The molecular basis for the broad substrate specificity of human sulfotransferase 1A1

Cytosolic sulfotransferases (SULTs) are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.     

Spatial regulation of histidine kinases governing biofilm formation in Bacillus subtilis

Bacillus subtilis is able to form architecturally complex biofilms on solid medium due to the production of an extracellular matrix. A master regulator that controls the expression of the genes involved in matrix synthesis is Spo0A, which is activated by phosphorylation via a phosphorelay involving multiple histidine kinases. Here we report that four kinases, KinA, KinB, KinC, and KinD, help govern biofilm formation but that their contributions are partially masked by redundancy. We show that the kinases fall into two categories and that the members of each pair (one pair comprising KinA and KinB and the other comprising KinC and KinD) are partially redundant with each other. We also show that the kinases are spatially regulated: KinA and KinB are active principally in the older, inner regions of the colony, and KinC and KinD function chiefly in the younger, outer regions. These conclusions are based on the morphology of kinase mutants, real-time measurements of gene expression using luciferase as a reporter, and confocal microscopy using a fluorescent protein as a reporter. Our findings suggest that multiple signals from the older and younger regions of the colony are integrated by the kinases to determine the overall architecture of the biofilm community.     

Drug delivery systems employing 1,6-elimination: releasable poly(ethylene glycol) conjugates of proteins

Using lysozyme as a representative protein substrate that loses its activity when PEGylation takes place on the epsilon-amino group of lysine residues, various amounts of a novel releasable PEG linker (rPEG) were conjugated to the protein. rPEG-lysozyme conjugates were relatively stable in pH 7.4 buffer for over 24 h. However, regeneration of native protein from the rPEG conjugates occurred in a predictable manner during incubation in high pH buffer or rat plasma, as demonstrated by enzymatic activity and structural characterization. The rates of regeneration were also correlated with PEG number: native lysozyme was released more rapidly from the monosubstituted conjugate than from the disubstituted conjugate, suggesting possible steric hindrance to the approach of cleaving enzymes. Recovery of normal activity and structure for the regenerated native lysozyme was shown by a variety of assays.     

CFTR directly mediates nucleotide-regulated glutathione flux

Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.     

Effective surgical treatment of cubital tunnel syndrome based on provocative clinical testing without electrodiagnostics

This study evaluated the effectiveness of identifying surgically correctable ulnar nerve compression at the elbow based on provocative clinical testing alone in patients with cubital tunnel syndrome after failure of conservative treatment. Twenty-four patients were included in the study (mean age, 60 years). Three patients underwent bilateral procedures. Patients complaining of symptoms in the distribution of the ulnar nerve were tested by elicitation of Tinel's sign and combined flexion and pressure testing at the elbow and wrist. Two-point discrimination was determined. After a failed 6-week trial of conservative therapy patients underwent anterior submuscular transposition of the ulnar nerve with carpal tunnel release. Postoperatively, the change in two-point discrimination as measured at 6 months was significantly improved, with a mean improvement per digital nerve of 2.52 mm (p<0.001). Mean time to relief was 7.2 weeks. Complications included one hematoma and one seroma. A total of 26 of the 27 limbs chosen for surgical treatment by provocative clinical testing alone experienced relief of symptoms with anterior submuscular transposition of the ulnar nerve and carpal tunnel release. This study demonstrates the effectiveness of surgical therapy in patients with lesions identified by clinical examination without electrodiagnostic testing.     

Orbital roof fractures in the pediatric population

Twenty-three patients aged 3.3 +/- 1.6 years (mean +/- SD) presented between January of 1984 and September of 1987 with fronto-orbital trauma resulting in fractures of one (N = 20) or both (N = 3) orbital roofs. All patients had computed tomography (CT) with axial and coronal sections that revealed three fracture patterns of the orbital roof (nondisplaced, superiorly displaced, and inferiorly displaced fractures). Orbital dystopia was exhibited in 35 percent (N = 8) of the patients. Exophthalmos was noted in 61 percent (N = 14) of the patients. Only 30 percent of the patients (N = 7) sustained associated maxillofacial fractures. Eight percent of fractures exhibited orbital encephaloceles. All patients lacked frontal sinus pneumatization. The majority of children with orbital roof fractures do not exhibit concomitant facial fractures. CT utilizing both axial and coronal sections is valuable in defining the extent and pattern of the fracture as well as in identifying associated neurologic injuries. Large, displaced orbital roof fractures, which occurred in 3 of 13 patients with displaced fractures in our series, should undergo early reduction to avoid late development of encephalocele.     

The cytochrome oxidase II gene in mitochondria of the sugar-beetBeta vulgaris L.

We have cloned and analyzed the sugar-beet mitochondrial gene for cytochrome oxidase subunit II (coxII). The sugar-beet and its deduced amino acid sequence were compared to its homologouscoxII gene sequences from both monocot and dicot plants. It was found to be highly conserved (89–95%) compared to homologue in other plant species. The 780 bp coding sequence of the sugar beetcoxII gene is interrupted at position 383 by a 1463 bp intron. This intron contains an additional 107 bp sequence that is not found in any of the plantcoxII genes studied thus far. The structure of the intron suggests that a large intron existed in an ancestralcoxII gene before monocots and dicots diverged in evolution. Three CGG codons in the sugar-beetcoxII coding sequence align with conserved tryptophan residues in the homologous gene of other species, suggesting that RNA editing takes place also in sugar-beet mitochondria. In 13 out of 24 codons ofcoxII mRNA that were found to be edited in four other plants, the sugar-beet gene already utilizes the edited codons. This phenomenon may indicate that the mitochondrial genome in sugar-beet is phylogenetically more archaic relative to these plants. An additional sequence of 279 bp that is identical to the first exon ofcoxII was identified in the mtDNA of the sugar-beet. This pseudo-gene is transcribed and its existence in the mitochondrial genome is unexplained.     

The Disulfide Bond Arrangement in the Extracellular Domain of the Activin Type II Receptor

The initial step in the signaling cascade of the growth factor activin involves its binding to the extracellular domain of the activin type II receptor. This receptor domain contains 10 cysteine residues which are engaged in intramolecular disulfide bonds. To elucidate the structural framework of this domain we have characterized its disulfide-bonding pattern using an extracellular fragment of the receptor which binds activin A with high affinity. By combining proteolysis with mass spectroscopy and chemical sequence analysis, the disulfide connectivity was determined to be as follows: C1–C3, C2–C4, C5–C8, C6–C7, and C9–C10. A similar disulfide arrangement occurs in a family of snake toxins for which the three-dimensional structure is known.     

Tetrahydrocurcumin Protects against Cadmium-Induced Hypertension,Raised Arterial Stiffness and Vascular Remodeling in Mice

Background

Cadmium (Cd) is a nonessential heavy metal, causing oxidative damage to various tissues and associated with hypertension. Tetrahydrocurcumin (THU), a major metabolite of curcumin, has been demonstrated to be an antioxidant, anti-diabetic, anti-hypertensive and anti-inflammatory agent. In this study, we investigated the protective effect of THU against Cd-induced hypertension, raised arterial stiffness and vascular remodeling in mice.

Methods

Male ICR mice received CdCl₂ (100 mg/l) via drinking water for 8 weeks. THU was administered intragastrically at dose of 50 or 100 mg/kg/day concurrently with Cd treatment.

Results

Administration of CdCl₂ significantly increased arterial blood pressure, blunted vascular responses to vasoactive agents, increased aortic stiffness, and induced hypertrophic aortic wall remodeling by increasing number of smooth muscle cells and collagen deposition, decreasing elastin, and increasing matrix metalloproteinase (MMP)-2 and MMP-9 levels in the aortic medial wall. Supplementation with THU significantly decreased blood pressure, improved vascular responsiveness, and reversed the structural and mechanical alterations of the aortas, including collagen and elastin deposition. The reduction on the adverse response of Cd treatment was associated with upregulated eNOS and downregulated iNOS protein expressions, increased nitrate/nitrite level, alleviated oxidative stress and enhanced antioxidant glutathione. Moreover, THU also reduced the accumulation of Cd in the blood and tissues.

Conclusions

Our results suggest that THU ameliorates cadmium-induced hypertension, vascular dysfunction, and arterial stiffness in mice through enhancing NO bioavailability, attenuating oxidative stress, improving vascular remodeling and decreasing Cd accumulation in other tissues. THU has a beneficial effect in moderating the vascular alterations associated with Cd exposure.