Issues of academic integrity in U.S. dental schools.

Evidence of violations of academic integrity can be identified at all levels of education. A survey on academic integrity was mailed in 1998 to the academic deans of all fifty-five U.S. dental schools, with a response rate of 84 percent. This survey showed that reported incidents of academic dishonesty occur in most dental schools, with the average school dealing with one or two cases a year. The most common incidents of dishonest behavior involved copying or aiding another student during a written examinations; the second most common involved writing an untrue patient record entry or signing a faculty member's name in a patient chart. Respondents indicated the major reason for failure to report academic dishonesty was fear of involvement because of time and procedural hassles and fear of repercussions from students and peers.

Renal function following cardiac arrest and resuscitation in the canine.

Organ malfunction often occurs following cardiac arrest and resuscitation. Renal function, inulin clearance (Cln), was examined in 27 dogs before and after (days 2, 4, and 6) cardiac arrest and resuscitation. Group A (n = 7) had no ventricular fibrillation (VF), but cardiopulmonary support was applied for 20 min, and three transthoracic countershocks were delivered. In groups B (n = 7), C (n = 7), and D (n = 6) VF was induced for 2.5, 5.0, and 7.5 min, respectively, followed by cardiopulmonary support for 20, 20, and 15 min, respectively. When necessary, epinephrine and sodium bicarbonate were given during resuscitation. Countershock was applied for defibrillation. Kidneys were examined histologically in groups C and D. Following cardiac arrest, Cln was significantly less in the arrested groups compared to the nonarrested group. Within group C, which received the most epinephrine, Cln correlated negatively with epinephrine administration, and with the energy applied for defibrillation. Histologically, group C showed the highest incidence of cortical tubular cytoplasmic vacuolization, regeneration, inflammation, and tubular casts. Groups C and D showed outer medullary tubular cytoplasmic vacuolization, renal vascular changes, and calcification. In conclusion, cardiac arrest and resuscitation may precipitate acute renal hypofunction as well as reversible and irreversible morphological changes in normal functioning canine kidneys. The confounding effect of pre-existing renal disease remains to be examined experimentally.

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Identification of essential sulfhydryl residues in the primary sequence of the enzyme.

The kinase and sugar phosphate exchange reactions of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by treatment with 5'-p-fluorosulfonylbenzoyladenosine or 8-azido-ATP, but activity could be restored by the addition of dithiothreitol. This inactivation was accompanied by incorporation of 5'-p-sulfonylbenzoyl[8-14C]adenosine into the enzyme that was not released by the addition of dithiothreitol. The lack of effect of ATP analogs on the ATP/ADP exchange or on bisphosphatase activity and reversal of their effects on the kinase and sugar phosphate reactions by dithiothreitol suggest that 1) they reacted with sulfhydryl groups important for sugar phosphate binding in the kinase reaction, and 2) the inactivation of the kinase by these analogs involves a specific reaction that is not related to their general mechanism of attacking nucleotide-binding sites. In addition, alkylation of the enzymes' sulfhydryls with iodoacetamide prevented inactivation by 5'-p-fluorosulfonylbenzoyladenosine, suggesting that the same thiols were involved. o-Iodosobenzoate inactivated the kinase and sugar phosphate exchange; the inactivation was reversed by dithiothreitol; but there was no effect on the bisphosphatase or nucleotide exchange, indicating that oxidation occurred at the same sulfhydryl that are associated with sugar phosphate binding. ATP or ADP, but not fructose 6-phosphate, protected these groups from modification by 5'-p-fluorosulfonylbenzoyladenosine, 8-azido-ATP, and o-iodosobenzoate. ATP also induced dramatic changes in the circular dichroism spectrum of the enzyme, suggesting that adenine nucleotide protection of thiol groups resulted from changes in enzyme secondary structure. Analysis of cyanogen bromide fragments of 14C-carboxamidomethylated enzyme showed that all radioactivity was associated with cysteinyl residues in a single cyanogen bromide fragment. Three of these cysteinyl residues are clustered in a 38-residue region, which probably plays a role in maintaining the conformation of the kinase sugar phosphate-binding site.

Tissue distribution, immunoreactivity, and physical properties of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

6-Phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46) activities were determined in various rat tissues, the latter by using a method based on the formation of a phosphorylated enzyme intermediate during the course of catalysis. Both activities from liver, skeletal muscle, lung, kidney, and testis copurified during polyethylene glycol fractionation, anion-exchange and blue Sepharose chromatography, and gel filtration. The Stokes radius of these enzymes and of the liver bifunctional enzyme was 45 A. Extrahepatic tissues had only 10% or less of the kinase activity found in liver. The results indicate that a liver-type bifunctional enzyme is present in most extrahepatic tissues but that it is minimally expressed. However, the ratio of kinase to bisphosphatase activity in most extrahepatic tissues was 4- to 6-fold higher than in liver, whereas heart 6-phosphofructo-2-kinase had no associated bisphosphatase activity, although its Stokes radius was also 45 A. The heart enzyme was not precipitated by an antiserum to the liver enzyme, whereas only a fraction of the kidney and testis activities was precipitated by this antiserum. The data support the existence of a distinct form of extrahepatic 6-phosphofructo-2-kinase, most readily demonstrated in heart, which may not be bifunctional.

Effect of short-term energy intake level and exercise on oxygen consumption in men.

The influence of short-term energy intake and cycle exercise on oxygen consumption in response to a 1.5 MJ test meal was investigated in ten young, adult men. On the morning after a previous day's "low-energy" intake (LE regimen) of 4.5 MJ, the mean resting oxygen consumption increased by 0.7 ml X kg-1 X min-1 after the test meal (P less than 0.025). After a "high-energy" intake (HE regimen) of 18.1 MJ, the resting measurement was unchanged (+0.4 ml X kg-1 X min-1) after the meal (n.s.). These trends are the reverse of what would be expected if oxygen consumption in response to feeding is a factor in the acute control of body weight. The mean fasting oxygen consumption during cycle exercise at 56% of VO2max (constant work) for both LE and HE prior intakes was not different at 31.1 ml X kg-1 X min-1. Oxygen consumption during exercise increased after feeding by 0.5 ml X kg-1 X min-1 on the LE regimen (n.s.) and decreased by 1.2 ml X kg-1 X min-1 on the HE regimen (n.s.). These results are also the reverse of what would be expected if oxygen consumption in response to exercise is related to short-term energy intake.

Changes in key regulatory enzymes of hepatic carbohydrate metabolism after glucose loading of starved rats.

When glucose was given to starved rats there was an increase in both 6-phosphofructo 2-kinase and pyruvate kinase activity and a decrease in fructose 2,6-bisphosphatase activity 30 min and 60 min later. These changes were accompanied by an increase in glycogen deposition and by modest, but significant increases in fructose 2,6-bisphosphate levels at the same time. Metabolite measurements indicated that flux through 6-phosphofructo 1-kinase and pyruvate kinase were increased. These results suggest that although glycogen deposition may occur via the gluconeogenic pathway, glycolysis is activated at the same time by changes in the phosphorylation state of key regulatory enzymes as well as by the small rise in fructose 2,6-bisphosphate.

Differential effects of proteolysis and protein modification on the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes exchange reactions between ADP and ATP and between fructose-6-P and fructose-2,6-P2 at histidyl residues. Limited proteolysis of the enzyme with thermolysin yielded an enzyme core with a subunit molecular weight of 35,000-38,000. This enzyme core had no kinase activity and a 2-fold activated bisphosphatase activity whose sensitivity to the product inhibitor fructose-6-P was unchanged. The thermolysin-treated enzyme also did not catalyze the fructose-6-P/fructose-2,6-P2 exchange reaction but did catalyze the ADP/ATP exchange. These results suggest that 1) the enzyme's reactions may be catalyzed at two active sites, 2) there are at least two fructose-6-P binding sites, 3) the fructose-6-P/fructose-2,6-P2 exchange is catalyzed only at the kinase site, and 4) inactivation of the exchange and kinase reactions by thermolysin digestion is due to the loss of the fructose-6-P binding site of the kinase. Also consistent with these conclusions was the finding that oxidation of the enzyme with ascorbate/Fe3+ or H2O2 resulted in complete loss of the kinase activity as well as the fructose-6-P/fructose-2,6-P2 exchange but did not affect the bisphosphatase activity or the ADP/ATP exchange. Dithiothreitol could completely reactivate the ascorbate/Fe3+-inactivated enzyme, suggesting that oxidation occurred at a sulfhydryl group(s) essential for fructose-6-P binding in the kinase reaction. In addition, the kinase and fructose-6-P/fructose-2,6-P2 exchange reactions were more sensitive to inactivation by diethylpyrocarbonate than was the bisphosphatase. The different responses of the kinase and bisphosphatase reactions to the action of these various protein-modifying agents and to thermolysin digestion support the existence of a separate site for each reaction and an essential role for sulfhydryl groups at the sugar-phosphate-binding site(s) of the kinase.

Effect of sulfhydryl modification on the activities of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Alkylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with p-mercuribenzoate caused a rapid stimulation of the kinase and an inhibition of the bisphosphatase. At later times, the kinase activity also became inhibited. In contrast, treatment with N-ethylmaleimide abolished kinase activity but had no effect on the bisphosphatase. Selective modification of residues involved in the kinase reaction was also seen with iodoacetamide, which caused a 10-fold stimulation of the kinase Vmax without affecting the bisphosphatase. The stimulatory effect of carboxyamidomethylation was seen when the kinase was assayed in the presence of inorganic phosphate, an allosteric activator of the enzyme. The iodoacetamide-treated enzyme had a 10-20-fold higher Km for fructose 6-phosphate than the native enzyme and the Ki for fructose 2,6-bisphosphate was also increased. However, the adenine-nucleotide site did not seem to be affected since there was no change in the Km for ATP, the Ki for ADP, or the adenine-nucleotide exchange. There was also a direct correlation between the incorporation of [14C]acetamide into the enzyme and activation of the kinase. The residues modified by iodoacetamide were shown to be cysteines by the exclusive appearance of carboxymethylcysteine in protein hydrolysates. Activation was associated with alkylation of 2 cysteines/subunit, of the 12 which could be alkylated after denaturation/reduction. Iodoacetamide-activated kinase was inhibited by ascorbate/Fe3+, which has been shown to modify sulfhydryl groups in the native enzyme, with concomitant loss of kinase activity.

Characterization of the exchange reactions of rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase.

The Fru-6-P/Fru-2,6-P2 exchange reaction of rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was almost entirely dependent on the presence of Pi. This exchange was not due to a reversal of the bisphosphatase nor to trace amounts of adenine nucleotide in the enzyme. Exchange activity was maximal at pH 7, activated by ADP, and equal to 10-15 percent of the kinase Vmax. The ADP/ATP exchange reaction was more resistant to various protein modifying agents than the kinase. These studies confirm the existence of both exchange reactions but do not prove they are related to the kinase reaction.

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.