenes: 30-hydroxyfriedelan-3-one, 29-hydroxyfriedelan-3-one and 3-oxo-friedelan-29-oic acid. The structures ofthese were determined by spectral studies and correlations, and were confirmed by X-ray analysis of 29-hydroxyfriedelan-3-one acetate.     

Thermotropic behavior of monoglucocerebroside--dipalmitoylphosphatidylcholine multilamellar liposomes

The thermotropic behavior of multilamellar liposomes prepared from mixtures of glucocerebroside and dipalmitoylphosphatidylcholine has been studied by high-sensitivity scanning calorimetry. It is shown that glucocerebroside has a marked effect on the gel--liquid crystalline transition of dipalmitoylphosphatidylcholine. The pretransition seen in pure samples of dipalmitoylphosphatidylcholine is undetectable at small mode fractions of glucocerebrosides (less than 10%). The main transition is shifted to higher temperatures and becomes broader and less cooperative in the presence of glucocerebroside. The enthalpy change of the main transition decreases with increasing the glucocerebroside content. However, this decrease is not linear with the glucocerebroside/phospholipid mole ratio. Glucocerebroside itself does not show a separate transition in the temperature range of these studies (10--75 degree C). The origin of these effects and their dependence on the glucocerebroside content suggest that the in-plane distribution of glucocerebroside molecules is affected by the physical state of the lipid bilayer and by the glucocerebroside/phospholipid mole ratio.     

Thermodynamics of transfer ribonucleic acids: the effect of sodium on the thermal unfolding of yeast tRNAPhe.

The thermal unfolding of yeast phenylalanine-specific tRNA (tRNA^Phe) has been calorimetrically investigated at several salt concentrations in the absence of magnesium. Application of the deconvolution theory of macromolecular conformational transitions allows calculation of the thermodynamic parameters of unfolding. It is demonstrated that the unfolding of tRNA^Phe occurs in a sequential fashion and that four separate transitions or five macromolecular thermodynamic states exist in the temperature range 8–72°C under the experimental conditions of these studies (0.067–0.52M Na⁺). The enthalpy and entropy changes between states and the relative population of each state as a function of temperature and salt concentration have been obtained. Sodium stabilizes the low-temperature conformations of tRNA^Phe. The increase in the melting temperatures of each transition is shown to be linearly dependent on the logarithm of sodium concentration. These results allow calculation of the “phase” diagram for the transitions as a function of salt concentration.