Initiation of transcription of a gene from a core promoter region by RNA polymerase II requires the assembly of several initiation factors to form a preinitiation complex. Assembly of this complex is thought to be nucleated exclusively by the sequence-specific binding of the TFIID transcription factor complex, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s), to the different promoters. Here we isolate and characterize a new multiprotein complex that does not contain either TBP or a TBP-like factor but is composed of several TAF(II)s and other proteins. This complex can replace TFIID on both TATA-containing and TATA-lacking promoters in in vitro transcription assays. Moreover, an anti-TBP antibody that inhibits TBP- and TFIID-dependent transcription does not inhibit activity of this new complex. These results indicate that TBP-free RNA polymerase II mediated transcription may be able to occur in mammalian cells and that multiple preinitiation complexes may play an important role in regulating gene expression.

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The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.

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A juvenile‐hormone‐binding protein (juvenile‐hormone carrier), isolated fromGalleria mellonella haemolymph, was treated with trypsin, chymotrypsin, carboxypeptidase A and subtilisin. Among these enzymes, only subtilisin was able to affect juvenile‐hormone‐binding activity of this protein. With SDS/PAGE it was shown that juvenile‐hormone‐binding protein, a 32‐kDa peptide, is first slowly converted into a 30‐kDa molecule, then into two or three smaller‐molecular‐mass species (20–25 kDa), which in turn were further digested to small peptides undetectable in PAGE. The 30‐kDa peptide has a 2.4‐times‐higher dissociation constant for juvenile hormone than the native protein. No binding activity was detected for 20–25‐kDa peptides. The rate of proteolysis of juvenile‐hormone‐binding protein was decreased by more than twofold in the presence of hormone, however, the overall cleavage pattern was unchanged. Under non‐denaturing conditions, free binding‐protein molecules could be separated from juvenile‐hormone–binding‐protein complex due to a slower electrophoretic mobility of the complex. As judged from ultracentrifugation and cross‐linking experiments, binding of the hormone to its haemolymph carrier does not induce formation of oligomers, but shifts the sedimentation coefficient from 2.30S to 2.71S. It is conclude that juvenile‐hormone binding induces a conformational transition of its carrier protein. This hormone‐induced change might have a physiological significance for signal transmission.

Transthyretin (TTR) is a tetrameric protein transporting hormones in the plasma and brain, which has many other activities that have not been fully acknowledged. TTR is a positive indicator of nutrition status and is negatively correlated with inflammation. TTR is a neuroprotective and oxidative-stress-suppressing factor. The TTR structure is destabilized by mutations, oxidative modifications, aging, proteolysis, and metal cations, including Ca2+. Destabilized TTR molecules form amyloid deposits, resulting in senile and familial amyloidopathies. This review links structural stability of TTR with the environmental factors, particularly oxidative stress and Ca2+, and the processes involved in the pathogenesis of TTR-related diseases. The roles of TTR in biomineralization, calcification, and osteoarticular and cardiovascular diseases are broadly discussed. The association of TTR-related diseases and vascular and ligament tissue calcification with TTR levels and TTR structure is presented. It is indicated that unaggregated TTR and TTR amyloid are bound by vicious cycles, and that TTR may have an as yet undetermined role(s) at the crossroads of calcification, blood coagulation, and immune response.

The apparent vitamin B-6 status of 31 children with familial hypophosphatemic rickets (FHR) was determined. All children had alkaline phosphatase activity that was high-normal to elevated for their ages. A sensitive assay for pyridoxal 5'-phosphate (PLP) indicated that 15 of the 31 children had an undetectable (less than 0.2 nmol/L) concentration of the vitamer--the lowest values yet reported in human serum. The 16 remaining children had concentrations of the vitamer so low that they indicated a potential severe vitamin B-6 deficiency. However, none of the children had ever presented with any of the classical vitamin B-6-deficiency symptoms. Treatment of three additional FHR children with 100 mg pyridoxine.HCl/d resulted in a moderate and transient elevation of their serum PLP concentrations, a dramatic elevation of their erythrocyte PLP concentrations, and no improvement in clinical condition. Serum or plasma PLP concentrations are an inappropriate index for determining vitamin B-6 status in people with FHR and perhaps in others with elevated alkaline phosphatase activity.