A cis/trans Test of the Effect of the First Enzyme for Histidine Biosynthesis on Regulation of the Histidine Operon

Previous studies showed that when triazolalanine was added to a derepressed culture of a histidine auxotroph, repression of the histidine operon occurred as though histidine had been added (6). However, when triazolalanine was added to a derepressed culture of a strain with a mutation in the first gene of the histidine operon which rendered the first enzyme for histidine biosynthesis resistant to inhibition by histidine, repression did not occur. The studies reported here represent a cis/trans test of this effect of mutations to feedback resistance. Using specially constructed merodiploid strains, we were able to show that the wild-type allele is dominant to the mutant (feedback resistant) allele and that the effect operates in trans. We conclude that the enzyme encoded by the first gene of the histidine operon exerts its regulatory effect on the operon not by acting locally at its site of synthesis, but by acting as a freely diffusible protein.     

Repression of the Histidine Operon: Effect of the First Enzyme on the Kinetics of Repression

Kinetic studies on repression of the enzymes for histidine biosynthesis in Salmonella typhimurium showed that, upon addition of histidine to a derepressed culture, the enzymes became repressed in a temporal sequence which corresponds with the positional sequence of the genes in the histidine operon. This serial pattern of repression occurred under conditions in which the feedback site of the first enzyme for histidine biosynthesis is intact. When this site was rendered nonfunctional the pattern of repression was changed so that all of the enzymes became repressed concomitantly. These results suggest that the first enzyme for histidine biosynthesis plays a hitherto unrecognized role in control of the histidine system.     

Preventive and therapeutic vaccination with PAP-3, a novel human prostate cancer peptide, inhibits carcinoma development in HLA transgenic mice

Conventional treatment of recurrent and metastasized prostate cancer (CaP) remains inadequate; this fact mandates development of alternative therapeutic modalities, such as specific active or passive immunotherapy. Previously, we reported the identification of a novel highly immunogenic HLA-A*0201-restricted Prostatic Acid Phosphatase-derived peptide (PAP-3) by a two-step in vivo screening in an HLA-transgenic (HHD) mouse system. In the present study we aimed at elucidating the efficiency of PAP-3-based vaccine upon active antitumor immunization. To this end we established preventive and therapeutic carcinoma models in HHD mice. The 3LL murine Lewis lung carcinoma clone D122 transduced to express HLA-A*0201 and PAP served as a platform for these models. The HLA-A*0201–PAP-3 complex specific recombinant single chain scFV-PAP-3 antibodies were generated and used to confirm an endogenous PAP processing resulting in PAP-3 presentation by HLA-A*0201. PAP-3 based vaccines significantly decreased tumor incidence in a preventive immunization setting. Therapeutic vaccination of HHD mice with PAP-3 led to rejection of early established tumors and to increase of mouse survival. These results strongly support a therapeutic relevance of the identified CTL epitope upon active antitumor immunization. The newly established carcinoma model presented herein might be a useful tool for cancer vaccine design and optimization.     

Invalidation of a commercially available human 5α-dihydrotestosterone immunoassay

Enzyme immunoassays (EIA) are commonly utilized for the evaluation of androgens in biological fluids; however, careful consideration must be given to cross-reactivity with other endogenous sex-steroid hormones. Our purpose was to determine the validity of a commonly-utilized commercially-available dihydrotestosterone (DHT) EIA. Serum samples obtained from older hypogonadal men who participated in a 12-month randomized controlled trial evaluating the effects of testosterone-enanthate (125 mg/week) or vehicle in combination with finasteride (5 mg/day) or placebo were assayed for DHT via EIA and using a validated gold-standard LC–MS/MS approach. Additionally, commercially-available (DHT-free) buffer containing graded testosterone doses was evaluated by DHT immunoassay. DHT concentrations measured via EIA were 79% to >1000% higher than values obtained by LC–MS/MS (p < 0.05), with the largest differences (415–1128%) occuring in groups receiving finasteride. Both LC–MS/MS and EIA indicated that testosterone-enanthate increased serum DHT to a similar magnitude. In contrast, finasteride-induced reductions in DHT were detected by LC–MS/MS, but not EIA (p < 0.05). No significant associations were present for DHT concentrations between measurement techniques. Cross-reactivity of testosterone with the immunoassay ranged from 18% to 99% and DHT concentrations measured by EIA were highly associated with the spiked testosterone concentrations in DHT-free buffer (r = 0.885, p < 0.001). In conclusion, we provide evidence invalidating a commonly-utilized commercially-available DHT immunoassay because significant cross-reactivity exists between testosterone and the EIA and because the changes in DHT observed via EIA were not associated with a validated gold-standard measurement technique. The cross-reactivity of testosterone is particularly concerning because testsoterone is present in 100-fold greater concentrations than is DHT within the circulation.     

Atropine unmasks bed-rest effect: a spectral analysis of cardiac interbeat intervals

Bed-rest deconditioning is suspected to reduce cardiac reserve, possibly by impairing autonomic function. Heart rate response in normal subjects reveals considerable variability, reflected by a relatively broadband interbeat interval power spectrum. A reduction in this autonomically modulated variability would be predicted to cause a narrowing of the spectrum. We retrospectively analyzed data from 10 aerobically conditioned men (age range 35-49 yr) who had undergone orthostatic tolerance testing with lower body negative pressure pre-bed rest and after 7-10 days of bed rest, while on placebo and after intravenous atropine. Spectra were derived by Fourier analysis of 128 interbeat interval data sets. Spectral power was estimated by computing the root-mean-square (rms) values (mean +/- SD) for the band encompassing the 2nd to 64th harmonics from subjects with a sufficient number of beats: placebo rms is 93 +/- 33 ms for pre-bed rest and 84 +/- 38 ms for bed rest (NS, n = 6); atropine rms is 63 +/- 24 ms for pre-bed rest and 40 +/- 23 ms for bed rest (P less than 0.01; n = 7). These data suggest that atropine "unmasks" a deconditioning effect of bed rest in athletic men, evidenced by a reduction in interbeat interval spectral power not apparent with placebo. Spectral analysis offers a useful means of quantitating the effects of bed-rest deconditioning and autonomic perturbations on cardiac dynamics.