Crystal structure of the Campylobacter jejuni Cj0090 protein reveals a novel variant of the immunoglobulin fold among bacterial lipoproteins

Bacterial lipoproteins play an important role in bacterial pathogenesis and physiology. The genome of Campylobacter jejuni, a major foodborn pathogen, is predicted to contain over 20 lipoproteins. However, the functions of the majority of C. jejuni lipoproteins remain unknown. The Cj0090 protein is encoded by a lipoprotein operon composed of cj0089, cj0090, and cj0091. Here, we report the crystal structure of Cj0090 at 1.9 Å resolution, revealing a novel variant of the immunoglobulin fold with β‐sandwich architecture. The structure suggests that Cj0090 may be involved in protein‐protein interactions, consistent with a possible role for bacterial lipoproteins. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40mm in shell length) and small (3-15mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.     

Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in immunity, inflammation, angiogenesis, and cancer. S1P lyase (SPL) is the essential enzyme responsible for S1P degradation. SPL augments apoptosis and is down-regulated in cancer. SPL generates a S1P chemical gradient that promotes lymphocyte trafficking and as such is being targeted to treat autoimmune diseases. Despite growing interest in SPL as a disease marker, antioncogene, and pharmacological target, no comprehensive characterization of SPL expression in mammalian tissues has been reported. We investigated SPL expression in developing and adult mouse tissues by generating and characterizing a β-galactosidase-SPL reporter mouse combined with immunohistochemistry, immunoblotting, and enzyme assays. SPL was expressed in thymic and splenic stromal cells, splenocytes, Peyer's Patches, colonic lymphoid aggregates, circulating T and B lymphocytes, granulocytes, and monocytes, with lowest expression in thymocytes. SPL was highly expressed within the CNS, including arachnoid lining cells, spinal cord, choroid plexus, trigeminal nerve ganglion, and specific neurons of the olfactory bulb, cerebral cortex, midbrain, hindbrain, and cerebellum. Expression was detected in brown adipose tissue, female gonads, adrenal cortex, bladder epithelium, Harderian and preputial glands, and hair follicles. This unique expression pattern suggests SPL has many undiscovered physiological functions apart from its role in immunity.     

Monozygotic twins concordant for Kleine-Levin syndrome

ABSTRACT: BACKGROUND: Kleine-Levin syndrome is a rare idiopathic form of episodic hypersomnia that typically occurs during adolescence. The cardinal clinical features are recurrent hypersomnia, accompanied by cognitive disturbances and behavioral abnormalities [1]. The most typical form of classical Kleine-Levin syndrome is associated with hyperphagia [2, 3], although hyperphagia is now optional after change of the criteria. Hypersexuality, behavioral disinhibition, delusions, autonomic alteration and hallucinations have also been described, but the patients show normal cognitive function and behavior between attacks. The pathogenesis of Kleine-Levin syndrome is not yet known. Although most cases of recurrent hypersomnia are sporadic, the occurrence of nine familial cases indicate that there may be a genetic predisposition to the syndrome [4-8] However, no cases of twins affected with Kleine-Levin syndrome have been reported [9]. In this case study we describe monozygotic twins suffering from the syndrome. This is the first case report describing twins affected with Kleine-Levin syndrome thereby supporting the theory that there is an underlying genetic predisposition to the syndrome.     

Structural and functional characterization of <Emphasis Type="Italic">IbMYB1</Emphasis> genes in recent Japanese purple-fleshed sweetpotato cultivars

To elucidate further the genetic mechanism underlying anthocyanin accumulation in the storage roots of recent Japanese purple-fleshed sweetpotato cultivars, we compared the structure of the IbMYB1 gene in cultivar Ayamurasaki and its spontaneous mutant, AYM96, whose storage roots do not accumulate anthocyanin. Amplification of the IbMYB1 genomic fragment covering the coding sequences suggested that the genome of Ayamurasaki contained three types of IbMYB1 sequences, named IbMYB1-1, IbMYB1-2a and IbMYB1-2b, whereas AYM96 had only IbMYB1-1. Although these three IbMYB1 sequences had identical coding sequences, IbMYB1-1 had a 7-bp insertion in the first intron. IbMYB1-2a and IbMYB1-2b were characterized by a single nucleotide polymorphism in the second intron. Further cloning and sequencing of the flanking regions of these IbMYB1 sequences showed that the promoter and 3′ flanking regions of IbMYB1-2a and IbMYB1-2b were different from those of IbMYB1-1. Genetic analysis using an F₁ population derived from a cross between the purple-fleshed cultivar Murasakimasari and AYM96 suggested that IbMYB1-2 sequences are responsible for anthocyanin accumulation in the storage roots. The structural features of these three IbMYB1 sequences and identification of the IbMYB1-2null sequence, which contained sequences very similar to those of the flanking regions of IbMYB1-2a and IbMYB1-2b, but which lacked the sequence around the coding region, suggested that IbMYB1 genes in recent Japanese purple-fleshed cultivars had been established through multiple gene-duplication events.     

Entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved S protein as revealed by pseudotype virus bearing cleaved S protein

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is known to take an endosomal pathway for cell entry; however, it is thought to enter directly from the cell surface when a receptor-bound virion spike (S) protein is affected by trypsin, which induces cleavage of the S protein and activates its fusion potential. This suggests that SARS-CoV bearing a cleaved form of the S protein can enter cells directly from the cell surface without trypsin treatment. To explore this possibility, we introduced a furin-like cleavage sequence in the S protein at amino acids 798 to 801 and found that the mutated S protein was cleaved and induced cell fusion without trypsin treatment when expressed on the cell surface. Furthermore, a pseudotype virus bearing a cleaved S protein was revealed to infect cells in the presence of a lysosomotropic agent as well as a protease inhibitor, both of which are known to block SARS-CoV infection via an endosome, whereas the infection of pseudotypes with an uncleaved, wild-type S protein was blocked by these agents. A heptad repeat peptide, derived from a SARS-CoV S protein that is known to efficiently block infections from the cell surface, blocked the infection by a pseudotype with a cleaved S protein but not that with an uncleaved S protein. Those results indicate that SARS-CoV with a cleaved S protein is able to enter cells directly from the cell surface and agree with the previous observation of the protease-mediated cell surface entry of SARS-CoV.     

Determination of the number of active GroES subunits in the fused heptamer GroES required for interactions with GroEL

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.     

Design, synthesis and characterization of podocarpate derivatives as openers of BK channels

We found that the podocarpic acid structure provides a new scaffold for chemical modulators of large-conductance calcium-activated K(+) channels (BK channels). Structure-activity analysis indicates the importance of both the arrangement (i.e., location and orientation) of the carboxylic acid functionality of ring A and the hydrophobic region of ring C for expression of BK channel-opening activity.