Molecular paleontology: a biochemical model of the ancestral ribosome

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.     

Structure based design of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors from a phenotypic screen

Nicotinamide phosphoribosyltransferase is a key metabolic enzyme that is a potential target for oncology. Utilizing publicly available crystal structures of NAMPT and in silico docking of our internal compound library, a NAMPT inhibitor, 1, obtained from a phenotypic screening effort was replaced with a more synthetically tractable scaffold. This compound then provided an excellent foundation for further optimization using crystallography driven structure based drug design. From this approach, two key motifs were identified, the (S,S) cyclopropyl carboxamide and the (S)-1-N-phenylethylamide that endowed compounds with excellent cell based potency. As exemplified by compound 27e such compounds could be useful tools to explore NAMPT biology in vivo.     

The Microtubule-Associated Protein CLASP Sustains Cell Proliferation through a Brassinosteroid Signaling Negative Feedback Loop

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Plastid structure and chlorophyll content in leaf primordia of onion bulbs

Angiosperms do not synthesize chlorophyll in the dark. Here we show that leaf primordia in onion bulbs are green, though they developing in dark conditions. We present results that show plastids in green primordia are chloroplasts, and that they contain chlorophyll as well as embryos in seeds of certain angiosperms.     

DNA-cation interactions: The major and minor grooves are flexible ionophores.

Several crystallographic, solution-state and theoretical studies carried out this past year provide new support for the sequence-specific nature of monovalent and divalent cation coordination within the DNA major and minor grooves. Correlations observed between groove width and cation coordination indicate that the grooves are flexible and respond to cation binding.     

Changes in photosynthetic apparatus of mustard (<Emphasis Type="Italic">Sinapis alba</Emphasis>) cotyledons following cycloheximide and kinetin treatments

Biochemical and accompanying structural characteristics of the photosynthetic process were studied in mustard seedlings cultivated on medium with increasing concentrations of cycloheximide alone as well as in combination with various kinetin concentrations. After 7 days of cultivation the contents of total chlorophyll, carotenoids and content of Rubisco in mustard cotyledons were determined. The content of chlorophyll pigments and carotenoids decreased in dependence of cycloheximide concentration. Following antibiotic treatment the content of both Rubisco subunits markedly decreased. In addition cycloheximide caused disturbance in mesophyll organization and chloroplast ultrastructure. Kinetin applied with cycloheximide increased the amount of photosynthetic pigments as well as of Rubisco, compared to the cycloheximide alone. In the seedlings treated with cycloheximide+kinetin the structure of leaf mesophyll and chloroplast membrane system was similar to control. Our results indicate that kinetin diminished the negative effects of cycloheximide on photosynthetic pigments and Rubisco as well as on the structural traits of the cotyledons.     

Effects of 1800 MHz GSM-like exposure on the gonadal function and hematological parameters of male mice

The aim of this study was to evaluate the possible effects of in vivo 1800 MHz GSM-like exposure on male reproduction. In five separate experiments, male NMRI mice (35-41 g) were exposed (11-12 mice each) to 1800 MHz GSM-like radiation. The average power density was 100 microW/cm2, the estimated SAR was 0.018-0.023 W/kg. The animals were exposed ten times (over two weeks on workdays) and the duration of exposure was 2 h/day. On the day of the last treatment, mice were anesthetized with i.p. pentobarbital, and blood samples were taken for hematology, serum chemistry and serum testosterone (T) determinations (ELISA). Testicles, epididymes, adrenals, prostates and pituitary glands were removed for histology. One testicle of each animal was used for culture of Leydig cells. The cells were cultured for 48 h in the presence or absence of human chorionic gonadotropin (hCG) to evaluate the in vitro steroidogenic response of Leydig cells. In the exposed animals red blood cell count (RBC: 8.59+/-0.10 T/l, n=37) and volume of packed red cells (VPRC: 42.29+/-0.43%, n=37) were significantly higher (p<0.01) compared with the controls (RBC: 8.12+/-0.08 T/l, n=36; VPRC: 39.76+/-0.36%, n=36). The serum testosterone level of the exposed animals (7.85+/-1.08 ng/ml, n=56) was also significantly elevated (p<0.05) compared to the controls (5.12+/-0.79, n=52), while the in vitro steroidogenic capacity of the Leydig cells was unaltered. No significant differences in the other investigated variables were found between controls and exposed mice. Our results indicate that the applied GSM-like microwave exposure may induce slight, but statistically significant alterations in some hematological and endocrine parameters of male mice within the physiological range. Further investigations are required to establish the biological significance of these phenomena.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

Effects of whole-body 50-Hz magnetic field exposure on mouse Leydig cells

The main goal of this study was to evaluate the possible effect of whole-body magnetic field (MF) exposure on the steroidogenic capacity of Leydig cells in vitro. In four separate experiments, male CFLP mice were exposed to sinusoidal 50-Hz, 100-microT MF. The duration of exposure was 23.5 h/day over a period of 14 days. At the end of the exposure, interstitial (Leydig) cells were isolated from the testicles of the sham-exposed and exposed animals. The cells were cultured for 48 h in the presence or absence of 1, 10, or 100 mIU/ml human chorionic gonadotropin (hCG). The luteinizing hormone (LH) analog hCG was used to check the testosterone (T) response of the sham-exposed controls and to evaluate the possible effect of the whole-body MF exposure on the steroidogenic capacity of Leydig cells in vitro. Testosterone content of the culture media and blood sera was measured by radioimmunoassay (RIA). In the cultures obtained from MF-exposed animals, the hCG-stimulated T response was significantly higher (p < 0.01) compared with the sham-exposed controls, while the basal T production of cells and the level of serum T remained unaltered. No MF exposure-related histopathological alterations were found in testicles, epididymes, adrenals, prostates, and pituitary glands. The MF exposure did not affect the animal growth rate and the observed hematologic and serum chemical variables. Our results indicate a presumably direct effect of whole-body MF exposure on the hCG-stimulated steroidogenic response of mouse Leydig cells.