Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo

Pathogens or pathogen-associated molecular patterns can signal to cells of the innate immune system and trigger effective adaptive immunity. However, relatively little is known about how the innate immune system detects tissue injury or necrosis. Evidence suggests that the release of heat-shock proteins (HSPs) may provide adjuvant-like signals, but the ability of HSPs to promote activation or tolerance in vivo has not been addressed. In this study we show that Hsp70 promotes dendritic cell (DC) function and, together with antigen, triggers autoimmune disease in vivo.     

Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.     

A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons

We tested the contribution of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation in cultured cerebellar granule neurons. p160ROCK inhibition, presumably by reducing the stability of the cortical actin network, triggered immediate outgrowth of membrane ruffles and filopodia, followed by the generation of initial growth cone-ike membrane domains from which axonal processes arose. Furthermore, a potentiation in both the size and the motility of growth cones was evident, though the overall axon elongation rate remained stable. Conversely, overexpression of dominant active forms of Rho or ROCK was suggested to prevent initiation of axon outgrowth. Taken together, our data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics.     

Deletion of the yhhP gene results in filamentous cell morphology in Escherichia coli

The Escherichia coli yhhP gene was predicted to encode a small hypothetical protein of 81 amino acids, the cellular function of which is not known. To gain insight into the function of this uncharacterized YhhP protein, genetic and biochemical studies were done. We first tried to express and purify the YhhP protein to prepare an anti-YhhP antiserum. Western blotting showed that the hypothetical yhhP gene is indeed transcribed and translated as a minor cytoplasmic protein. YhhP-deficient (delta yhhP) cells formed colonies poorly on a rich medium (e.g., Luria-Bertani medium) containing a relatively low concentration of NaCl, while they can grow normally either in LB containing 3% NaCl or in a synthetic medium (e.g., M9-glucose). During exponential growth in rich medium, an early step of cell division was inhibited in delta yhhP cells, forming filaments. For the YhhP-deficient filamentous cells, the FtsZ-ring formation was analyzed with immunofluorescence microscopy. The FtsZ-ring formation did not occur normally in the delta yhhP filaments, although the filamentous cells contained the FtsZ protein at a certain level comparable to that in the wild-type cells. The ftsZ gene was found to function as a multicopy suppressor of the delta yhhP mutant. Another multicopy suppressor gene was identified as the dksA gene. Provided that either the ftsZ or dksA gene was introduced into the mutant cells with its multicopy state, the resulting transformants were capable of growing in rich medium, formed wild-type short rods. These results are discussed with regard to the presumed function of this ubiquitous protein.     

Radical-capturing reaction of 5,7,3',4'-tetramethylquercetin with the AIBN radical initiator

In order to clarify the mechanism for the radical-capturing reaction which is initiated at the C3-hydroxyl group of flavonols, 5,7,3',4'-tetramethylquercetin (TMQ) was reacted with the 2,2'-azobis-isobutyronitrile (AIBN) radical initiator in benzene. Six products, one depside and its two hydrolytic products, one nitrile adduct, and two others, were isolated from the reaction mixture, and their structures were determined by instrumental analyses. The quantitative change to the four main products against the reaction time was measured by an HPLC method. The radical-capturing reaction pathway for TMQ with AIBN is proposed from these products and their quantitative changes. The pathway dividing into two clearly reveals that one subpath formed the depside and its hydrolytic products, while the other formed the nitrile adduct. The reactivity of each two sub-path was nearly the same, different from the case of TMQ and the 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) radical initiator.     

Identification of a gene disrupted by inv(11)(q13.5;q25) in a patient with left-right axis malformation

An inv(11)(q13.5;q25) inversion was previously identified in a 9-month-old male patient with complex cyanotic heart defects, altered lung lobation, symmetric liver, and abnormally lobulated spleen (polysplenia). This chromosomal rearrangement was inherited from the phenotypically normal father. We termed these regions DHTX-A (disrupted in heterotaxy)-- A at 11q13.5 and DHTX-B at 11q25. Here, we report the isolation and characterization of the inversion breakpoints and the gene that is disrupted by the DHTX-A breakpoint. The putative DHTX is identical to the UVRAG gene, which was originally identified as a gene that complements the UV sensitivity of xeroderma pigmentosum complementation group C. The 4-kb mRNA was found to be encoded by a large gene, at least 300 kb long, composed of 15 exons. The function of the gene product remains largely unknown. However, the near central portion of the UVRAG protein is predicted to contain a coiled-coil domain, which has been implicated in mediating protein-protein interactions. Southern analyses and fluorescence in situ hybridization (FISH) revealed that the DHTX-A breakpoint in the patient and his father lies within the intron between exons 6 and 7 of UVRAG. Northern blot analysis indicated strong expression in human fetal and adult tissues and in mouse embryonic day-7 and adult tissues, respectively. Whole mount in situ hybridization also showed that the Uvrag gene is expressed in the presomite-stage embryo. Several hypotheses are discussed to explain the relationship between the chromosomal inversion and the accompanying phenotypes.     

Temporal contribution of body movement to very long-term heart rate variability in humans

A newly developed, very long-term ( approximately 7 days) ambulatory monitoring system for assessing beat-to-beat heart rate variability (HRV) and body movements (BM) was used to study the mechanism(s) responsible for the long-period oscillation in human HRV. Data continuously collected from five healthy subjects were analyzed by 1) standard auto- and cross-spectral techniques, 2) a cross-Wigner distribution (WD; a time-frequency analysis) between BM and HRV for 10-s averaged data, and 3) coarse-graining spectral analysis for 600 successive cardiac cycles. The results showed 1) a clear circadian rhythm in HRV and BM, 2) a 1/f (beta)-type spectrum in HRV and BM at ultradian frequencies, and 3) coherent relationships between BM and HRV only at specific ultradian as well as circadian frequencies, indicated by significant (P < 0.05) levels of the squared coherence and temporal localizations of the covariance between BM and HRV in the cross-WD. In a single subject, an instance in which the behavioral (mean BM) and autonomic [HRV power >0.15 Hz and mean heart rate (HR)] rhythmicities were dissociated occurred when the individual had an irregular daily life. It was concluded that the long-term HRV in normal humans contained persistent oscillations synchronized with those of BM at ultradian frequencies but could not be explained exclusively by activity levels of the subjects.