THE DIRECT MEASUREMENT OF THE AXOPLASMIC TRANSPORT OF INDIVIDUAL RNA SPECIES: TRANSFER BUT NOT RIBOSOMAL RNA IS TRANSPORTED

Analysis of chick retinal and tectal RNA revealed that in addition to the major cytoplasmic RNAs (rRNA and tRNA), a number of the small mol wt nuclear RNAs (snRNAs) can also be detected. Subfractionation data indicated that one of these molecules, DD′, is of at least 95% nuclear location within the retina. Thus, very little, if any, of the retinal DD′ is available for axoplasmic transport from the retina into the optic nerve and tectum. Following intraocular injection of [³H]uridine, considerable incorporation of isotope into DD′ was observed within the optic tectum after 4, 8 and 16 days. This result indicates the presence of considerable local (i.e. tectal) synthesis. The specific activities of 29S, 18S and 5S rRNA and 4s tRNA relative to that of DD′ were measured in the optic tectum 8 and 16 days after the intraocular introduction of [³H]uridine. The same measurements were also made in intracranially injected animals. While the 29S/DD′, 18S/DD′ and 5S/DD′ specific activity ratios obtained were independent of the injection route, the 4S/DD′ ratio obtained from intraocularly injected animals was significantly greater (at least 2-fold) than that obtained from intracranially injected animals. Similar analysis was also performed with the optic nerve complex at 16 days post-injection with identical results. These results demonstrate that tRNA, but not rRNA, is transported from the retina into the optic nerve and tectum in the 2-day-old chicken.     

Neutralization of native human gamma interferon (HuIFN gamma) by antibodies to a synthetic peptide encoded by the 5' end of HuIFN gamma cDNA

A synthetic peptide corresponding to the N-terminal amino acid sequence of human gamma-interferon (HuIFN gamma), based on the cDNA sequence, was used to produce antibodies in rabbits that were reactive with native HuIFN gamma. Antibodies from all immunized rabbits neutralized the antiviral activity of HuIFN gamma. Significant neutralization of other HuIFN and mouse IFN was not observed. The peptide had the sequence Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Lys-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala ,and was coupled to keyhole limpet hemocyanin by disulfide linkage with the use of cystamine. The specificity of the antibodies produced to the peptide was compared to that of antibodies produced to native HuIFN gamma by neutralization of HuIFN gamma and by reactivity with peptide in the enzyme-linked immunosorbent assay (ELISA). The ratio of anti-peptide antibody neutralization of HuIFN gamma vs reactivity with peptide in the ELISA was at least 28-fold lower than for anti-HuIFN gamma antibody. Thus the antibodies to peptide and to HuIFN gamma were directed primarily against different determinants on native HuIFN gamma or the anti-HuIFN gamma antiserum probably contained antibodies to additional determinants. The anti-peptide antibodies should be useful for further characterization and purification of HuIFN gamma.     

Nonsensitized lymphocytes produce leukocyte interferon when cultured with foreign cells.

Nonsensitized human lymphocytes produce interferon when cultured with either transformed or normal heterologous cells. De novo RNA and protein synthesis was not required in a foreign cell for it to act as the inducer. The interferon produced has been characterized as being leukocyte type rather than immune or fibroblast type.     

Effects of induction of parturition in ewes with dexamethasone or oestrogen on concentrations of immunoglobulins in colostrum, and absorption of immunoglobulins by lambs

Immunoglobulins were measured in colostral whey from untreated ewes or ewes treated on days 142-145 of gestation with 15 mg oestradiol benzoate (ODB) or 10, 17.5 or 25 mg dexamethasone m-sulfobenzoate (DEX) to induce parturition. DEX at all levels reduced the concentrations of immunoglobulins in colostrum, but ODB had no effect. In a second experiment, similar treatments of ODB and DEX were applied on day 143 of gestation. Lambs were removed from their dams at birth and fed a standard volume of pooled colostrum taken from untreated ewes. They were injected with an antigen (Brucella abortus) at 12 h and 4 weeks of age. Concentrations of immunoglobulins in blood plasma at 24 h after birth of lambs from DEX-treated ewes were lower than for lambs from untreated ewes or from those treated with ODB. Response to the antigen, monitored to 12 weeks of age, was unaffected by either drug. The impairment of transfer of immunoglobulins from ewe to lamb offers a possible explanation for the higher mortality among older lambs found previously after treatment of ewes with DEX.     

Analysis of Serum Inflammatory Mediators Identifies Unique Dynamic Networks Associated with Death and Spontaneous Survival in Pediatric Acute Liver Failure

Background

Tools to predict death or spontaneous survival are necessary to inform liver transplantation (LTx) decisions in pediatric acute liver failure (PALF), but such tools are not available. Recent data suggest that immune/inflammatory dysregulation occurs in the setting of acute liver failure. We hypothesized that specific, dynamic, and measurable patterns of immune/inflammatory dysregulation will correlate with outcomes in PALF.

Methods

We assayed 26 inflammatory mediators on stored serum samples obtained from a convenience sample of 49 children in the PALF study group (PALFSG) collected within 7 days after enrollment. Outcomes were assessed within 21 days of enrollment consisting of spontaneous survivors, non-survivors, and LTx recipients. Data were subjected to statistical analysis, patient-specific Principal Component Analysis (PCA), and Dynamic Bayesian Network (DBN) inference.

Findings

Raw inflammatory mediator levels assessed over time did not distinguish among PALF outcomes. However, DBN analysis did reveal distinct interferon-gamma-related networks that distinguished spontaneous survivors from those who died. The network identified in LTx patients pre-transplant was more like that seen in spontaneous survivors than in those who died, a finding supported by PCA.

Interpretation

The application of DBN analysis of inflammatory mediators in this small patient sample appears to differentiate survivors from non-survivors in PALF. Patterns associated with LTx pre-transplant were more like those seen in spontaneous survivors than in those who died. DBN-based analyses might lead to a better prediction of outcome in PALF, and could also have more general utility in other complex diseases with an inflammatory etiology.     

Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Haemophilus parasuis is the causative agent of Glässer''s disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3′ end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer''s disease.     

Fatty acyl benzamido antibacterials based on inhibition of DnaK-catalyzed protein folding

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. N(alpha)-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 +/- 20 microg/ml. The compounds were shown to compete with fluorophore-labeled sigma(32)-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.     

The role of GRAS proteins in plant signal transduction and development

GRAS proteins are a recently discovered family of plant-specific proteins named after GAI, RGA and SCR, the first three of its members isolated. Although the Arabidopsis genome encodes at least 33 GRAS protein family members only a few GRAS proteins have been characterized so far. However, it is becoming clear that GRAS proteins exert important roles in very diverse processes such as signal transduction, meristem maintenance and development. Here we present a survey of the different GRAS proteins and review the current knowledge of the function of individual members of this protein family.