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Earlier experimental data in our laboratory showed that introduction of an exogenous protein into early chicken embryonic blood leads to immunotolerance of hatched chicken to that protein. However, the underlying mechanism is yet unknown. In the present study, we show that the blood cells collecting circulating antigen might contribute to the establishment of immunotolerance. In this experiment, most of the chicken embryo blood cells took up injected fluorescein isothiocyanate-BSA at approximately embryonic d 3. At the same stage, 1 microL of embryo blood was taken out and incubated with BSA. After being loaded with BSA in vitro and washed, these cells were injected back into the original embryo. The BSA-specific lymphocytes were depleted in chickens whose early embryo cells had been loaded with BSA, as evidenced by a significant decrease in anti-BSA antibody after challenge with BSA when the chickens were 3 wk old. In addition, by direct injection of BSA to embryonic d 3 embryo blood, the hatched chickens had decreased amounts of anti-trinitrophenol antibody after the chickens were challenged with trinitrophenol-BSA, indicating that the helper function of BSA-specific T cells was impaired. In conclusion, these observations suggest that some early embryo blood cells possibly collect and store antigen for the establishment of self-tolerance before the maturation of B and T cells.

Site-directed mutagenesis was used to evaluate the effects on structure and function of selected substitutions within and N-terminal to the core "alpha-crystallin" domain of the small heat-shock protein (sHsp) and molecular chaperone, human alphaB-crystallin. Five alphaB-crystallin mutants containing single amino acid substitutions within the core alpha-crystallin domain displayed a modest decrease in chaperone activity in aggregation assays in vitro and in protecting cell viability of E. coli at 50 degrees C in vivo. In contrast, seven alphaB-crystallin mutants containing substitutions N-terminal to the core alpha-crystallin domain generally resembled wild-type alphaB-crystallin in chaperone activity in vitro and in vivo. Size-exclusion chromatography, ultraviolet circular dichroism spectroscopy and limited proteolysis were used to evaluate potential structural changes in the 12 alphaB-crystallin mutants. The secondary, tertiary and quaternary structures of mutants within and N-terminal to the core alpha-crystallin domain were similar to wild-type alphaB-crystallin. SDS-PAGE patterns of chymotryptic digestion were also similar in the mutant and wild-type proteins, indicating that the mutations did not introduce structural modifications that altered the exposure of proteolytic cleavage sites in alphaB-crystallin. On the basis of the similarities between the sequences of human alphaB-crystallin and the sHsp Mj HSP16.5, the only sHsp for which there exists high resolution structural information, a three-dimensional model for alphaB-crystallin was constructed. The mutations at sites within the core alpha-crystallin domain of alphaB-crystallin identify regions that may be important for the molecular chaperone functions of sHsps., (Copyright 1999 Academic Press.)

To know the changes in plasma endothelin of patients with pulmonary hypertension, we studied 32 patients with valvular heart disease. Among them, 22 patients had pulmonary hypertension (group I) and 10 had pulmonary arterial pressures in the normal range (group II). Plasma endothelin-1 concentrations of the patients in group I were significantly greater than those of the patients in group II (p < 0.05). No significant difference in plasma endothelin-3 concentrations existed between the two groups. Cardiac output and pulmonary capillary wedge pressure had a linear correlation with plasma endothelin-1 levels. There was also a significant correlation between plasma endothelin-1 levels and hemodynamic indicators of severity of pulmonary hypertension, such as mean pulmonary arterial pressure and pulmonary vascular resistance (p < 0.05). All patients in this study underwent surgical procedures for the correction of valvular lesions. All patients in group I showed a decrease in pulmonary arterial pressures, and their plasma endothelin-1 levels decreased from 3.84 +/- 0.20 pg/mL to 1.66 +/- 0.07 pg/mL (p < 0.05), whereas the plasma endothelin-3 levels had only slight variation from 0.64 +/- 0.11 pg/mL to 0.75 +/- 0.06 pg/mL (p > 0.05) between the preoperative and the postoperative stages. The results demonstrated that plasma endothelin-1 rather than endothelin-3 had a role in pulmonary hypertension. Several pieces of evidence pointed out that endothelin-1 functioned as a reactive mediator during vasoconstriction in the case of pulmonary hypertension rather than as a triggering factor of pulmonary hypertension.

We constructed mutants with a deletion of either half of the 18 base-pair B block palindrome in the VARNA1 gene, mutants with different intra-palindromic spacings, a complete set of mutants with single base substitutions, and mutants with double and triple base substitutions in the palindrome. The transcription efficiencies of these mutants were determined in human KB cell-free cytoplasmic S100 extracts. The relative competing strength of each mutant, as determined by a sequential competition experiment, was used to assess each mutant's ability to sequester factors into formation of a stable preinitiation complex. The ability of each mutant to assemble transcriptionally active preinitiation complexes was also determined by direct transcription of the isolated complexes. Finally, the ability of each mutant to interact with the transcription factor(s) TFIIIC and form a distinct gel-resolved complex was also determined. From the results of the above assays, we concluded that the two seemingly identical halves of the palindrome did not contribute equally to transcription, or to assembly of the functional preinitiation complex, nor to interaction with TFIIIC. The anterior half (B1) of the B block palindrome, which is proximal to the A block promoter element, played a stronger role in transcription and in assembly of the functional preinitiation complex than the posterior half (B2) of the palindrome. Consistent with this observation, the point mutations in four base-pairs, GTTC, from +60 to +63 in the anterior half of the B block palindrome, has the most severe effect on transcription. In contrast, we showed that the central sequence and the posterior half (B2) played a stronger role than the anterior half (B1) of the B block palindrome in the interaction of the promoter with TFIIIC. This was corroborated by the observation that base substitutions in the central four base-pair sequence of the palindrome, TCGA, from +62 to +65, had the most severe effect on interaction with TFIIIC, and that mutations in most of the sequences in the posterior half of the B block palindrome had more drastic effects than mutations in the anterior half of the palindrome in this interaction. Furthermore, the spacing between the two halves of the B block palindrome had a drastic effect on the overall transcription efficiency and the interaction of the promoter with TFIIIC, suggesting that the interaction between the two halves of the B block palindrome is not only essential, but also synergistic for the interaction with TFIIIC as well as the assembly of a transcriptionally active preinitiation complex and efficient transcription.

Four or more consecutive thymidine residues on the non-template strand and G + C-richness of flanking DNA are the two necessary characteristics of efficient RNA polymerase-III-dependent transcriptional terminators. We have identified, from the study of in vitro transcription of a human Alu family repeat, a new type of RNA polymerase-III-dependent transcriptional terminator. A 258 base-pair Alu repeat located on the 3' side of the human alpha 1 globin gene can be transcribed in a HeLa S-100 extract to generate three RNA species of lengths 404 to 408, 252 to 255 and 173 to 174 nucleotides, respectively. Kinetics, pulse-chase and RNA incubation experiments showed no significant internal processing of the longer transcripts into shorter ones. These data plus detailed RNA mapping demonstrated conclusively that the multiple Alu RNA species resulted from accurate initiation at the first base (5' end) of the repeat, and multiple termination downstream. The 3' end(s) of the major transcript (252 to 255 nucleotides) maps at the 3' end of the Alu repeat sequence where there are not four or more consecutive thymidine residues on the non-template strand. The functional domain of the terminator has been mapped to a 45 base-pair segment that includes 36 base-pairs of the 3' end sequence of the Alu repeat plus nine base-pairs downstream. The high efficiency of termination (greater than 90%), the lack of consecutive T residues, the richness in A + T content, and the proposed ability of the RNA to form an imperfect hairpin structure in the 3' region of the transcript, thus identify a new type of eukaryotic class III terminator. We compare the structure of this class III terminator with that of the bacterial rho-dependent terminator. We also discuss its implication in the mechanism(s) of amplification and dispersion of Alu sequences in the primate genomes.

The outer boundaries of the internal transcriptional control region in the VARNA1 gene have been located from positions +10 to +69. To further define the detailed organization of the functional domains in this region and the function(s) of the 5' flanking sequence, and to obtain a more detailed insight into other transcriptionally important sequences, we have constructed 77 mutants with deletion endpoints at almost every one to five base-pairs in the entire region from -30 to +160 for transcriptional studies. Using our highly active crude extract under our assay conditions, and quantitatively measuring the transcriptional efficiency and competing strength of each mutant, we have revealed new features of important transcriptional control sequences and defined the transcriptional functions of several functional domains in this gene. The essential domain is from +59/+63 to +66/+68, which corresponds to the B block sequence. This is smaller than that defined previously. The second most important domain is the region from +12/14 to +40, which includes the A block sequence that dictates the wild-type major start site and amplifies the events started by the B block region, mediated through factors and RNA polymerase III. Furthermore, the domain from -5 to +11 affects the use of certain start site(s). Moreover, the 5' flanking region from -30 to +1 contributes 80 to 90% of the overall transcriptional efficiency of the gene. Finally, our transcriptional studies of mutants deleted of the A block sequence and all of the upstream sequence indicated that an intimate interaction between the two blocks is essential for initiation of transcription. Furthermore, the B block sequence is more important than the A block sequence in the transcription reaction. The mechanism and control of transcriptional initiation in the VARNA1 gene is similar to that in some tRNA genes, but differs from that in others.