Designing proteins to crystallize through beta-strand pairing

Inherent difficulties in growing protein crystals are major concerns within structural biology and particularly in structural proteomics. Here, we describe a novel approach of engineering target proteins by surface mutagenesis to increase the odds of crystallizing the molecules. To this end, we have exploited our recent triad-hypothesis using proteins with crystallographically defined beta-structures as the principal models. Crystal packing analyses of 182 protein structures belonging to 21 different superfamilies implied that the propensities to crystallize could be engineered into target proteins by replacing short segments, 5-6 residues, of their beta-strands with 'cassettes' of suitable packing motifs. These packing motifs will generate specific crystal packing interactions that promote crystallization. Key features of the primary and tertiary structures of such packing motifs have been identified for immunoglobulins. Further, packing motifs have been engineered successfully into six model antibodies without disturbing their capabilities to be produced, their immunoreactivity and their overall structure. Preliminary crystallization analyses have also been performed. Taken together, the procedures outline a rational protocol for crystallizing proteins by surface mutagenesis. The importance of these findings is discussed in relation to the crystallization of proteins in general.     

The parasitome of the phytonematode Heterodera glycines

Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.     

Synthesis and visualization of a membrane-permeable MRI contrast agent

The study of in vivo developmental events has undergone significant advances with the advent of biological molecular imaging techniques such as computer enhanced light microscopy imaging, positron emission tomography (PET), micro-CT, and magnetic resonance imaging (MRI). MRI has proven to be a particularly powerful tool in clinical and biological settings. Images can be acquired of opaque living animals, with the benefit of tracking events of extended periods of time on the same specimen. Contrast agents are routinely used to enhance regions, tissues, and cells that are magnetically similar but histologically distinct. A principal barrier to the development of MR contrast agents for investigating developmental biological questions is the ability to deliver the agent across cellular membranes. As part of our research, we are investigating a number of small molecules that facilitate transport of charged and uncharged species across cell membranes. Here we describe the synthesis and testing of a Gd(III)-based MR contrast agent conjugated to polyarginine that is able to permeate cell membranes. We confirmed cellular uptake of the agent using two-photon laser microscopy to visualize a Eu(III) derivative of the contrast agent in cell culture, and verified this uptake by T₁ analysis of the Gd(III) agent in cells.Abbreviations DOTA 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid - DOTA(tris-t-Bu ester) 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid-tert-butyl ester)-10-acetic acid - DO3A(tris-t-Bu ester) 1,4,7-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane - MRI magnetic resonance imaging - PET positron emission tomography - TPLM two-photon laser microscopy     

FGF-dependent generation of oligodendrocytes by a hedgehog-independent pathway

During development, spinal cord oligodendrocyte precursors (OPCs) originate from the ventral, but not dorsal, neuroepithelium. Sonic hedgehog (SHH) has crucial effects on oligodendrocyte production in the ventral region of the spinal cord; however, less is known regarding SHH signalling and oligodendrocyte generation from neural stem cells (NSCs). We show that NSCs isolated from the dorsal spinal cord can generate oligodendrocytes following FGF2 treatment, a MAP kinase dependent phenomenon that is associated with induction of the obligate oligogenic gene Olig2. Cyclopamine, a potent inhibitor of hedgehog signalling, did not block the formation of oligodendrocytes from FGF2-treated neurosphere cultures. Furthermore, neurospheres generated from SHH null mice also produced oligodendrocytes, even in the presence of cyclopamine. These findings are compatible with the idea of a hedgehog independent pathway for oligodendrocyte generation from neural stem cells.     

Dehydroepiandrosterone with other neurosteroids preserve neuronal mitochondria from calcium overload

This current study was designed to test whether the dehydroepiandrosterone (DHEA) and other neurosteroids could improve mitochondrial resistance to ischemic damage and cytoplasmic Ca(2+) overload. To imitate these mechanisms at mitochondrial level we treated the saponin permeabilized neurons either with the respiratory chain inhibitor, 1-methyl-4-phenylpyridinium or raised free extra-mitochondrial [Ca(2+)]. Loss of mitochondrial membrane potential (as an indicator of loss of function) was detected by JC-1. The results demonstrate that DHEA partly prevented Ca(2+) overload induced loss of mitochondrial membrane potential but not the loss of potential induced by the inhibitor of the respiratory chain. A similar effect was observed in the presence of other neurosteroids, pregnenolone, pregnanolone and allopregnanolone. DHEA inhibited also the Ca(2+) accumulation to the mitochondria in the presence of Ca(2+) efflux inhibitors. Thus, in the present work we provide evidence that DHEA with several other neurosteroids protect the mitochondria against intracellular Ca(2+) overload by inhibiting Ca(2+) influx into the mitochondrial matrix.     

The genetics of species differences

Species are separated by reproductive isolation as well as by more 'ordinary' differences in morphology and behavior that play no necessary role in blocking gene flow. Although a great deal is now known about the genetics of reproductive isolation, we are only beginning to understand the genetic basis of ordinary phenotypic differences between species. I review what is known about the number of genes involved in such differences, as well as about the role of major genes and epistasis in the evolution of these differences. I also compare and contrast these findings with those on the genetics of reproductive isolation.     

Bioinformatics and discovery: induction beckons again

With the flood of information from genomics, proteomics, and microarrays, what we really need now is the computer software to tell us what it all means. Or do we?