Two methods for rapid assembly and oligomerization of synthetic genes: construction of human calcitonin-encoding sequences

Two fast, simple and reliable methods for construction of synthetic genes are described. The first represents a one-vial method for assembly and cloning of monomeric genes, and the second allows a simultaneous assembly, oligomerization and cloning of synthetic genes. Both methods consist of ligation of the synthetic oligodeoxyribonucleotides (oligos) building up the gene directly into the cloning vector, followed by screening of the recombinant clones for the presence of specific gene constructs. The average length of the oligomeric genes thus constructed is directly proportional to the molar ratio between the vector DNA and the synthetic oligos used. Both methods are illustrated by the construction of a series of human calcitonin-encoding genes.     

Increased proteolysis in chromatin of terminally differentiated and quiescent cells

Endogenous proteolysis in chromatin of terminally differentiated, quiescent, and actively proliferating cells was studied by measuring the released acid-soluble radioactivity of [³H]tryptophan-prelabelled nuclear proteins, and by following the specific quantitative and qualitative changes in electrophoregrams of chromosomal proteins. The experiments suggest that the chromatin of differentiated mouse kidney and liver cells, as well as chromatin from Friend cells induced to commit terminal differentiation, exhibit increased proteolysis in comparison with that of chromatin isolated from actively proliferating cells. Enhanced proteolysis was found also for the slowly renewing and quiescent cells from adult mice. The control experiments designated to discriminate between the two possible alternatives explaining the difference—increased activity of the proteolytic enzymes associated with chromatin, or increased susceptibility of the chromosomal proteins to proteases—supported the latter alternative.     

Differential expression of wheat genes during cold acclimation

Overwintering crops such as winter wheat display significant increase in freezing tolerance during a period of cold acclimation (CA). To gain better understanding of molecular mechanisms of CA, it is important to unravel functions and regulations of CA-associated genes. Differential screening of a cDNA library constructed from cold acclimated crown tissue of winter wheat identified three novel CA-associated cDNA clones. Nucleotide sequence analysis showed that the clones encode a high mobility globular protein (HMGB1), a glycine-rich RNA-binding protein (TaGRP2), and a LEA D-11 dehydrin (DHN14). Accumulation of the three mRNAs during 14 days of CA was differentially regulated. In response to drought, and ABA, DHN14 mRNA rapidly accumulated while HMGB1 and TaGRP2 mRNA levels remained unchanged. The possible functions of each of these genes in cold acclimation are discussed.     

Effects of abscisic Acid treatment on the thermostability of the photosynthetic apparatus in barley chloroplasts

Thermostability of the photosynthetic apparatus of abscisic acid (ABA)-treated seedlings of barley (Hordeum vulgare) was studied by light-scattering and by fluorescence measurements of isolated chloroplasts. ABA treatment markedly decreased heat damage of the chloroplast ultrastructure; an exogenous ABA concentration of 10⁻⁵ molar was most effective. Heat-induced increase of the 77 kilodalton fluorescence ratio F₇₄₀/F₆₈₅ was also smaller at this ABA concentration. The heat-induced increase of the initial chlorophyll fluorescence level (F_o) was virtually eliminated in ABA-treated (10⁻⁵ molar) chloroplasts up to 45°C and slightly increased at 50°C, relative to control chloroplasts where F_o increased even at 35°C and reached its maximal value at 45°C. In control chloroplasts, F_o increased with a 5-minute pretreatment temperature, an effect observed as low as 35°C. F_o was maximal at 45°C. In contrast, chloroplasts treated with 10⁻⁵ molar ABA did not exhibit a heat-induced increase in F_o until 50°C.     

The symptomless leaf infection with grapevine leafroll associated virus 3 in grown in vitro plants as a simple model system for investigation of viral effects on photosynthesis

The photosynthetic changes evaluated by oxygen evolution, chlorophyll fluorescence, photoacoustics, and delayed fluorescence (DF) were studied in leaves of grown in vitro for 8 weeks grapevine plants (Vitis vinifera) infected by grapevine leafroll-associated virus 3 (GLRaV-3). The infected leaves were characterized during the viral infection without visible disease symptoms. The symptomless infection led to a decrease in plant biomass. The non-photochemical fluorescence quenching, qN, declined, whereas the photochemical quenching, qP, and the Chl a/b ratio were not significantly affected. Photoacoustic and oxygen evolution measurements showed that the energy storage and oxygen evolution rate decreased in the infected leaves. Enhanced alternative electron sinks during the symptomless viral infection were also estimated. The changes in fluorescence and DF temperature curves demonstrated an enhanced stability of the thylakoid membranes in the infected leaves. This effect was clearly expressed at high actinic light intensities. The viral infected in vitro grown grapevine plants were used in the present study as a simplified model system that allow to avoid the involvement of different environmental factors that could interfere with the GLRaV infection and the virus-grapevine interactions. Thus, the 'pure' impact of the viral infection on photosynthesis could be investigated.     

Effect of Moringa oleifera leaves on hematological profile of fluorosis affected rats

Fluorosis is a metabolic disease that is endemic in nearly 25 countries with India being one of the most affected. It primarily affects the bone and the teeth. Moringa oleifera (MO) leaves are known to reduce the effect of fluorosis on various tissues. Therefore, it is of interest to document the effect of Moringa oleifera leaves on the hematological profile of fluorosis affected rats. Twenty four Sprague Dawley rats were housed two per cage in a room with 12 hours light and 12 hours dark cycle. The rats were allowed to adjust to the laboratory environment for about one to two weeks before the beginning of the study. This study reveals that MO leaves is effective in reducing the plasma fluoride content. It also helps in improving the Hb % and RBC count in fluorosis affected rats. Data shows that Moringa olifera leaves powder is effective in reducing the plasma fluoride content. It also helps in improving the Hemoglobin percentage & Red Blood Cell count in fluorosis affected rats.     

Conformational Effects on the Circular Dichroism of Human Carbonic Anhydrase II: A Multilevel Computational Study

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions.     

Catalysis in glycine N-methyltransferase: testing the electrostatic stabilization and compression hypothesis

Glycine N-methyltransferase (GNMT) is an S-adenosyl-l-methionine dependent enzyme that catalyzes glycine transformation to sarcosine. Here, we present a hybrid quantum mechanics/molecular mechanics (QM/MM) computational study of the reaction compared to the counterpart process in water. The process takes place through an SN2 mechanism in both media with a transition state in which the transferring methyl group is placed in between the donor (SAM) and the acceptor (the amine group of glycine). Comparative analysis of structural, electrostatic, and electronic characteristics of the in-solution and enzymatic transition states allows us to get a deeper insight into the origins of the enzyme's catalytic power. We found that the enzyme is able to stabilize the substrate in its more active basic form by means of a positively charged residue (Arg175) placed in the active site. However, the maximum stabilization is attained for the transition state. In this case, the enzyme is able to form stronger hydrogen bonds with the positively charged amine group. Finally, we show that in agreement with previous computational studies on other methyltransferases, there is no computational evidence for the compression hypothesis, as was formulated by Schowen (Hegazi, M. F., Borchardt, R. T., and Schowen, R. L. (1979) J. Am. Chem. Soc. 101, 4359-4365).     

Fragment-based screening of programmed death ligand 1 (PD-L1)

The PD-1 immune checkpoint pathway is a highly validated target for cancer immunotherapy. Despite the potential advantages of small molecule inhibitors over antibodies, the discovery of small molecule checkpoint inhibitors has lagged behind. To discover small molecule inhibitors of the PD-1 pathway, we have utilized a fragment-based approach. Small molecules were identified that bind to PD-L1 and crystal structures of these compounds bound to PD-L1 were obtained.