Biomimetic assembly and activation of [FeFe]-hydrogenases.

Hydrogenases are the most active molecular catalysts for hydrogen production and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN(-) ligands. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.

Thromboxane A2 receptors in equine monocytes: identification of a new subclass of TXA2 receptors.

Thromboxane (TX) A2 has been implicated as an important pathophysiologic mediator of a variety of cardiovascular diseases. Monocytes synthesize TXA2 and it modulates their function. This study sought to characterize monocyte TXA2 receptors. Radioligand binding studies were performed on membranes prepared from equine peripheral blood monocytes using [125I]BOP, a TXA2 receptor agonist. [125I]BOP bound to a single class of binding sites (Kd = 1.0 +/- 0.3 nM and Bmax = 389 +/- 191 fmol/mg protein; n = 5). Several TXA2 receptor agonists and antagonists competed for binding with [125I]BOP. I-BOP produced a concentration-dependent inhibition of endotoxin-induced tumor necrosis factor (TNF) activity (IC50 = 9.6 +/- 2.5 nM; n = 5). In contrast to its effects in platelets and vascular smooth muscle, I-BOP significantly increased cAMP formation in monocytes (EC50 = 22 +/- 3.6 nM; n = 4). The TXA2 receptor antagonists SQ29548 (5.6 microM) and L657925 (0.13 microM) significantly blocked I-BOP-stimulated cAMP formation but did not block 250 nM prostaglandin E2-stimulated cAMP formation. These data support the presence of a TXA2 receptor in equine peripheral blood monocytes. Activation of this receptor results in suppression of endotoxin-induced TNF formation and stimulation of cAMP production. Increased cAMP production after receptor activation suggests that this receptor may represent a unique subclass of TXA2 receptors.

Differentiation of chronic lymphocytic leukemia in the horse. A report of two cases.

Chronic lymphocytic leukemia (CLL) was diagnosed in two horses: an 18-year-old Quarter Horse gelding that was examined because of edema of the prepuce and ventral abdomen; and a 20-year-old mixed breed gelding that was referred because of lymphocytosis, ventral edema, and weight loss. The first horse had enlarged peripheral lymph nodes and cool nonpainful pitting edema of the ventral abdomen and prepuce. The second horse had enlarged peripheral lymph nodes, cool nonpainful pitting edema of the ventral thorax and cranial ventral abdomen, and a 3/5 holosystolic heart murmur. The diagnosis of CLL was based on increased blood lymphocyte counts and infiltration of marrow and other tissues by lymphocytes. In horse 1, the lymphocytosis persisted for 2 months between initial examination and death. The results of flow cytometric analysis on blood lymphocytes using anti-lymphocyte antibodies suggested that horse 1 had T-cell CLL, and horse 2 had B-cell CLL. In addition, the second horse had a monoclonal gammopathy (IgG), with light-chain proteinuria.

Treatment of right dorsal ulcerative colitis in a horse.

Excessive administration of phenylbutazone was associated with development of right dorsal ulcerative colitis. The clinical signs of right dorsal colitis include chronic colic and weight loss. The laboratory abnormalities include panhypoproteinemia and a high WBC count in the abdominal fluid. Medical management of the chronic colic and protein-losing enteropathy associated with the ulcerative lesions in the right dorsal colon and surgical bypass of the right dorsal colon did not result in long-term resolution of clinical signs. Resection of the ulcerated right dorsal colon through a right lateral approach at the 16th rib resulted in resolution of intestinal protein loss and colic. The results of this case suggest that surgical resection of the ulcerated right dorsal colon may be the recommended treatment for right dorsal ulcerative colitis.

Primary cholangiohepatitis in a horse.

An 8-year-old mare was presented to the New York State College of Veterinary Medicine Large Animal Clinic for evaluation of anorexia, fever and icterus. The mare had a 5-day history of anorexia, depression and tongue protrusion. Diagnostic procedures included serum hepatic enzyme activities, serum bile acid concentrations, blood ammonia evaluations and hepatic ultrasound and ultrasound guided biopsy. The history, clinical pathology and histopathology in this case supported a probable diagnosis of primary septic cholangiohepatitis.