The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through proteinprotein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.In prokaryotic organisms, the PII protein is recognized as the key mediator of energy, carbon, and nitrogen interactions and is referred to as the central processing unit of carbon:nitrogen metabolism (1-4). Escherichia coli PII is a 112-amino acid protein that as a homotrimer senses the cellular status of both ATP and the carbon skeleton 2-oxoglutarate (2KG) 3 via allosteric means. Nitrogen status is assessed through glutamine levels by covalent modification (uridylylation) of PII. This metabolic information is signaled to other proteins by proteinprotein interaction and produces an appropriate response that alters gene expression and the activity of glutamine synthetase (3, 5). In terms of metabolic sensing, cyanobacterial PII plays a similar role, but in this case, covalent modification is by phosphorylation (6). To date, the processes known to be regulated by PII in cyanobacteria are: ammoniumdependent nitrate/nitrite uptake (7), high affinity bicarbonate transport (8), regulation of the global transcriptional activation by NtcA (9, 10), and arginine biosynthesis (11).In eukaryotes, PII has only been identified in plants and red algae (12), and its sequence is highly conserved when compared with prokaryotic PIIs, with Arabidopsis thaliana PII being 50 and 55% identical to E. coli and Synechococcus elongatus PII, respectively. Plant PII proteins have a conserved N-terminal extension that functions as a chloroplast transit peptide, which is consistent with biochemical data indicating that PII resides in this compartment. We have previously shown that the plant PII protein is not regulated by phosphorylation (13). Like the bacterial...

Primary hepatic carcinoid and neuroendocrine carcinoma (NEC) are rare tumors. We experienced three carcinoids and two NEC originating in the liver during the past 25 years and attempted to elucidate the clinicopathological and immunohistochemical features of these tumors. The patients had no endocrine symptoms despite two of them having elevated plasma serotonin. Three of the five patients died of the tumor after operation with an average survival time of 20.6 months. All tumors were large (up to 26 cm in diameter), four of them solitary and one multinodular, and were not associated with liver cirrhosis. The carcinoid tumors showed insular, trabecular or glandular arrangement of argyrophilic cells, whereas in the NEC this histological pattern was distorted. Immunohistochemically the tumors showed expression of chromogranin A (all cases), chromogranin B (three cases), pancreastatin and chromostatin (four cases, respectively), prohormone convertase PC3 (three cases), carcinoembryonic antigen (CEA) and CA19-9 (two cases), cytokeratin 56 kDa (three cases), 160 kDa neurofilament (two cases) and neuron-specific enolase (two cases). Serotonin and glucagon were sporadically detected in two tumors. The most useful marker to confirm the diagnosis was chromogranin A, which was cleaved to pancreastatin and chromostatin in the tumor tissue, and was more reliable than other markers of neuroendocrine differentiation.

PII is a highly conserved regulatory protein found in organisms across the three domains of life. In cyanobacteria and plants, PII relieves the feedback inhibition of the rate-limiting step in arginine biosynthesis catalyzed by N-acetylglutamate kinase (NAGK). To understand the molecular structural basis of enzyme regulation by PII, we have determined a 2.5-Å resolution crystal structure of a complex formed between two homotrimers of PII and a single hexamer of NAGK from Arabidopsis thaliana bound to the metabolites N-acetylglutamate, ADP, ATP, and arginine. In PII, the T-loop and Trp 22 at the start of the ␣1-helix, which are both adjacent to the ATP-binding site of PII, contact two ␤-strands as well as the ends of two central helices (␣E and ␣G) in NAGK, the opposing ends of which form major portions of the ATP and N-acetylglutamate substratebinding sites. The binding of Mg 2؉ ⅐ATP to PII stabilizes a conformation of the T-loop that favors interactions with both open and closed conformations of NAGK. Interactions between PII and NAGK appear to limit the degree of opening and closing of the active-site cleft in opposition to a domain-separating inhibitory effect exerted by arginine, thus explaining the stimulatory effect of PII on the kinetics of arginine-inhibited NAGK.PII (GlnB) is now recognized as one of the most ancient and conserved signal transduction proteins known, with orthologs spread across the three domains of life (1, 2). Originally discovered as a factor necessary for the inactivation of Escherichia coli glutamine synthetase, PII is now known to play roles in the regulation of gene transcription, enzyme activity, and membrane channel function, all in response to cellular carbon, nitrogen, and energy status (3-6). PII interprets the metabolic status of the cell by directly binding to ATP and 2-ketoglutarate, and in certain bacteria, nitrogen status is sensed via covalent modification of PII. PII then directly interacts with a variety of proteins to regulate their function in response to the detected metabolic conditions.A eukaryotic PII protein has been discovered in several algae and higher plants. Arabidopsis thaliana PII is Ͼ50% identical to proteobacterial and cyanobacterial PII (7), but unlike these orthologs, it does not appear to be regulated by phosphorylation or uridylylation in response to nitrogen metabolites (8). Plant PII has a conserved chloroplast transit peptide that is cleaved upon entry into the chloroplast, where PII performs its function. PII knock-out plants display altered carbon and nitrogen metabolite levels as well as increased sensitivity to nitrite (9).To date, the sole interacting protein discovered for plant PII is the chloroplast enzyme N-acetylglutamate kinase (NAGK), 2 which catalyzes the second and rate-limiting step in the pathway of arginine biosynthesis (10). We (11) and others (12) have performed enzyme kinetics to define the role of PII in NAGK activity: although PII activates NAGK slightly, the primary purpose of binding appears to be relief of feedback inhi...

To investigate the chemopreventive effects of seaweed on breast cancer, we have been studying the relationship between iodine and breast cancer. We found earlier that the seaweed, wakame, showed a suppressive effect on the proliferation of DMBA (dimethylbenz(a)anthracene)-induced rat mammary tumors, possibly via apoptosis induction. In the present study, powdered mekabu was placed in distilled water, and left to stand for 24 h at 4°C. The filtered supernatant was used as mekabu solution. It showed an extremely strong suppressive effect on rat mammary carcinogenesis when used in daily drinking water, without toxicity. In vitro, mekabu solution strongly induced apoptosis in 3 kinds of human breast cancer cells. These effects were stronger than those of a chemotherapeutic agent widely used to treat human breast cancer. Furthermore, no apoptosis induction was observed in normal human mammary cells. In Japan, mekabu is widely consumed as a safe, inexpensive food. Our results suggest that mekabu has potential for chemoprevention of human breast cancer.

The effects of 1.0, 1.5, and 2.0 minimum alveolar concentration (MAC) of sevoflurane on hemodynamic, pulmonary and blood chemistry variables were measured during spontaneous and controlled ventilation in healthy horses. Sevoflurane was the only anesthetic drug administered to the horses. In a dose-dependent manner, sevoflurane significantly decreased (P < .05) mean arterial blood pressure, cardiac output, and stroke volume. There was a progressive decrease in peripheral vascular resistance and an increase in heart rate as the concentration of sevoflurane was increased, but the differences were not significant. During spontaneous ventilation there was a dose-dependent decrease in respiratory rate that caused a decrease in the minute volume. As the dose of sevoflurane increased, the arterial carbon dioxide tension also increased (P < .05). All blood chemistries remained within normal limits. Recovery from anesthesia was without incident. In conclusion, sevoflurane induces a dose-dependent decrease in hemodynamic variables and pulmonary function in horses that is not greatly different from that of other approved inhalant anesthetics.

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ABSTRACT. To determine a safe and efficacious dose of flecainide acetate for treating equine atrial fibrillation (Af), the safe dosage level was determined by injecting 1, 2, or 3 mg/kg i.v. of 1% flecainide acetate solution at a rate of 0.2 mg/kg/min to five clinically healthy horses. Clinical signs and the ECG were monitored (HR, PR, QRS, and QT intervals) and blood was taken to measure the plasma flecainide concentration pre-and post-administration. No abnormal signs were observed in the 1-or 2-mg/kg groups, while agitation was observed in three of five horses in the 3-mg/kg group. The QRS, and QT intervals for the 3-mg/kg group increased significantly. The peak plasma flecainide concentrations were 1,316 ± 358 (SD) ng/ml, 1,904 ± 314 ng/ml, and 2,251 ± 387 ng/ml for the 1-, 2-, and 3-mg/kg groups, respectively. To evaluate the efficacy of flecainide, Af was induced by right atrial pacing in six clinically healthy horses, and 1% flecainide acetate solution was then administered until they converted to sinus rhythm. All horses with induced Af converted. For the conversion, a total dose of 1.40 ± 0.63 mg/kg flecainide was required, the duration of administration was 7.00 ± 3.15 min and plasma flecainide concentration at conversion was 1,303 ± 566 ng/ml. In conclusion, flecainide acetate is a safe and effective antiarrhythmic agent for equine Af, and the clinically effective dosage is 1 to 2 mg/kg. Atrial fibrillation (Af) is one of the most common arrhythmias encountered in horses. It is thought that treatment is necessary for Af that does not recover spontaneously [1,4]. There have been a number of reports on Af in horses. Most of these studies used quinidine for treatment [1,11,14]. However, quinidine is known to have a number of side effects, including nasal mucosal edema, dyspnea, diarrhea, colic, tachycardia and laminitis [6,9]. Successful treatment has not been reported using any drug other than quinidine [10]. It would be highly desirable to convert equine Af, using an antiarrhythmic agent with less severe side effects than quinidine.In humans, a wide variety of antiarrhythmic agents have been developed and clinically applied to Af. Flecainide is an antiarrhythmic agent of Vaughan-Williams class Ic, a different class than quinidine [7]. In humans, flecainide has been shown to be effective in treating both supraventricular and ventricular arrhythmias. The rate of cardioversion of paroxysmal Af exceeds 90%, which is higher than that for other antiarrhythmic agents [2,8]. With side effects seldom involving organs other than the heart, flecainide is a relatively safe drug [5]. Nonetheless, flecainide has not been tried in horses, nor has its ability to convert equine Af been evaluated.In this study, we examined the safety and effectiveness of flecainide acetate in treating induced Af in horses. The efficacious dosage and rate of administration of flecainide was determined using clinical observations, ECG, and measurement plasma flecainide concentrations as indicators of side effects. MATERIALS AND METHODSStu...

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