Neisseria gonorrhoeae is capable of utilizing host iron-binding proteins, such as transferrin, lactoferrin, and hemoglobin, as the sole source of iron. The receptor involved in transferrin iron acquisition is composed of two distinct transferrin-binding proteins, TbpA and TbpB. The genes that encode these proteins are linked on the chromosome in the order tbpB-tbpA but are separated by an inverted repeat of unknown function. In this study, we sought to understand the transcriptional organization and regulation of the tbp genes, using a combination of lacZ transcriptional fusion analysis and reverse transcriptase PCR (RT-PCR). First, we demonstrated that tbpB and tbpA are cotranscribed and coregulated from the common upstream promoter that precedes tbpB. Using ␤-galactosidase activity as a surrogate for tbp-specific transcription, we found that tbpB-specific transcripts were more prevalent than tbpA-specific transcripts after 2 h of growth under iron stress conditions. We confirmed the results obtained by fusion analysis by using RT-PCR applied to native RNA isolated from wild-type gonococci. Three different varieties of RT-PCR were employed: relative, competitive, and real time quantitative. The results of all analyses indicated that tbpB-specific transcripts were approximately twofold more prevalent than tbpA-specific transcripts at steady state. In iron-stressed cultures, the ratio of tbpB-to tbpA-specific message was approximately 2; however, in iron-replete cultures, this ratio dropped to 1. Using these techniques, we also quantitated the effects of iron, external pH, and presence of ligand on tbp mRNA levels.Pathogenic bacteria must obtain essential nutrients in order to establish an infection; of these nutrients, iron plays a critical role (for a recent review, see reference 53).

Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5= endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon.

ABSTRACT. Although the precise causes of psoriasis remain to be elucidated, psoriasis has been known as a disorder in which factors in the immune system, enzymes and other biochemical substances that regulate skin cell division are functionally imbalanced, thereby resulting in rapid proliferation of keratinocytes and incomplete keratinization. The expression of candidate genes such as E2A and caspase-9, which have been recognized to play a critical role in cellular proliferation/ differentiation and apoptosis, is of great interest. They may be therapeutically targeted by the antipsoriatic drug, dithranol. We examined the molecular effects of dithranol on the mRNA and protein expression levels of E2A and caspase-9 in the HaCaT keratinocyte cell line. The HaCaT cells were treated with 0-0.5 µg/mL dithranol for 30 min. After dithranol was washed out, the HaCaT cells were cultured for 2 h, and their total cellular RNA and proteins were isolated. Quantitative realtime reverse transcriptase-polymerase chain reaction and Western blot were performed to determine the mRNA and protein levels of these two genes. We found that dithranol treatment in the range of 0.25-0.5 µg/ mL slightly upregulated the mRNA expression of E2A and caspase-9 approximately 1.5-and 1.2-fold, respectively. However, undetectable Expression of E2A and caspase-9 gene in response to dithranol change and minor downregulation of the protein expression levels were observed for E2A and caspase-9, respectively. Consequently, these genes appear not to be viable therapeutic targets for dithranol.

ABSTRACT. Expression of serotonin 2A receptor (5-HTR2A) is known to increase in psoriasis, a chronic inflammatory skin disease. We investigated a possible association between the -1438A/G single nucleotide polymorphism (rs6311) in the promoter region of 5-HTR2A gene and psoriasis in a Thai population. One hundred and twelve psoriatic patients and 151 unrelated healthy controls were included in our study. Genotyping was performed using the polymerase chain reaction and restriction fragment length polymorphism techniques. We found no overall differences in genotype distributions and allele frequencies when comparing between the two groups. When we analyzed a subset of psoriatic patients classified by onset and severity, only the -1438A allele was significantly increased in patients with lateonset psoriasis when compared with the healthy control group (c 2 = 4.77, d.f. = 1, P = 0.029, odds ratio = 2.298 [95% confidence interval =

ABSTRACT. Although the precise causes of psoriasis are unclear, it is widely accepted that psoriasis is a disorder in which factors in the immune system, enzymes, and other biochemical substances that regulate skin cell division are impaired, leading to rapid proliferation of keratinocytes and incomplete keratinization. Expression of the helix-loop-helix transcription factor Id1 (inhibitor of differentiation/DNA binding), functioning as an inhibitor of differentiation, is known to increase in psoriatic skin. However, the molecular involvement of this particular biomarker in the psoriatic immune system remains to be elucidated. We measured Id1 mRNA expression in peripheral blood mononuclear cells (PBMCs) of psoriatic patients and healthy controls using semi-quantitative reverse transcriptase- PCR. The normalized level of Id1 transcripts in psoriatic patients was about 2-fold higher than that in controls (P < 0.05). When we examined the proliferation rate of PBMCs, the stimulation index obtained from the phytohemagglutinin stimulation assay was not significantly different in psoriatic patients. In patients with psoriasis, there was no correlation between the stimulation index and the psoriasis area severity index. We suggest that Id1 has a role in causing psoriatic immune cell symptoms.