A novel signaling pathway controlling induced systemic resistance in Arabidopsis

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P. syringae pv tomato. In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation. Using the jasmonate response mutant jar1, the ethylene response mutant etr1, and the SAR regulatory mutant npr1, we demonstrate that signal transduction leading to P. fluorescens WCS417r-mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1. Similar to P. fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P. s. tomato in salicylic acid-nonaccumulating NahG plants. Moreover, methyl jasmonate-induced protection was blocked in jar1, etr1, and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate-induced protection was affected in etr1 and npr1 plants but not in jar1 plants. Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1. We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.     

Anaphylactic shock and its implication for nurses

We report the case of a voluminous melanotic neuroectodermic tumor, also termed melanotic progonoma, which developed in a 5 month old infant, recalling the main aspects of such uncommon non-odontogenic tumors with characteristic histological features. The large size of the tumor can hinder feeding in infants. Surgical cure is generally easy and outcome quite satisfactory without recurrence.     

Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation

The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.     

Activation of extracellular matrix metalloproteinases in equine laminitis

Samples of connective tissue obtained from the hoof of six laminitic and eight non-laminitic adult horses were analysed zymographically to investigate whether connective tissue matrix metalloproteinases are activated or induced during laminitis. The activity or matrix metalloproteinases was substantially greater in the tissues from the laminitic horses than in the tissues from the non-laminitic horses. A comparison of the collagenolytic activity in the laminitic and control tissues showed that collagenolytic activities corresponding to the 92 kDa (P < 0.001), 72 kDa (P < 0.01) and 66 kDa (P < 0.01) bands were induced in the laminitic tissues.     

Neuroleptic use and behavioral disturbance in nursing homes: a 1-year study

This article discusses a longitudinal study of change in disruptive behaviors among nursing home residents treated with neuroleptics compared with those not treated with neuroleptics. Observations were made of 201 participants on admission to and after 1 year in eight skilled nursing facilities. Nine disruptive behaviors were measured using the Psychogeriatric Dependency Rating Scale with nursing assistants. Neuroleptic use was documented from medication records. Odds ratios are reported for the association of behavior at baseline and use of neuroleptics on nine problem behaviors. For those who received neuroleptics during the year, there was greater change in both developing and resolving disruptive behaviors than for those not receiving neuroleptics. For both groups, restless or pacing behavior and belligerent behavior manifested by refusing instructions changed the most, both in developing and in apparently resolving. Our results show that change in disruptive behaviors occurs among nursing home residents regardless of neuroleptic use, but it occurs more frequently among those who receive neuroleptic medication. Knowledge of which disruptive behaviors are most likely to resolve or develop is important in training nursing home staff to cope with the behaviors as well as in planning interventions that may modify such behaviors.     

Beta 2-adrenergic receptor and angiotensin II receptor modulation of sympathetic neurotransmission in human atria

BACKGROUND: Cultures of epidermal cells are commonly used to study skin biology and differentiation. Recently a method to culture nail matrix cells has been established. OBJECTIVE: We report the biologic characteristics of nail matrix cells in vitro compared with those of epidermal keratinocytes. METHODS: Human nail matrix cells were isolated and cultured in defined medium. Electron-microscopic examination, growth rate, integrin expression and keratin synthesis pattern were evaluated. In addition, the cells were cultured in serum-containing medium. RESULTS: Nail matrix cells appear to be larger than human epidermal keratinocytes and, at the ultrastructural level, they contain a higher euchromatin/heterochromatin ratio and a lower nucleus/cytoplasm ratio and have a higher growth rate. The synthesis of "hard" keratins was detected at all calcium concentrations. Immunofluorescence analyses showed the expression of alpha 2, alpha 3, and alpha 6 integrin subunits. When cultured in serum-containing medium, nail matrix cells produced an outgrowth of epithelium and a spontaneous migration phenomenon associated with a tendency to stratify in a semilunar area that resembles the architecture of the nail matrix. The pluristratified epithelium showed characteristic markers of nail differentiation. CONCLUSION: Culture of nail matrix cells may represent a useful model to study the biologic properties of nail structure, alterations in some nail diseases and the effects of drugs.     

The whistle and the rattle: the design of sound producing muscles

Vertebrate sound producing muscles often operate at frequencies exceeding 100 Hz, making them the fastest vertebrate muscles. Like other vertebrate muscle, these sonic muscles are "synchronous," necessitating that calcium be released and resequestered by the sarcoplasmic reticulum during each contraction cycle. Thus to operate at such high frequencies, vertebrate sonic muscles require extreme adaptations. We have found that to generate the "boatwhistle" mating call (approximately 200 Hz), the swimbladder muscle fibers of toadfish have evolved (i) a large and very fast calcium transient, (ii) a fast crossbridge detachment rate, and (iii) probably a fast kinetic off-rate of Ca2+ from troponin. The fibers of the shaker muscle of rattlesnakes have independently evolved similar traits, permitting tail rattling at approximately 90 Hz.     

Effects of substitutions in the binding surface of an antibody on antigen affinity

The interactions between the Fab and single-chain Fv (scFv) fragments of an antibody (NC10) and its antigen, influenza virus neuraminidase, were analysed in the crystal structures of the Fab-neuraminidase and scFv-neuraminidase complexes. To investigate the contribution to binding made by cavities, salt links and hydrogen bonds in the antibody- antigen interface, 14 single amino acid replacements were made at six contact residues in the scFv fragment by site-directed mutagenesis. The binding affinity of each mutant scFv antibody for neuraminidase was determined with a BIAcore optical biosensor. Four of the mutations resulted in large changes in the free energy of binding to neuraminidase (deltadeltaG > 1 kcal/mol) and together may account for approximately 70% of the free energy of binding. Hence these data support the theory that a small number of residues form the 'functional epitope' and are most important for binding of NC10 to neuraminidase. The salt link between antibody residue (Asp)H56 and (Lys)N432 from neuraminidase was demonstrated to be important for affinity, since substitution of (Asp)H56 with Asn caused a large reduction in the free energy of binding (deltadeltaG = +2.8 kcal/mol). Hydrogen bonds provided by (Tyr)L32 and (Asp)H56 were also important for binding: mutation of (Tyr)L32 to Phe resulted in a significant reduction in binding affinity (deltadeltaG = +1.7 kcal/mol). Disruption of hydrophobic interactions (van der Waals contacts) led to significant reductions in affinity also ((Tyr)H99 to Ala, deltadeltaG = +1.5 kcal/mol; (Leu)L94 to Ala, deltadeltaG > +3.0 kcal/mol). An attempt to increase binding affinity by filling a cavity in the interface with a larger antibody side chain was unsuccessful, as the free energy gained by new antibody-antigen interactions did not compensate for the removal of cavity-bound water molecules.      

Expression of human aldehyde dehydrogenase-3 associated with hepatocellular carcinoma: promoter regions and nuclear protein factors related to the expression

Studies in our laboratory have shown that as early as day 8.5 of development, mouse yolk sac cells can generate T cells when placed in a thymic microenvironment. At this stage, yolk sac cells can also differentiate into myeloid cells in vitro. B cell differentiation in vitro was achieved with day 9 yolk sac by providing a bone marrow stromal feeder layer. We have now established endothelial cell lines and clones from yolk sacs of day 8-12 mouse embryos. These vary in their ability to support stem cell maintenance and differentiation. Our principal work has been carried out with day 12 cloned endothelial cell lines. One clone supported the > 100 fold expansion of yolk sac hematopoietic stem cells that subsequently could generate B cells, T cells and myeloid cells both in vitro and in vivo. Preliminary experiments with endothelial cells from younger embryos are also described.     

Molecular analysis of the third component of canine complement (C3) and identification of the mutation responsible for hereditary canine C3 deficiency

Genetically determined deficiency of the third component of complement (C3) in the dog is characterized by a predisposition to recurrent bacterial infections and to type 1 membranoproliferative glomerulonephritis. The current studies were undertaken to characterize the cDNA for wild-type canine C3 and identify the molecular basis for hereditary canine C3 deficiency. Amplification, cloning, and sequence analysis indicated that canine C3 is highly conserved in comparison with human, mouse, and guinea pig C3. Southern blot analysis failed to show any gross deletions or rearrangements of DNA from C3-deficient animals. Northern blot analysis indicated that the livers of these animals contain markedly reduced quantities of a normal length C3 mRNA. The full-length 5.1-kb canine C3 cDNA was amplified in overlapping PCR fragments. Sequence analysis of these fragments has shown a deletion of a cytosine at position 2136 (codon 712), leading to a frameshift that generates a stop codon 11 amino acids downstream. The deletion has been confirmed in genomic DNA, and its inheritance has been demonstrated by allele-specific oligonucleotide hybridization.