Teratogenesis by retinoic acid analogs positively correlates with elevation of retinoic acid receptor-beta 2 mRNA levels in treated embryos

The concentration/effect relationship of bunazosin, a selective alpha-1-adrenoceptor antagonist of the quinazoline class, was established in hypertensive patients after a first dose of bunazosin and after 8 weeks of chronic dosing. The study was part of a placebo controlled, multicenter dose-finding trial with parallel group design. After a wash-out period and a 2-week placebo run-in phase, each 4 patients received either placebo, 3 mg, 6 mg or 12 mg bunazosin o.d. in a slow-release formulation. Concentration/effect analysis based on a sigmoidal Emax-model and the drop in the mean arterial blood pressure application of this model was feasible in 9/11 patients. The EC50 after the first dose was 4.6 +/_ 2.0 ng/ml and increased to 9.1 +/- 7.3 ng/ml at steady-state. The decrease in the average of the responsiveness parameter was mainly due to about three-fold, right-shift in the concentration/effect curves at steady-state in 3 patients, the remaining patients showed no or considerably lesser changes of the EC50. Whereas a significant correlation was observed between the pretreatment mean arterial blood pressure and the height of the maximal effect under bunazosin, no relationship appears between the bunazosin dose, the resulting plasma levels at trough and the corresponding effect.     

Solution structure of the DNA-binding domain of a human papillomavirus E2 protein: evidence for flexible DNA-binding regions

The three-dimensional structure of the DNA-binding domain of the E2 protein from human papillomavirus-31 was determined by using multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. A total of 1429 NMR-derived distance and dihedral angle restraints were obtained for each of the 83-residue subunits of this symmetric dimer. The average root mean square deviations of 20 structures calculated using a distance geometry-simulated annealing protocol are 0.59 and 0.90 angstroms for the backbone and all heavy atoms, respectively, for residues 2-83. The structure of the human virus protein free in solution consists of an eight-stranded beta-barrel and two pairs of alpha-helices. Although the overall fold of the protein is similar to the crystal structure of the bovine papillomavirus-1 E2 protein when complexed to DNA, several small but interesting differences were observed between these two structures at the subunit interface. In addition, a beta-hairpin that contacts DNA in the crystal structure of the protein-DNA complex is disordered in the NMR structures, and steady-state 1H-15N heteronuclear NOE measurements indicate that this region is highly mobile in the absence of DNA. The recognition helix also appears to be flexible, as evidenced by fast amide exchange rates. This phenomenon has also been observed for a number of other DNA-binding proteins and may constitute a common theme in protein/DNA recognition.     

Effect of redox conditions on the DNA-binding efficiency of the retinoic acid receptor zinc-finger

All 24-hour endocrine rhythms partially reflect the interaction of circadian rhythmicity with sleep-wake homeostasis but their relative contributions vary from one system to another. In older adults, many 24-hour rhythms are dampened and/or advanced, including those of cortisol and GH. Amplitude reduction and phase advance of 24-hour rhythms may represent age-related changes in the central nervous systems underlying circadian rhythmicity and sleep-wake homeostasis. Age-related alterations in circadian function could also reflect decreased exposure and/or responsivity to the synchronizing effects of both photic (e.g. light exposure) and nonphotic (e.g. social cues) inputs. There are pronounced age-related alterations in sleep quality in aging which consist primarily of a marked reduction of slow-wave sleep, a reduction in REM stages and a marked increase in the number and duration of awakenings interrupting sleep. Alterations in slow-wave sleep occur abruptly in young adulthood (30-40 years of age) whereas disturbances in amounts of REM and wake appear more gradually. This article reviews evidence indicating that deficits in characteristics of sleep-wake homeostasis and circadian function may mediate age-related alterations in somatotropic and corticotropic function. Because sleep loss in young subjects results in endocrine disturbances which mimic those observed in aging, it is conceivable that the decrease in sleep quality which characterizes aging may contribute to age-related alterations in hormonal function and their metabolic consequences.     

Solution structure of the origin DNA-binding domain of SV40 T-antigen

The structure of the domain from simian virus 40 (SV40) large T-antigen that binds to the SV40 origin of DNA replication (T-ag-OBD131-260) has been determined by nuclear magnetic resonance spectroscopy. The overall fold, consisting of a central five-stranded antiparallel beta-sheet flanked by two alpha-helices on one side and one alpha-helix and one 3(10)-helix on the other, is a new one. Previous mutational analyses have identified two elements, termed A (approximately 152-155) and B2 (203-207), as essential for origin-specific recognition. These elements form two closely juxtaposed loops that define a continuous surface on the protein. The addition of a duplex oligonucleotide containing the origin recognition pentanucleotide GAGGC induces chemical shift changes and slows amide proton exchange in resonances from this region, indicating that this surface directly contacts the DNA.     

Secondary structure of the DNA-binding domain of the c-Myb oncoprotein in solution. A multidimensional double and triple heteronuclear NMR study

Euglycemic hyperinsulinemia evokes both sympathetic activation and vasodilation in skeletal muscle, but the mechanism remains unknown. To determine whether insulin per se or insulin-induced stimulation of carbohydrate metabolism is the main excitatory stimulus, we performed, in six healthy lean subjects, simultaneous microneurographic recordings of muscle sympathetic nerve activity, plethysmographic measurements of calf blood flow, and calorimetric determinations of carbohydrate oxidation rate. Measurements were made during 2 h of: (a) insulin/glucose infusion (hyperinsulinemic [6 pmol/kg per min] euglycemic clamp), (b) exogenous glucose infusion at a rate matched to that attained during protocol a, and (c) exogenous fructose infusion at the same rate as for glucose infusion in protocol b. For a comparable rise in carbohydrate oxidation, insulin/glucose infusion that resulted in twofold greater increases in plasma insulin concentrations than did glucose infusion alone, evoked twofold greater increases in both muscle sympathetic nerve activity and calf blood flow. Fructose infusion, which increased carbohydrate oxidation comparably, but had only a minor effect on insulinemia, did not stimulate either muscle sympathetic nerve activity or calf blood flow. These observations suggest that in humans hyperinsulinemia per se, rather than insulin-induced stimulation of carbohydrate metabolism, is the main mechanism that triggers both sympathetic activation and vasodilation in skeletal muscle.     

The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain

Replication protein A (RPA) is a conserved nuclear single-stranded DNA (ssDNA)-binding protein. Human RPA (hRPA) comprises three subunits of approximately 70, 32, and 14 kDa (hRPA70, hRPA32 and hRPA14). RPA is known to bind ssDNA through two ssDNA-binding domains in the RPA70 subunit. Here, we demonstrate that the complex of hRPA32 and hRPA14 has an ssDNA-binding domain. Limited proteolysis of the hRPA14.32 complex defined a core dimer composed of the central region of hRPA32 (amino acids 43-171) and RPA14. The core dimer bound ssDNA with an affinity of approximately 10-50 microM, which is at least 100-fold more avid than the DNA-binding affinity of the intact dimer. Analysis of the predicted secondary structure of hRPA32 suggests that amino acids 63-150 of hRPA32 form an ssDNA-binding domain similar in structure to each of those in hRPA70. The complex of hRPA14 and hRPA32-(43-171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436-616). The ssDNA-binding affinity of this trimeric complex was 3 to 5-fold higher than hRPA14.32-(43-171) alone, suggesting a role for the C terminus of hRPA70 in ssDNA binding.     

The regulation of retinoic acid formation

Rat liver microsomal as well as cytosolic retinol dehydrogenases from cellular retinal-binding protein (CRBP)-retinal from CRBP-retinol. A cytosolic dehydrogenase transforms retinal into retinoic acid. Apo-CRBP and ethanol inhibit the cytosolic but not the microsomal retinol dehydrogenase, the latter being the primary enzyme in the rate-limiting step in retinoic acid synthesis.     

NMR solution structure of domain 1 of human annexin I shows an autonomous folding unit

Annexins are excellent models for studying the folding mechanisms of multidomain proteins because they have four-eight homologous helical domains with low identity in sequence but high similarity in folding. The structure of an isolated domain 1 of human annexin I has been determined by NMR spectroscopy. The sequential assignments of the 1H, 13C, and 15N resonances of the isolated domain 1 were established by multinuclear, multidimensional NMR spectroscopy. The solution structure of the isolated domain 1 was derived from 1,099 experimental NMR restraints using a hybrid distance geometry-simulated annealing protocol. The root mean square deviation of the ensemble of 20 refined conformers that represent the structure from the mean coordinate set derived from them was 0. 57 +/- 0.14 A and 1.11 +/- 0.19 A for the backbone atoms and all heavy atoms, respectively. The NMR structure of the isolated domain 1 could be superimposed with a root mean square deviation of 1.36 A for all backbone atoms with the corresponding part of the crystal structure of a truncated human annexin I containing all four domains, indicating that the structure of the isolated domain 1 is highly similar to that when it folded together with the other three domains. The result suggests that in contrast to isolated domain 2, which is largely unfolded in solution, isolated domain 1 constitutes an autonomous folding unit and interdomain interactions may play critical roles in the folding of annexin I.     

High-resolution solution NMR structure of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein

The retroviral nucleocapsid (NC) protein is a multifunctional protein essential for RNA genome packaging and viral infectivity. The NC protein, NCp8, of the human immunodeficiency virus type-II (HIV-2) is a 49 amino acid peptide containing two zinc fingers, of the type C-X2-C-X4-H-X4-C, connected by seven amino acid residues, called the "basic amino acid cluster." It has been shown that the N-terminal zinc finger flanked by the basic amino acid cluster is the minimal active domain for the specific binding to viral RNA and other functions. However, the structure-activity relationships of NCp8 have not been investigated in detail. In the present study, the three-dimensional structure of a 29 amino acid peptide, including the minimal active domain (NCp8-fl), was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 15 converged structures of NCp8-fl were obtained on the basis of 355 experimental constraints, including 343 distance constraints obtained from nuclear Overhauser effect connectivities, 12 torsion angle (phi, chi1) constraints, and four constraints for zinc binding. The root-mean-square deviation of the 15 converged structures was 0.29 +/- 0.04 A for the backbone atoms (N, C(alpha), C) and 1.27 +/- 0.13 A for all heavy atoms. Interestingly, the basic amino acid cluster itself was defined well, with a loop-like conformation in which three arginine residues in the cluster and one arginine residue in the zinc finger are located approximately in the same plane of the molecule and are exposed to the solvent. The structure-activity relationships are discussed on the basis of the comparison of this well-defined structure with those of other NC proteins.     

Retinoic acid receptor-gamma in human epidermis preferentially traps all-trans retinoic acid as its ligand rather than 9-cis retinoic acid

Brain injury in the premature infant is an extremely important problem, in part because of the large absolute number of infants affected yearly. The 2 principal brain lesions that underlie the neurological manifestations subsequently observed in premature infants are periventricular hemorrhagic infarction and periventricular leukomalacia. The emphases of this article are the neuropathological features, pathogenesis, and potential means of prevention of these 2 lesions. Recent work suggests that the ultimate goal, prevention of the lesions, is potentially achievable. Periventricular hemorrhagic infarction may be avoidable by prevention of germinal matrix-intraventricular hemorrhage, and periventricular leukomalacia by detection of impaired cerebrovascular autoregulation, prevention of impaired cerebral blood flow, and interruption of the cascade to oligodendroglial cell death by such agents as free radical scavengers.     

Retinoic acid receptors regulate expression of retinoic acid 4-hydroxylase that specifically inactivates all-trans retinoic acid in human keratinocyte HaCaT cells

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), 7beta-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-Ibeta and/or tumor necrosis factor (TNF)-alpha secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7beta-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1beta secretion. TNF-alpha secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the alpha- or beta-hydroxyl radical position plays a key role in apoptosis and IL-1beta secretion.     

Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain

The equilibrium unfolding reaction of the C-terminal 80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, delta GH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 x 10(-8) M. Equilibrium sedimentation experiments give a Kd of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.     

The roles of cytokines and retinoic acid in the regulation of human thyroid cancer cell growth

The purpose of this study was to investigate the effects of cytokines and retinoic acid in human thyroid cancer cell growth. Cellular proliferation studies of the CGTH W-1 and SW 579 cell lines were performed with various cytokines and all-trans retinoic acid (RA). Cell number was determined by cell counting and incorporation of [3H]thymidine into DNA. Inhibitory effects of tumour necrosis factor alpha (TNF-alpha) were found in both cell lines. SW 579 was more sensitive to TNF-alpha. The SW 579 cell line revealed gradually decreased cell proliferation in [3H]thymidine incorporation studies as TNF-alpha concentration increased. In contrast, the CGTH W-1 cell line revealed prominent suppressive effects when the TNF-alpha concentrations increased over 1 ng/ml. An inhibitory effect of interleukin 1 beta (IL-1 beta) on CGTH W-1 cells was noted at the concentration of 1 ng/ml, however, IL-1 beta failed to demonstrate an inhibitory effect in SW 579 cells.