Tumor necrosis factor alpha regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kappa B-like factor to a promoter element.

Expression of the major histocompatibility complex (MHC) class I and class II antigens and the class II-associated invariant chain (Ii) is strongly increased by treatment of cells with tumor necrosis factor alpha (TNF-alpha) and gamma interferon. We investigated elevation of expression of the invariant chain gene by TNF-alpha. Rat fibroblast cells transfected with the mouse Ii gene containing 802 base pairs of 5' sequences could be stimulated for Ii expression by treatment with TNF-alpha. Analysis of 5'-deleted Ii gene promoter-CAT constructs provided evidence for the presence of a TNF-alpha response box (TRB). Cloning of TRB in front of a non-TNF-alpha-responsive promoter could transfer the TNF-alpha stimulatory effect. We demonstrate binding of a TNF-alpha-induced factor to a kappa B-like motif within TRB. Mutations introduced into the kappa B element of the Ii promoter-CAT plasmid abolished the TNF-alpha-mediated stimulatory effect. Comparison of the TNF-alpha-induced factor and lipopolysaccharide-induced NF-kappa B in gel mobility shift assays upon partial protease digestion suggests similar DNA-binding protein cores. Further support for the NF-kappa B-like nature of the TNF-alpha-induced factor was obtained in methylation interference assays. The TNF-alpha-induced nuclear factor comprises DNA contact sites that are identical to those described for NF-kappa B. This TNF-alpha-induced factor also interacts with kappa B-like sequences of the MHC Kb, Ek alpha, and beta 2-microglobulin promoter, suggesting a common TNF-alpha-mediated regulatory signal for expression of MHC antigens and Ii.     

Identifiability of DNA distribution from flow cytometric data with cell debris

In flow cytometric measurement of cell DNA distribution one of the major problems is accounting for the effect of fragmentation in the staining process. This work considers a recent probabilistic model that has been proposed for the fragmentation process and species under which conditions it is possible to uniquely identify the DNA distributions of the original population using flow cytometric data. Attention is given both to the normal and to the polyploid case. This work was partially supported by a grant of the Italian National Research Council, Special Project “Oncology”, contract number 84.00632.44.     

Distribution, quantitation, and origin of immunoreactive neuropeptide Y in the human gastrointestinal tract

A radioimmunoassay for measurement of immunoreactive neuropeptide Y has been developed using antiserum from a rabbit (221) immunized with porcine neuropeptide Y. Antibody 221 has been characterized for both sensitivity and specificity. To determine the distribution of neuropeptide Y in the human gastrointestinal tract, fresh tissue specimens were separated by microdissection into the muscularis externa and the mucosa-submucosa. To examine the origin of neuropeptide Y in human colon, specimens of aganglionic and ganglionic colon were obtained from patients with Hirschsprung's disease. Immunoreactive neuropeptide Y in human gut was present in highest concentrations in the muscularis externa of the stomach and in lowest concentrations in the muscularis externa of the ileum and descending colon. Neuropeptide Y in the stomach was present in higher concentrations in the muscularis externa than in the mucosa-submucosa, but in the descending colon there were lower concentrations of neuropeptide Y in the muscularis externa than in the mucosa-submucosa. In Hirschsprung's disease, concentrations of neuropeptide Y were increased in aganglionic colon in both the muscularis externa and the mucosa-submucosa, compared to corresponding layers from proximal ganglionic colon. Extracts of the gastric muscularis externa and the colonic mucosa-submucosa were separated by C18 reverse-phase high-performance liquid chromatography. One major immunoreactive species was identified by radioimmunoassay which eluted in a position similar to synthetic human neuropeptide Y. These results demonstrated both regional and layer differences in concentrations of neuropeptide Y in human gut. Increased concentrations of neuropeptide Y in aganglionic colon from Hirschsprung's disease most likely result from enlargement of neuropeptide Y-containing extrinsic nerve fibers in both the mucosa-submucosa and the muscularis externa.     

Partition of autolysins between the medium, the internal part of the wall, and the surface of the wall of gram-positive rods

Autolysins are exoenzymes formed by bacteria to aid in wall expansion and in cell division. For the Gram-positive rod, these enzymes function predominately on the periphery of the wall. As they destroy their substrate they rebind to peptidoglycan when the cell density is high. Only then is the concentration of autolysins high enough on the external surface to favor cell division and prevent the formation of chains of cells (filaments). It is suggesting that circumstance is important for the kinetics of turnover of the wall and the splitting of the septum.     

The reproducibility of resting and post exercise plasma beta-endorphins

This investigation examined the reproducibility of resting and post exercise plasma beta-endorphin levels. Twenty subjects (10 men and 10 women) had their resting endorphin levels measured under controlled conditions on four separate occasions. Concomitantly, the endorphin response of eight trained runners completing three similar ten mile runs was also determined. For the resting data, there was no significant overall variation among trials, but the intra-subject variability was substantial; the within subject variance was 6.16, and it corresponded to an intra-class reliability coefficient of r = 0.239. No gender effect was noted for the average beta-endorphin values for the four occasions (men = 4.6 +/- 1.7; women = 4.4 +/- 2.1 pM/l); however, the males' within-subject variance of 8.548 (r = 0.080) was significantly larger than that of 3.719 (r = 0.485) for females. Of the runners, one outlier subject had a uniquely high average beta endorphin level of 85.67. Analysis including and excluding the outlier subject yielded within-subject variances of 29.61 (r = 0.960) and 34.47 (r = 0.176), respectively; variances for differences in confidence limits for random variation, they must exceed 7 pM/l at rest, 17 pM/l post exercise, and 20 pM/L difference from rest to post exercise.     

Fast one-step procedure for the detection of nucleic acids in situ by primer-induced sequence-specific labeling with fluorescein-12-dUTP.

We provide fast, simple, one-step procedures for sequence-specific detection of nucleic acids in situ. Tandem repeat sequences in DNA are stained within 30 min, and mRNA is stained within 2 h. The procedures are based on the incorporation of the newly available fluorescein-labeled dUTP into DNA synthesized in situ by primed in situ labeling, with denatured fragments of cloned DNA or oligonucleotides as primers. The extreme speed and simplicity of the reaction make it attractive for automatization in routine laboratory procedures and opens up new diagnostic possibilities.     

A portable EAG system for the measurement of pheromone concentrations in the field

A robust and portable apparatus for the measurement of pheromoneconcentrations under field conditions has been developed. Ituses the insect antenna (Lobesia botrana Hb.) itself as a sensitiveand specific pheromone detector. Shock-proof contact with theelectrodes is maintained by fixing the antenna in a specially-shapedplexiglass holder mounted within a glass tube. This allows measurementsto be made while moving the apparatus. Continuous airflow throughthe tube is generated by a suction pump and the incoming aircan be purified by passage through a charcoal filter. This allowsto readjust the offset and to calibrate the instrument by theapplication of pheromone pulses of known concentrations. Removalof the filter allows the direct access of ambient air over theantenna which responds by generating an electro-antennogram(EAG) as a measure of the pheromone concentration. Using the calibration curve, relative pheromone concentrationsin ambient air in a vineyard can be determined. Sample measurementsfrom areas treated with artificial pheromone for pest controlare presented.     

Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle.

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.