Chiral ureas with two electronegative substituents at 1-N: an unusual case of coexisting pyramidal and almost planar 1-N atoms in the same crystal

XRD studies of structure of N-acetoxy-N-methoxyurea and N,N-bis(methoxycarbonyl)-N-methoxyimide have revealed that in N-methoxy-N-X-ureas (X = OAc, Cl, OMe, N(+)C(5)H(5)) the additional shortening of N-OMe bond took place, which arising from an n(O(Me))-sigma*(N-X) anomeric orbital interaction. XRD studies of N-chloro-N-ethoxyurea crystal have revealed the presence of two kinds of anomeric nitrogen configuration in the O-N-Cl group in the form of a pyramidal configuration and a planar configuration for same 1-N nitrogen atom. XRD studies of N-4-chlorobenzoyloxy-N-ethoxyurea have revealed that the degree of pyramidality of the 1-N nitrogen in N-aroyloxy-N-alkoxyureas is tuned by orientation of benzoyl group with respect to the N-O bond, which in turn depends of size of N-alkoxy group.     

How the body controls brain temperature: the temperature shielding effect of cerebral blood flow.

Normal brain functioning largely depends on maintaining brain temperature. However, the mechanisms protecting brain against a cooler environment are poorly understood. Reported herein is the first detailed measurement of the brain-temperature profile. It is found to be exponential, defined by a characteristic temperature shielding length, with cooler peripheral areas and a warmer brain core approaching body temperature. Direct cerebral blood flow (CBF) measurements with microspheres show that the characteristic temperature shielding length is inversely proportional to the square root of CBF in excellent agreement with a theoretical model. This "temperature shielding effect" quantifies the means by which CBF prevents "extracranial cold" from penetrating deep brain structures. The effect is crucial for research and clinical applications; the relationship between brain, body, and extracranial temperatures can now be quantitatively predicted.     

Chromosome composition of interspecies hybrid embryonic stem cells in mice

Chromosome complements of 20 hybrid clones obtained by fusing Mus musculus embryonic stem cells (ESCs) and Mus caroli splenocytes were studied. The use of two-color fluorescence hybridization in situ with chromosome- and species-specific probes has allowed us to reliably reveal the parental origin of homologs of any chromosome in hybrid cells. Depending on the ratio of parental chromosome homologs, all 20 hybrid clones were separated in several groups ranging from the clones that contain cells that are nearly tetraploid with two diploid sets of M. musculus and single M. caroli chromosomes to clones with a marked predominance of the M. caroli chromosome. In eight hybrid cell clones, we observed the pronounced prevalence of chromosomes of the pluripotent partner over chromosomes of the somatic partner in a ratio of 5: 1 to 3: 1. In other hybrid cell clones, the ratio of M. musculus to M. caroli chromosomes was either equal (1: 1; 2: 2) or with the prevalence of the pluripotent (2: 1) or differentiated (1: 2) partner. In three hybrid cell clones, for the first time, we observed the predominant segregation of ESC-derived pluripotent chromosomes. This might indicate the compensation for the epigenetic differences between parental chromosomes of the ESC and splenocyte origin.     

Identification of MicroRNAs controlling human ovarian cell proliferation and apoptosis

Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre‐miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir‐15a and mir‐188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir‐15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir‐188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre‐mir‐10a, mir‐105, and mir‐182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome‐wide miRNA screen. J. Cell. Physiol. 223: 49–56, 2010. © 2009 Wiley‐Liss, Inc.     

Fast and precise protein tracking using repeated reversible photoactivation

Photoactivatable fluorescent proteins opened principally novel possibilities to study proteins' movement pathways. In particular, reversibly photoactivatable proteins enable multiple tracking experiments in a long-drawn work with a single cell. Here we report 'protein rivers tracking' technique based on repeated identical rounds of photoactivation and subsequent images averaging, which results in dramatic increase of imaging resolution for fast protein movement events.     

Structural basis for the fast maturation of Arthropoda green fluorescent protein

Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.     

Matrix metalloproteinase triple-helical peptidase activities are differentially regulated by substrate stability

Matrix metalloproteinases (MMPs) are involved in physiological remodeling as well as pathological destruction of tissues. The turnover of the collagen triple-helical structure has been ascribed to several members of the MMP family, but the determinants for collagenolytic specificity have not been identified. The present study has compared the triple-helical peptidase activities of MMP-1 and MMP-14 (membrane-type 1 MMP; MT1-MMP). The ability of each enzyme to efficiently hydrolyze the triple helix was quantified using chemically synthesized fluorogenic triple-helical substrates that, via addition of N-terminal alkyl chains, differ in their thermal stabilities. One series of substrates was modeled after a collagenolytic MMP consensus cleavage site from types I-III collagen, while the other series had a single substitution in the P(1)' subsite of the consensus sequence. The substitution of Cys(4-methoxybenzyl) for Leu in the P(1)' subsite was greatly favored by MMP-14 but disfavored by MMP-1. An increase in substrate triple-helical thermal stability led to the decreased ability of the enzyme to cleave such substrates, but with a much more pronounced effect for MMP-1. Increased thermal stability was detrimental to enzyme turnover of substrate (k(cat)), but not binding (K(M)). Activation energies were considerably lower for MMP-14 hydrolysis of triple-helical substrates compared with MMP-1. Overall, MMP-1 was found to be less efficient at processing triple-helical structures than MMP-14. These results demonstrate that collagenolytic MMPs have subtle differences in their abilities to hydrolyze triple helices and may explain the relative collagen specificity of MMP-1.     

5-Aminolevulinic acid synthase: mechanism,mutations and medicine

5-Aminolevulinic acid synthase (ALAS), the first enzyme of the heme biosynthesis pathway, catalyses the pyridoxal 5'-phosphate-dependent condensation between glycine and succinyl-CoA to yield 5-aminolevulinic acid (5-amino-4-oxopentanoate). A three-dimensional structural model of Rhodobacter spheroides ALAS has been constructed and used to identify amino acid residues at the active site that are likely to be important for the recognition of glycine, the only amino acid substrate. Several residues have been investigated by site-directed mutagenesis and enzyme variants have been generated that are able to use alanine, serine or threonine. A three-dimensional structure model of 5-aminolevulinic acid synthase from human erythrocytes (ALAS 2) has also been constructed and used to map a range of naturally occurring human mutants that give rise to X-linked sideroblastic anemia. A number of these anemias respond favourably to vitamin B(6) (pyridoxine) therapy, whereas others are either partially responsive or completely refractory. Detailed investigations with selected human mutants have highlighted the importance of arginine-517 that is implicated in glycine carboxyl group binding.