Acute improvement in histological outcome by MK-801 following focal cerebral ischemia and reperfusion in the cat independent of blood flow changes. The present study reports on the acute effects of MK-801 on the histopathological outcome and blood flow changes during focal cerebral ischemia and reperfusion. In addition, acute changes in the EEG and blood pressure are also reported. In 16 halothane-anesthetized cats, the left middle cerebral artery (MCA) was occluded for 2 h followed by 4 h of reperfusion. Thirty minutes after the onset of ischemia, eight animals were treated with 1 mg/kg of MK-801, while eight animals received saline. Blood flow from the peripheral MCA territory was measured with H2 clearance. There was a comparable reduction in blood flow (down to 20% of control) in the ischemic gyri of the two groups followed by a partial recovery after recirculation. There was a similar decrease in the EEG amplitude over the ischemic central MCA territory in the treated and the untreated group. Treatment with MK-801 induced a burst suppression in the EEG and a transient drop (11.4 +/- 6.5 mm Hg) in the mean arterial pressure. The volume of early ischemic damage decreased by one-third in the MK-801-treated group compared to the untreated one, both in the total hemisphere (from 29 +/- 10 to 20 +/- 5%) and in the hemispheric cortex (range 36 +/- 8 to 24 +/- 13%). A major fraction of this improvement was localized to the middle and posterior parietal (mainly perifocal) regions of the MCA territory. These results show that in our model, MK-801 improves histopathological outcome despite the lack of apparent effect on the cortical blood flow, and an adverse effect on the systemic blood pressure. This is the first report that describes data on a reproducible model of reperfusion after temporary occlusion of the MCA in a cat, extending the findings of the Glasgow group, who observed similar neuroprotection in models of permanent MCA occlusion.


Alteration of voltage-dependent calcium channels in canine brain during global ischemia and reperfusion. Elevated intracellular calcium (iCa2+) plays an important role in the pathophysiology of ischemic brain damage. The mechanisms by which iCa2+ increases are uncertain. Recent evidence implicates the voltage-dependent calcium channel (VDCC) as a likely site for the alteration in Ca2+ homeostasis during ischemia. The purpose of this study was to determine whether VDCCs are altered by global ischemia and reperfusion in a canine cardiac arrest, resuscitation model. We employed the radioligand, [3H]PN200-110, to quantitate the equilibrium binding characteristics of the VDCCs in the cerebral cortex. Twenty-five adult beagles were separated into four experimental groups: (a) nonischemic controls, (b) those undergoing 10-min ventricular fibrillation and apnea, (c) those undergoing 10-min ventricular fibrillation and apnea followed by spontaneous circulation and controlled respiration for 2 and (d) 24 h. Brain cortex samples were taken prior to killing of the animal, frozen immediately in liquid nitrogen, and crude synaptosomal membranes isolated by differential centrifugation/filtration. After 10 min of ischemia the maximal binding (Bmax) of [3H]PN200-110 increased to greater than 250% of control values (control Bmax 11.16 +/- 0.98; ischemic 28.35 +/- 2.78 fmol/mg protein; p less than 0.05). Bmax returned to near control values after 2 h of reperfusion but remained significantly greater than the control at 24 h. Although the affinity constant (Kd) (control = 0.12 +/- 0.03 nM) appeared to increase with ischemia and normalize with reperfusion, the changes were not statistically significant. We conclude that the binding of [3H]PN200-110 to L-type VDCCs is increased after 10 min of global ischemia/anoxia produced by ventricular fibrillation and apnea in the dog.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of sumatriptan, a selective 5-HT1-like receptor agonist, on pial vessel diameter in anaesthetised cats. The action of sumatriptan, a selective 5-HT1-like receptor agonist that is effective for the acute treatment of migraine, was compared on pial vessel diameter following perivascular or intravenous administration to anaesthetised cats. Sumatriptan (0.01-10 microM), when microinjected perivascularly, caused a decrease in pial artery diameter (maximum change of -19 +/- 9%; mean +/- SD) but had no effect on the diameter of pial veins. Sumatriptan (1 microM)-induced pial artery vasoconstriction was unaffected by coadministration of ketanserin (1 microM) or ondansetron (1 microM) but was significantly (p less than 0.01) attenuated by methiothepin (1 microM). Intravenous infusion of a clinically effective dose of sumatriptan (64 micrograms/kg/10 min) caused selective carotid vasoconstriction (22 +/- 6% increase in carotid vascular resistance with little or no change in blood pressure or heart rate) and no change in pial artery diameter, although sumatriptan (1 microM) administered perivascularly in these animals before and after the infusion caused pial artery vasoconstriction. These results demonstrate that perivascularly administered sumatriptan causes pial artery vasoconstriction via activation of 5-HT1-like receptors. However, intravenously administered sumatriptan does not cause pial artery vasoconstriction, which suggests that sumatriptan does not readily penetrate the cerebrovascular intima.


Recovery of responses to ovine corticotropin-releasing hormone after withdrawal of a short course of glucocorticoid. To characterize the recovery of the hypothalamic-pituitary-adrenal axis from suppression by short-term glucocorticoid treatment, we examined the responses to ovine CRH (oCRH) before and after prednisolone administration. Eight normal male volunteers were studied before (control) and after administration of 25 mg prednisolone twice daily orally for 14 days. Data are mean +/- SEM. The ACTH basal level was suppressed 24 h after prednisolone withdrawal (1.7 +/- 0.4 pmol/L vs. control, 3.5 +/- 0.6, P less than 0.02), but the ACTH response to oCRH was not significantly different from control (peak 12.8 +/- 2.0 pmol/L vs. 13.5 +/- 12.1, respectively). Seventy-two h post prednisolone basal ACTH levels had recovered to pretreatment values. Cortisol levels, both basal and in response to oCRH, were significantly suppressed 24 h post prednisolone (P less than 0.001). By 72 h post prednisolone, both basal and oCRH-stimulated cortisol had recovered to pretreatment levels. Dehydroepiandrosterone (DHEA), both basal and stimulated, was significantly suppressed 24 h post prednisolone (P less than 0.001). In contrast to cortisol, basal and peak DHEA remained suppressed 72 h post prednisolone (basal DHEA 9.1 +/- 1.1 nmol/L, P less than 0.05 vs. control; peak DHEA 20.0 +/- 3.3 nmol/L, P less than 0.01 vs. control). When expressed as percent rise, however, the DHEA response to oCRH was not significantly different from control. DHEA sulfate (DHEAS) was significantly lower than control at both 24 and 72 h post prednisolone (1.8 +/- 0.3 and 3.3 +/- 0.4 mumol/L respectively; control 7.2 +/- 0.7 mumol/L; P less than 0.001). The ratio of basal DHEA to DHEAS was significantly higher than control 72 h post prednisolone, indicating that DHEAS was more profoundly suppressed than DHEA. We conclude that after a short course of prednisolone pituitary ACTH secretion is the first parameter of the hypothalamic-pituitary-adrenal axis to recover. Hypothalamic secretion of CRH recovers next, followed by recovery of cortisol secretion. Secretion of DHEA and DHEAS remain suppressed after recovery of cortisol. This suppression may be caused by inhibition of sulfokinase activity by glucocorticoid.


Identification and initial characterization of insulin receptor-like immunoreactivity in human plasma. With a two-step purification procedure employing sequential affinity chromatography with insulin receptor monoclonal antibody followed by wheat germ agglutinin, we isolated from the plasma of two healthy individuals a material that reacted in a specific RIA for insulin receptors. This material produced dilution curves that were parallel to a human placental insulin receptor standard. This material also bound [125I]insulin; competition-inhibition curves revealed an ED50 of 0.3 nM, a value similar to that obtained with placental insulin receptors. The material purified from plasma was then labeled with [125I] Bolton-Hunter reagent, followed by polyacrylamide gel electrophoresis under reducing conditions and autoradiography. A band at 135 kilodaltons (kDa) was observed, corresponding to the alpha-subunit of the insulin receptor. Several bands ranging from 82-46 kDa were also detected. One or more of these fragments had intrinsic autophosphorylation activity, but only the 82-kDa band activity was responsive to insulin. In addition, employing the synthetic substrate poly(Glu4:Tyr1), no insulin-sensitive tyrosine kinase activity was present. These studies demonstrate, therefore, that insulin receptor-derived material is present in human plasma. This material retains high affinity insulin binding, but has an altered beta-subunit that is devoid of insulin-responsive tyrosine kinase activity.


An arginine to histidine mutation in codon 315 of the c-erbA beta thyroid hormone receptor in a kindred with generalized resistance to thyroid hormones results in a receptor with significant 3,5,3'-triiodothyronine binding activity. Generalized resistance to thyroid hormones results from diverse mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor, and different kindreds have variable phenotypes. However, the T3-binding affinities of these mutant receptors studied in vitro have all been severely reduced compared to wild type. We report here a new kindred, CL, with a mutation further upstream than previously reported, a guanine to adenine base substitution at nucleotide 1244 in codon 315 changing an arginine to histidine. This base substitution was the only one found in codons 90-456 of genomic sequence and was formally shown to be a mutation by screening 51 random individuals. The kindred CL receptor complementary DNA was recreated, and the mutant receptor synthesized with rabbit reticulocyte lysate had a T3-binding affinity of 2.4 +/- 0.9 x 10(10) M-1 compared to the wild-type human placental receptor affinity of 5.2 +/- 1.6 x 10(10) M-1. Affected members of this kindred appeared clinically to have a relatively mild degree of resistance with mean total thyroxine of only 192 +/- 24 nmol/L and inappropriately normal TSH levels. Kindred CL is an example of mild generalized resistance to thyroid hormones correlated with a mutation in the beta-receptor that resulted in only a modest deficiency in T3-binding activity.


Androgenic hormones during prolonged physical stress, sleep, and energy deficiency. Androgenic hormones were investigated during two separate 5-day military endurance training courses, with physical activities around the clock corresponding to a daily energy consumption of about 40,000 kilojoules, but with an intake of only 2,000 kilojoules. Altogether, the cadets slept for 1-3 h in the 5 days. Eleven male cadets participated in course I, and 10 in course II. Plasma levels of testosterone, free testosterone, dehydroepiandrosterone, 17 alpha-hydroxyprogesterone, and androstenedione decreased by 60-80% during the course. In contrast, plasma cortisol, aldosterone, progesterone, and dehydroepiandrosterone sulfate increased. LH, FSH, and ACTH decreased to about 50-80% of precourse levels. Weak correlations between plasma levels of hypophyseal and levels of adrenal and testicular hormones indicate a multifactorial regulation. In conclusion, both adrenal and testicular androgens decrease during prolonged physical strain combined with energy and sleep deficiency.


Effect of a short-term treatment with recombinant growth hormone (GH) on adrenal responsiveness to corticotrophin stimulation in children affected by isolated GH deficiency. Recent evidence suggests that GH and insulin growth factor-I (IGF-I) play a role in adrenal steroidogenesis. On the other hand, it has been shown that ACTH stimulates IGF-I secretion by cultured fasciculata adrenal cells. Aim of the present study was to investigate the influence of GH administration on adrenal steroids and IGF-I responsiveness to ACTH in children affected with isolated GH deficiency. Ten children (seven males and three females, 5-10 yr old) affected with isolated GH deficiency underwent a synthetic ACTH 1-17 test before and after administration of human recombinant GH at a dose of 4 IU/day sc for 10 days. After the therapy, no significant differences were detected in the responses of cortisol, dehydroepiandrosterone-sulfate, androstenedione, and 17-hydroxyprogesterone to ACTH 1-17, whereas an increased 11-deoxycortisol responsiveness to ACTH 1-17 was noted (P less than 0.005). Surprisingly, IGF-I significantly increased in response to ACTH 1-17 after short-term rGH administration (P less than 0.006). In conclusion, our data indicate that in isolated GH deficiency a short-term GH therapy does not substantially modify the adrenal responsiveness to exogenous ACTH, even if an increased 11-deoxycortisol and an induced IGF-I responsiveness to ACTH were observed.


Mineralocorticoids and acidosis regulate H+/HCO3- transport of intercalated cells. The effects of acidosis and mineralocorticoids on cellular H+/HCO3- transport mechanisms were examined in intercalated cells of the outer stripe of outer medullary collecting duct (OMCDo) from rabbit. Intracellular pH (pHi) of intercalated cells was monitored by fluorescence ratio imaging using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). pHi recovered from an acid load at 2.8 +/- 0.5 x 10(-3) pHU/s in the absence of ambient Na+. This pHi recovery rate was similar in chronic acidosis induced by NH4Cl loading, but it was enhanced (+111%) by treatment with deoxycorticosterone acetate (DOCA). In a DOCA-treated group, luminal 10 microM SCH28080 and 0.1 mM omeprazole, H(+)-K(+)-ATPase inhibitors, did not change the pHi recovery rate, while luminal 0.5 mM N-ethylmaleimide blocked the rate by 68%. DOCA, but not acidosis, increased (approximately 40%) initial pHi response to bath HCO3- or Cl- reduction in Na(+)-free condition. After an acid load in the absence of Na+ and HCO3-, pHi response to basolateral Na+ addition was stimulated (+66%) by acidosis, but not by DOCA. Our results suggest that (a) mineralocorticoids stimulate H+/HCO3- transport mechanisms involved in transepithelial H+ secretion, i.e., a luminal NEM-sensitive H+ pump and basolateral Na(+)-independent Cl(-)-HCO3- exchange; and (b) acidosis enhances the activity of basolateral Na(+)-H+ exchange that may be responsible for pHi regulation.


Parathyroid hormone decreases HCO3 reabsorption in the rat proximal tubule by stimulating phosphatidylinositol metabolism and inhibiting base exit. The mechanism of inhibition of HCO3 transport by parathyroid hormone (PTH) in the proximal tubule is not clearly defined. Previous studies in vitro have suggested that this effect is mediated via cAMP generation, which acts to inhibit Na/H exchange, resulting in cell acidification. To examine this question in vivo, intracellular pH (pHi) was measured in the superficial proximal tubule of the rat using the pH-sensitive fluoroprobes 4-methylumbelliferone (4MU) and 2',7'-bis(carboxyethyl)-(5, and 6)-carboxyfluorescein (BCECF). PTH was found to alkalinize the cell. This alkalinization suggested inhibition of basolateral base exit, which was confirmed by in situ microperfusion studies: lowering HCO3 in peritubular capillaries acidified the cell, an effect blunted by PTH. Removal of luminal Na promoted basolateral base entry, alkalinizing the cell. This response was also blunted by PTH. Readdition of luminal Na stimulated the luminal Na/H exchanger, causing an alkalinization overshoot that was partially inhibited by PTH. cAMP inhibited luminal H secretion but did not alkalinize the cell. Stimulation of phosphatidylinositol-bis-phosphate turnover by PTH was suggested by the effect to the hormone to increase cell Ca. Blocking the PTH-induced rise in cell Ca blunted the effect of the hormone to alkalinize the cell, as did inhibition of phosphatidylinositol breakdown. Furthermore, stimulation of protein kinase C by a phorbol ester and a diacylglycerol applied basolaterally alkalinized the cell and inhibited luminal H secretion. The findings indicate that both arms of the phosphatidylinositol-bis-phosphate cascade play a role in mediating the effect of PTH on the cell pH. The results are consistent with the view that PTH inhibits base exit in the proximal tubule by activation of the phosphatidylinositol cascade. The resulting alkalinization may contribute, with cAMP, to inhibit apical Na/H exchange and the PTH-induced depression of proximal HCO3 reabsorption.


Priming of polymorphonuclear neutrophils by atrial natriuretic peptide in vitro. In ischemia-reflow states of coronary artery disease, the activation of PMN precedes the initiation of tissue damage. Release of atrial natriuretic peptide (ANP) from myocytes occurs within minutes after the onset of myocardial ischemia, which suggests a possible role of ANP in PMN activation. To investigate this possibility, we tested the effects of ANP on functions of PMN in vitro. ANP is a potent signal for priming the PMN respiration burst to secrete superoxide anion. Phorbol 12-myristate 13-acetate, opsonized zymosan, or FMLP could all be used as triggering stimuli to demonstrate the priming of PMN activation by ANP. Only ANP fragments 1-28 and 7-28 enhanced respiration burst activity but identical preparations of ANP fragments 13-18 or 1-11 failed to do so. This structure-activity relationship is typical of receptors for ANP found in other tissues. In addition, ANP stimulated the release of beta-glucuronidase From PMN triggered by FMLP. The observed inhibition by ANP of FMLP-stimulated chemotaxis of PMN may be due to their enhanced adhesiveness. These data show that a classic cardiac hormone is involved in regulating important functional activities of PMN. These data support the possibility that ANP could act as a preinflammatory substance in ischemia-reperfusion states and myocardial necrosis.


Identification of a thermolabile component of the human neutrophil NADPH oxidase. A model for chronic granulomatous disease caused by deficiency of the p67-phox cytosolic component. Mild heating of human neutrophils inactivates the respiratory burst oxidase, producing a defect in superoxide production and bacterial killing comparable to that seen in patients afflicted with chronic granulomatous disease (CGD). We have now investigated the mechanism and specificity of this inactivation by examining the effect of mild heating on the known oxidase components: the membrane-bound subunits of the cytochrome b558 (gp91-phox and p22-phox) and the two cytosolic oxidase factors (p47-phox and p67-phox). Heating (46 degrees C for 7.5 min) caused intact neutrophils to lose greater than 85% of their capacity to produce superoxide, a defect which was localized to the cytosolic, but not the membrane, fraction. Complementation studies with CGD cytosols deficient in either p47-phox or p67-phox suggested that the defective component of heat-inactivated cytosol was p67-phox. This was confirmed by experiments showing that recombinant p67-phox, but not p47-phox, exhibited lability at 46 degrees C and completely reconstituted oxidase activity of heat-treated cytosol. These studies indicate that mild heating of either intact neutrophils or normal neutrophil cytosol results in a selective inactivation of p67-phox, providing a model oxidase system for the extremely rare p67-phox-deficient form of CGD.


Platelet activating factor mediates interleukin-2-induced lung injury in the rat. Interleukin-2 was recently shown to cause acute lung injury characterized by microvascular permeability defect, interstitial edema, and leukosequestration. Similar responses can also be produced by platelet activating factor (PAF). Thus, the present study aimed to examine whether PAF plays a key role in the development of IL-2-induced lung injury in the anesthetized rat. Intravenous infusion (60 min) of recombinant human IL-2 at 10(5)-10(6) U/rat (n = 7-9) dose-dependently elevated lung water content (27 +/- 1%, P less than 0.01), myeloperoxidase activity (+84 +/- 23%, P less than 0.05), and serum thromboxane B2 (990 +/- 70%, P less than 0.01), but failed to alter blood pressure, hematocrit, serum tumor necrosis factor-alpha, and circulating leukocytes and platelets. Pretreatment (-30 min) with a potent and specific PAF antagonist, BN 50739 (10 mg/kg, intraperitoneally, n = 6) prevented the pulmonary edema (P less than 0.05) and thromboxane B2 production (P less than 0.01), and attenuated the elevation of lung myeloperoxidase activity (+18 +/- 16%, P less than 0.05) induced by IL-2. These data suggest that PAF is involved in the pathophysiological processes leading to IL-2-induced lung injury, and point to the potential therapeutic capacity of PAF antagonists in preventing pulmonary edema during IL-2 therapy.


High levels of circulating soluble receptors for tumor necrosis factor in hairy cell leukemia and type B chronic lymphocytic leukemia. The presence of soluble tumor necrosis factor (TNF) binding proteins (BP) was investigated in the sera of healthy volunteer blood donors and cancer patients. Two distinct types of TNFBP, types A and B, which are immunologically related to the cellular 75-kD TNF receptor (TNFR) and the cellular 55-kD TNFR, respectively, were assessed by immunoassays using nonblocking anti-receptor antibodies and 125I-recombinant human TNF alpha. As compared to the titers observed in 25 healthy controls, TNFBP types A and B titers were found to be elevated in almost all sera obtained from patients with underlying malignant disease. The highest amounts of TNFBP were seen in the sera of patients with B cell malignancies including hairy cell leukemia (HCL) and type B chronic lymphocytic leukemia. Treatment of HCL patients with recombinant human interferon-alpha was associated with decrease of circulating TNFBP.


In situ degradability of fresh grass and grass conserved under different harvesting methods. The effects of forage conservation and harvesting method on DM and CP degradability of grass cut at the boot stage were determined using nylon bags incubated in the rumen of two fistulated lactating cows. Samples of fresh grass, wilted grass prior to and after ensiling in a stack silo and cut with either a cylinder-type forage harvester (11.3 mm of length cut) or a self-loading wagon (42.4 mm of length cut), wilted grass prior to and after ensiling in large round bales, and grass hay were obtained from the same field and used for determination of DM and CP degradability. The DM-soluble fraction of fresh grass was significantly lower than that of wilted grass and silage, but it was higher than that of hay. In general, the potentially degradable fraction of all treatments was related inversely to the readily soluble fraction in water. The effective degradability of DM of fresh grass was similar to that of hay but was lower than that of wilted grass and silage. The effective degradability of DM of silage was higher than that of wilted grass and hay. The disappearance rate of CP of fresh grass was similar to that of wilted grass, hay, and silage. Silage had a lower disappearance rate of CP than wilted grass. The effective degradability of CP was similar for fresh and wilted grasses. Protein degradability was greater for silages than for fresh and wilted grasses. Protein degradability of hay was similar to that of fresh grass and wilted grass.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of hay making on in situ nitrogen degradability of forages in cows. The influence of hay making on in situ N degradability of forages was assessed by comparing the degradability of nine hays with that of standing forages at the time of harvest. Three ryegrass, two cocksfoot, and three alfalfa forages were used. The N degradability of hays was related closely to that of the corresponding standing forages (R = .91) and was, on average, lower (-2.5 points). This decrease in degradability after hay making was not constant; it increased when the degradability of the standing forage was high and when harvesting conditions were poor.


Nutrient digestion and lactation performance of dairy cows fed combinations of prilled fat and canola oil. Four combinations of prilled fat and canola oil were fed to 10 lactating Holstein cows in a replicated 5 x 5 Latin square to determine whether mixing plant oil with a rumen inert fat had additive effects on digestive and lactation responses. Five diets of concentrate and corn silage (1:1, DM basis) contained either no added fat (control) or 5% fat comprising 100, 67, 33, or 0% prilled fat and the remainder canola oil. The fat supplement containing 100% prilled fat appeared to be rumen-inert because it caused no changes in ruminal VFA concentration, acetate to propionate ratio, or total tract fiber digestion. Prilled fat increased milk production, FCM, and milk fat percentage but decreased milk protein percentage, including casein content. Increasing canola oil in the fat supplement caused linear declines in ruminal VFA, acetate to propionate ratio, and milk production. Milk production efficiency (weight FCM/weight DMI) exceeded the control diet when fat supplements contained 100 or 67% prilled fat but dropped below control for 33 and 0% prilled fat. This study demonstrates additive effects of combining canola oil with hydrogenated, prilled fat on ruminal fermentation but nonadditive effects on milk production efficiency and milk composition. At low levels of supplementation, plant oils, such as the canola oil used in this study, can inhibit ruminal fermentation but still maintain milk production efficiency.


A buffer value index to evaluate effects of buffers on ruminal milieu in cows fed high or low concentrate, silage, or hay diets. Our objective was to develop a buffer value index that would incorporate alterations in both ruminal fluid pH and buffering capacity as indicators of the influence of dietary buffering and alkalinizing agents on ruminal acid-base status. This index was evaluated using ruminal fluid from four lactating Holstein cows fed either sorghum silage or alfalfa hay in high or low concentrate diets. Ruminal fluid was incubated in vitro for 1, 2, 3, 4, or 5 h with no buffer or with 7.1 g of either NaHCO3, sodium sesquicarbonate, or a multielement buffer added per liter of ruminal fluid. Ruminal fluid pH was lower for diets based on high concentrate or alfalfa; buffering capacity between pH 5 and 7 was greater for high concentrate diets but was not affected by forage type. Ruminal fluid pH was higher for sesquicarbonate than for NaHCO3, the multielement buffer, or the control; however, ruminal fluid H+ concentration was similar between sesquicarbonate and NaHCO3, and both were lower than for the multielement buffer. Hydrogen ion concentration for the multielement buffer was lower than for the control. Buffering capacity was highest for NaHCO3, followed by sesquicarbonate, the multielement buffer, and the control. The buffer value index, which accounted for alterations in both H+ concentration and buffering capacity, was highest for NaCHO3, followed by sesquicarbonate, the multielement buffer, and the control. The poor response to the multielement buffer may be attributable to our relatively short incubation interval (less than 5 h). Dietary buffers increase both ruminal fluid pH and buffering capacity; both of these responses are beneficial.(ABSTRACT TRUNCATED AT 250 WORDS)


Dehydroepiandrosterone sulphate plasma levels in normal women and patients with benign breast disease. Dehydroepiandrosterone sulphate plasma levels were measured in patients with benign breast disease and in healthy women. In addition the adrenal secretion of dehydroepiandrosterone sulphate was assessed by means of an ACTH stimulation test in some patients and control subjects. The results obtained demonstrate that dehydroepiandrosterone sulphate plasma levels of patients with benign breast disease overlap those found in controls and that the adrenal response to ACTH of patients with breast pathology does not differ from that of healthy women.


Epstein-Barr virus serology in the chronic fatigue syndrome. The antibody profiles against Epstein-Barr virus were studied in 136 patients presenting with chronic fatigue syndromes. These profiles were compared with a panel of sera from blood donors. The patients exhibited higher titres in a combined assay for antibodies to the Restricted (R) and Diffuse (D) components of the Early Antigen complex than controls (P less than 0.001) but titres against these antigens were not useful on an individual patient basis. The patients who displayed elevated titres of antibodies to Early Antigens did not differ clinically from those displaying titres in the control range. Four of nine patients who had increased antibodies to Early Antigens also had evidence of active enterovirus infection.


Increased frequency of coxsackie B virus IgM in women with spontaneous abortion. IgM antibodies to Coxsackie B virus serotypes 1-5 (CBV 1-5) were studied by radioimmunoassay (RIA) of blood samples from women who had undergone a spontaneous abortion or given birth to a stillborn child. Results were compared with those of controls comprising healthy pregnant women who had not suffered such experiences. Among 48 women with abortions, 16 (33%) had CBV-IgM, while among the controls only three of 37 (8%) had these antibodies, a statistically significant difference (P less than 0.025). Among 21 women with a stillborn child, 11 (52%) were CBV-IgM positive, while the corresponding proportion in their controls was four of 18 (22%), a difference which is not statistically significant. It is concluded that CBV may be an important causative agent in spontaneous abortions and possibly also in stillbirths.


Differential regulation of retinoic acid receptors and binding proteins in human skin. Many of the pleiotropic effects of retinoids are likely to be mediated by nuclear retinoic acid receptors (RAR) acting as ligand-dependent enhancer factors. However, in previous studies we have been unable to document altered RAR expression at the RNA level in response to retinoic acid (RA) treatment or in psoriatic lesions, conditions characterized by marked alterations in keratinocyte proliferation and differentiation, which are either caused by or responsive to RA. In an attempt to identify other potential regulators of RA responsiveness, we have used RNA blot hybridization to study the expression of the cellular retinoic acid binding proteins (CRABP) CRABP-I and CRABP-II, the RAR-gamma isoforms RAR-gamma 1 and RAR-gamma 2, and the low-affinity RAR homologue RXR in normal, RA-treated, and psoriatic human epidermis. CRABP-II is selectively and markedly induced by RA in adult human skin (J Biol Chem 266:17662-17666, 1991). However, in submerged, serum-free keratinocyte cultures, CRABP-II mRNA could not be induced by RA. Comparisons of intact human skin, submerged keratinocyte cultures, and human skin equivalent cultures indicated that induction of CRABP-II by RA requires epidermal stratification, dermal-epidermal interactions, or both. CRABP-II transcripts were also expressed in heat-separated human dermis at levels similar to those found in epidermal keratome biopsies, whereas CRABP-I transcripts were undetectable in dermal RNA. CRABP-II transcripts were markedly elevated in psoriatic lesions, as they were in RA-treated skin. In contrast, CRABP-I mRNA was undetectable and not increased in psoriatic lesions. Expression of RAR-gamma isoforms and RXR was not detectably altered in either psoriatic lesions or in RA-treated skin. Thus, altered expression of CRABP-II appears more likely to regulate the cutaneous actions of RA than does altered expression of CRABP-I, RXR, or RAR-gamma isoforms. From these and other results, a model for regulation of RA action involving sequestration of RA by CRABP-II is proposed.


Anthralin decreases keratinocyte TGF-alpha expression and EGF-receptor binding in vitro. Anthralin is an effective topical treatment for active psoriasis; however, its mechanism of action is unknown. Both TGF-alpha and its receptor, the EGF receptor, are overexpressed in active psoriatic plaques and might, therefore, play a role in psoriatic epidermal hyperplasia. In order to assess whether anthralin might act via alteration of this growth factor pathway, we examined the in vitro effects of pharmacologic concentrations of anthralin on cultured normal human keratinocytes. Keratinocyte proliferation was inhibited by 98% at an anthralin concentration of 10 ng/ml. In contrast, lymphocyte proliferation was inhibited by only 50% at an anthralin concentration of 10 micrograms/ml. Anthralin treatment did not induce cell-cycle-specific growth arrest as assessed by flow-cytometric analysis of acridine-orange-stained keratinocytes. Northern analysis of anthralin-treated keratinocytes demonstrated a marked decrease in TGF-alpha mRNA expression. Anthralin-treated keratinocytes showed decreased binding of 125I-EGF and 125I-IGF-I to their respective receptors, but EGF receptor binding was inhibited to a greater extent. Anthralin decreased ligand-binding affinity and cell-surface numbers of EGF receptors as assessed by Scatchard analysis of 125I-EGF binding to anthralin-treated keratinocytes. These results indicate that anthralin alters components of the EGF receptor pathway in cultured keratinocytes and that these effects might contribute to the clinical efficacy of anthralin in the treatment of active psoriasis.


Contact allergens modulate the expression of MHC class II molecules on murine epidermal Langerhans cells by endocytotic mechanisms. MHC class II molecules play an important role during the sensitization phase of allergic contact dermatitis. To study the influence of contact allergens on the expression of these molecules by murine epidermal Langerhans cells (LC), we performed a flow-cytofluorometric analysis of the Ia-antigen expression after in vivo application of contact allergens. A distinct decrease in the Ia-antigen expression of the entire LC population was noticed 3 h after in vivo application of the contact allergen 2,4-dinitrofluorobenzene (DNFB). This decrease was transient and balanced 24 h after in vivo application of DNFB. A downregulation was also detectable after in vivo application of the contact allergens 1-chloro-2,4-dinitrobenzene (DNCB), oxazolone, K2Cr2O7, 2,4,6-trinitrochlorobenzene (TNCB), and toxic concentrations of the irritant compound sodium dodecyl sulfate (SDS). In vitro studies showed that freshly prepared as well as 3-d cultured LC downregulated their Ia-antigen expression in the presence of DNFB, which was used as a model compound. This decrease was not inhibited by the MHC class II molecule transport-inhibitor brefeldin A nor by the ionophore monensin. The inhibition of receptor-mediated endocytosis with hypertonic media (0.45 M sucrose) abolished the DNFB-mediated downregulation of Ia-antigen expression. An accelerated clearance of cell-surface-expressed antibody-labeled IA molecules was detectable in the presence of DNFB. Internalization studies carried out with peroxidase-labeled anti-IA-antibody complexes showed remarkable alterations in the intracellular distribution of endocytosed material under the influence of subtoxic concentrations of DNFB, DNCB, K2Cr2O7, and TNCB. The irritant substance sodium dodecyl sulfate (SDS) influenced the intracellular distribution pattern of internalized material only when used in toxic concentrations. An augmented participation of MHC class II molecules in endocytotic processes is mediated by reactive substances like contact allergens and might contribute to the processing and presentation of these compounds.


Pentoxifylline inhibits certain constitutive and tumor necrosis factor-alpha-induced activities of human normal dermal fibroblasts. Pentoxifylline (PFN), analog of theobromine, which phenotypically and functionally alters various cell types including dermal fibroblasts, has been reported to inhibit tumor necrosis factor-alpha (TNF alpha) activation of neutrophils. We investigated the ability of PFN to alter constitutive and TNF alpha-induced biosynthetic activities of human normal dermal fibroblasts. The sixteenfold increase over constitutive intracellular 2'-5' oligo-adenylate synthetase (2'-5' A synthetase) activity induced by TNF alpha (400 U/ml) failed to occur when PFN (1 mg/ml) was added prior to cytokine treatment. This loss of biologic activity paralleled a reduction in 2'-5' A synthetase proteins and 2'-5' A synthetase-specific m-RNA. PFN failed to inhibit constitutive or TNF alpha-induced IL-6 hybridoma proliferative activity, IL-6 protein, or IL-6-specific m-RNA levels. The presence of PFN (1 mg/ml) in fibroblast cultures reduced constitutive synthesis of collagen and glycosaminoglycan (GAG) by 87% and 45%, respectively, and blocked induction of their synthesis by TNF alpha (10(4) U/ml). Total non-collagenous protein synthesis was not inhibited following PFN treatment (1 mg/ml). PFN did not inhibit TNF alpha induction of only those biosynthetic activities also susceptible to PFN in the constitutive state, with PFN failing to reduce constitutive collagenolytic activity but reducing TNF alpha-induced enhanced collagenolytic activity by 26% and collagenase m-RNA by 51%. Furthermore, PFN did inhibit, by 98%, TNF alpha-dependent murine and human fibroblast cytotoxicity. The selective nature of PFN inhibition of certain TNF alpha activities, the failure of PFN (1 mg/ml) to alter constitutive and TNF alpha-induced levels of type 1 and 2 TNF alpha receptor m-RNA, and the finding that PFN-treated fibroblasts express a similar number of receptors, of similar molecular weight and high affinity for TNF alpha as control, untreated cells, suggest that inhibitory activities of PFN are mediated at a locus other than receptors for TNF alpha.


Electron paramagnetic resonance (EPR) imaging in skin: biophysical and biochemical microscopy. Electron paramagnetic resonance (EPR) is a spectroscopic technique that allows detection of paramagnetic compounds, e.g., free radicals, in skin, and is used for measuring skin membrane fluidity and polarity. EPR imaging is concerned with spatially resolved EPR spectroscopy. We studied EPR images at X-band frequency (9 GHz) in the skin biopsies of hairless mice. Our particular imaging technique utilized a modulated field gradient to obtain cross-sectional images perpendicular to the skin surface. Employing nitroxide free radicals, this approach allows analysis of skin biophysical and biochemical features at the micrometer resolution level. By spin labeling drugs, pharmacokinetic properties of the labeled compound can be monitored in skin. We suggest that EPR imaging has a broad application potential in dermatologic research. In comparison to other spatially visualizing techniques, such as nuclear magnetic resonance (NMR) imaging and ultrasound, EPR imaging has significant advantages, such as high spatial resolution and providing specific biochemical and biophysical information that cannot be obtained by other methods in skin.


Subclinical Epstein-Barr virus infection of both the male and female genital tract--indication for sexual transmission. Epstein-Barr Virus (EBV) can infect B lymphocytes as well as epithelial cells of the oral cavity. Recently, infection of epithelial cells of the inflamed uterine cervix has been demonstrated, and EBV-DNA has been detected in urethral discharge of men suffering from genital infection. We investigated whether EBV can be found in the genital tract of both sexes independently from inflammatory disease states. Genital specimens of men and women of a sexually transmitted diseases outpatient clinic after excluding sexually transmitted diseases and clinically apparent signs of inflammation were investigated using the polymerase chain reaction to screen for EBV-DNA. In 13 of 47 samples (27.7%) swabbed from the uterine cervix, EBV-DNA could be detected. Similarly, 6 of 45 samples (13.3%) scraped from the sulcus coronarius contained EBV-DNA. Our study shows that the female genital tract and likewise the male genital tract can subclinically harbor EBV. These findings suggest i) that in addition to the oral cavity, the female and the male genital tract may be a reservoir for EBV and ii) that sexual transmission of this virus associated with an epidemiology different from that of oral infection may be possible.


Diminished interferon-gamma and lymphocyte proliferation in neonatal and postpartum primary herpes simplex virus infection. Interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and lymphocyte proliferation in response to herpes simplex virus (HSV) antigen were assessed in 13 neonates and 3 parturient women with primary HSV infection. In comparison with 9 nonparturient adults, the neonates and parturient women showed significantly (P less than .01) diminished HSV antigen-stimulated lymphocyte proliferation and IFN-gamma production in the first 3-6 weeks after onset of infection. TNF alpha production did not differ significantly among HSV-infected groups. The impairment in neonatal cellular immunity was due, at least in part, to a specific deficit in response to HSV antigen. Lymphocyte proliferation and TNF alpha production in response to the mitogen concanavalin A (ConA) were comparable in adults and infants, but ConA-stimulated IFN-gamma production in infants was diminished throughout the study period. In contrast, HSV antigen-stimulated IFN-gamma production was comparable in infants and adults after 6 weeks. Not all patients with diminished cellular immune responses to HSV antigen manifested severe clinical disease. Nevertheless, patients with significant clinical morbidity had diminished cellular immune responses to HSV antigen. These results suggest that delayed acquisition of antigen-specific cellular immunity in primary HSV infection predisposes to more severe clinical disease.


Role of hepatitis C virus infection in spontaneous hepatitis B surface antigen clearance during chronic hepatitis B virus infection. To examine the role of hepatitis C virus (HCV) infection in spontaneous hepatitis B surface antigen (HBsAg) clearance during the course of chronic hepatitis B virus (HBV) infection, serum specimens from 32 asymptomatic HBsAg carriers and 22 patients with chronic hepatitis type B who underwent spontaneous HBsAg clearance were studied for antibody to HCV (anti-HCV) using commercial EIAs. The results were compared with those of control groups matched for age, sex, hepatitis B e antigen, antibody to hepatitis delta virus, and cirrhosis. Eight (25%) of the asymptomatic carriers and 9 (41%) of the patients with chronic hepatitis were seropositive for anti-HCV in contrast to 1.6% and 9.1% of their respective control groups (P less than .01). Serum alanine aminotransferase level was persistently abnormal after HBsAg clearance in one asymptomatic carrier and in four patients with chronic hepatitis. These patients were seropositive for anti-HCV and at least one of them was negative for HBV-DNA by polymerase chain reaction. The data suggest that HCV superinfection may not only suppress HBV or terminate the HBsAg carrier state but may also assume the role of HBV as the cause of persistent hepatitis or transaminase elevation.


Altered histopathology in protein-deprived mice during Sendai virus pneumonia: evidence for delayed inflammatory response and recovery. The morphology of the lungs and airway during the course of respiratory infection caused by Sendai virus was examined in normal (20% protein diet) and malnourished (2% protein diet) BALB/c mice. Mortality in normal Sendai-infected mice was 0 compared with 71% in the infected malnourished group. Virus was isolated until day 6 in normally fed mice and until day 9 in the malnourished group. Pulmonary inflammation was largely mononuclear and began in the normally nourished animals on day 3, peaked at day 6, and reverted almost to normal by 30 days. In the malnourished group, inflammation was delayed by about 1 day and fell further behind during the first week. It peaked 10-13 days after infection and was still present with little resolution by day 30. These findings may have relevance to the high mortality of acute respiratory diseases in children of the developing world.


Kinetics and viral protein specificity of the cytotoxic T lymphocyte response in healthy adults immunized with live attenuated varicella vaccine. The cytotoxic T lymphocyte (CTL) response was evaluated in adults given live attenuated varicella vaccine, using target cells expressing varicella-zoster virus (VZV) immediate-early protein (IE62) or VZV glycoproteins gpI, gpIV, or gpV to determine viral protein specificity. The frequency of CTL that recognized IE62 was 1:171,000 +/- 46,000 SE in subjects tested 10 days to 8 weeks after the initial vaccine dose; the induction of CTL specific for gpI was equivalent. CTL recognition of VZV proteins was mediated by CD4+ or CD8+ cells. CTL recognition of IE62 and gpIV persisted in vaccinees (tested approximately 4 years later) and was comparable to that in the naturally immune. The mean frequency of CTL specific for gpV was lower (but not significantly) in vaccinees than in naturally immune subjects. Assay of responder cell frequencies showed persistence of equivalent numbers of T lymphocytes that recognized IE62 and gpI in vaccinees and naturally immune subjects. Immunization with this vaccine elicited memory T lymphocyte responses to VZV comparable to those induced by natural infection.


In vitro suppression of bone marrow progenitor cell differentiation by human herpesvirus 6 infection. Suppression of marrow function may be one of the most serious effects of human herpesvirus 6 (HHV-6) infection in marrow transplant patients. In this study, normal bone marrow mononuclear cells were infected in vitro with HHV-6, and a methylcellulose-based colony formation assay was used to evaluate the impact of the infection on marrow cell differentiation and proliferation. Results demonstrated that the outgrowth of colony-forming units of granulocyte and macrophage lineages (cfu-GM) was decreased by approximately 43%, that growth of cfu of granulocyte, erythrocyte, macrophage, and megakaryocyte lineages (cfu-GEMM) was inhibited by an average of 71%, and that the erythroid burst-forming unit (bfu-E) was decreased by approximately 73%. Further, outgrowth of the marrow stromal layer was reduced 74%. Direct infection of bone marrow monocytes was observed, although cell-free virus could not be detected in infected culture supernatants. Addition of a neutralizing monoclonal antibody specific for interferon-alpha to the infected cultures resulted in an almost complete reversal of the viral suppressive effects.


Sodium stibogluconate (Pentostam) overdose during treatment of American cutaneous leishmaniasis. A 27-year-old woman who acquired cutaneous leishmaniasis in Central America was inadvertently treated with 10 times the intended daily dose of the pentavalent antimonial compound sodium stibogluconate (Pentostam): 8500 mg (143 mg/kg) instead of 850 mg. The patient felt "wiped out" during the 4-h infusion of the drug. After the mistake in dosing was discovered, she was vigorously hydrated and carefully monitored in an intensive care unit for greater than 48 h. Her vital signs were stable, and no arrhythmias were noted. Her alanine aminotransferase level rose briefly to 2.4 times the upper limit of normal, and her white blood cell count briefly fell 43% to a low of 3700/microliter. Her skin lesions subsequently healed without further therapy. Although sodium stibogluconate has been associated with a variety of side effects, in this case, a single high dose of the drug was tolerated without serious toxicity.


Alveolar ridge augmentation with collagen tubes containing bone and hydroxylapatite. The use of collagen tube containers with implants of an autogenous bone and particulate hydroxylapatite composite is reported. The tubes were used to shape the mixture and to facilitate its implantation in cases of severe mandibular resorption. Following a minimum of 60 months after implant placement and a minimum of 48 months of denture wear, the implant and overall prosthodontic success were rated favorably by the patient and the prostodontist. Individual implants were evaluated with polarized and fluorescent light microscopy and with microradiography. While the clinical results were good, the microscopic evaluation showed the amount of bone present within the implant to be variable.


Relationship between cyclic AMP production and lipolysis induced by forskolin in rat fat cells. Forskolin (7 beta-acetoxy-8, 13-epoxy-1 alpha,6 beta,9 alpha-trihydroxy-labd-14-ene-11-one) induced both cyclic AMP production and lipolysis in intact fat cells, but stimulated lipolysis without increasing cyclic AMP at a concentration of 10(-5) M. Homogenization of fat cells elicited lipolysis without elevation of cyclic AMP. Forskolin did not stimulate lipolysis in the homogenate. Forskolin stimulated both cyclic AMP production and lipolysis in a cell-free system consisting of endogenous lipid droplets and a lipoprotein lipase-free lipase fraction prepared from fat cells. However, at a concentration of 10(-6) M, it induced lipolysis without increase in the cyclic AMP content in this cell-free system. In the cell-free system, homogenization of the lipid droplets resulted in marked increase in lipolysis to almost the same level as that with 10(-4) M forskolin without concomitant increase in cyclic AMP. Addition of forskolin to a cell-free system consisting of homogenized lipid droplets and lipase did not stimulate lipolysis further. Phosphodiesterase activities were found to be almost the same both in the presence and absence of forskolin in these reaction mixtures. Although 10(-3) M forskolin produced maximal concentrations of cyclic AMP: 6.7 x 10(-7) M in fat cells and 2.7 x 10(-7) M in the cell-free system, 10(-4) M cyclic AMP did not stimulate lipolysis in the cell-free system. In a cell-free system consisting of lipid droplets and the lipase, pyrophosphate inhibited forskolin-induced cyclic AMP production, but decreased forskolin-mediated lipolysis only slightly. Based on these results, mechanism of lipolytic action of forskolin was discussed.


Metabolism of oxygenated derivatives of arachidonic acid by Caco-2 cells. Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated separately with 3H8-labeled preparations of three different lipid mediators of inflammation: 5-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B4. Both [3H8]5-hydroxyeicosatetraenoic and [3H8]12-hydroxyeicosatetraenoic acids were taken up and metabolized by Caco-2 cells, but [3H]leukotriene B4 remained unmetabolized in the incubation medium. [3H]5-hydroxyeicosatetraenoic acid was esterified into cellular phospholipids (15%) and triglycerides (4%) but did not undergo beta-oxidation. When [3H]12-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, 14% underwent two cycles of beta-oxidation to form [3H]8-hydroxyhexadecatrienoic acid, and 3% underwent three cycles of beta-oxidation to form [3H]6-hydroxytetradecadienoic acid, both of which were released into the media. [3H]12-Hydroxyeicosatetraenoic acid was also esterified into cellular phospholipids (13%), but none was esterified into cellular triglycerides.


Effects of dietary fats and cholesterol on liver lipid content and hepatic apolipoprotein A-I, B, and E and LDL receptor mRNA levels in cebus monkeys. The effects of the long-term administration of the dietary fats coconut oil and corn oil at 31% of calories with or without 0.1% (wt/wt) dietary cholesterol on plasma lipoproteins, apolipoproteins (apo), hepatic lipid content, and hepatic apoA-I, apoB, apoE, and low density lipoprotein (LDL) receptor mRNA abundance were examined in 27 cebus monkeys. Relative to the corn oil-fed animals, no significant differences were noted in any of the parameters of the corn oil plus cholesterol-fed group. In animals fed coconut oil without cholesterol, significantly higher (P less than 0.05) plasma total cholesterol (145%), very low density lipoprotein (VLDL) + LDL (201%) and high density lipoprotein (HDL) (123%) cholesterol, apoA-I (103%), apoB (61%), and liver cholesteryl ester (263%) and triglyceride (325%) levels were noted, with no significant differences in mRNA levels relative to the corn oil only group. In animals fed coconut oil plus cholesterol, all plasma parameters were significantly higher (P less than 0.05), as were hepatic triglyceride (563%) and liver apoA-I (123%) and apoB (87%) mRNA levels relative to the corn oil only group, while hepatic LDL receptor mRNA (-29%) levels were significantly lower (P less than 0.05). Correlation coefficient analyses performed on pooled data demonstrated that liver triglyceride content was positively associated (P less than 0.05) with liver apoA-I and apoB mRNA levels and negatively associated (P less than 0.01) with hepatic LDL receptor mRNA levels. Liver free and esterified cholesterol levels were positively correlated (P less than 0.05) with liver apoE mRNA levels and negatively correlated (P less than 0.025) with liver LDL receptor mRNA levels. Interestingly, while a significant correlation (P less than 0.01) was noted between hepatic apoA-I mRNA abundance and plasma apoA-I levels, no such relationship was observed between liver apoB mRNA and plasma apoB levels, suggesting that the hepatic mRNA of apoA-I, but not that of apoB, is a major determinant of the circulating levels of the respective apolipoprotein. Our data indicate that a diet high in saturated fat and cholesterol may increase the accumulation of triglyceride and cholesterol in the liver, each resulting in the suppression of hepatic LDL receptor mRNA levels. We hypothesize that such elevations in hepatic lipid content differentially alter hepatic apoprotein mRNA levels, with triglyceride increasing hepatic mRNA concentrations for apoA-I and B and cholesterol elevating hepatic apoE mRNA abundance.


Effect of retinoids on nuclear retinoic acid receptors mRNA in adipose tissue of retinol-deficient rats. In this study, we examined effects of retinol deficiency and three retinoids, all-trans-retinoic acid, 13-cis-retinoic acid, and etretin, the aromatic derivative of retinoic acid on nuclear retinoic acid receptor isoforms alpha, beta, and gamma mRNA in rat adipose tissue. Retinol deficiency caused an almost 50% decrease in isoform beta mRNA levels of adipose tissue, whereas little change occurred in the abundance of alpha and gamma isoforms transcripts in this tissue. Intragastric administration of all-trans-retinoic acid to retinol-deficient rats increased the adipose tissue retinoic acid receptor isoforms beta and gamma mRNA levels sixfold and twofold, respectively, in 4 h as compared to adipose tissue of retinol-deficient rats that were administered cottonseed oil. In contrast to this, 13-cis-retinoic acid and etretin at equimolar doses were not effective in inducing beta or gamma isoforms mRNA in retinol-deficient rats. These results show that adipose tissue isoform beta responds to retinol deficiency, and all-trans-retinoic acid rapidly induces beta and gamma mRNA isoforms in this tissue. Thus, retinoic acid may regulate expression of specific genes through its interaction with retinoic acid receptors in adipocytes.


Recycling of vitamin E in human low density lipoproteins. Oxidative modification of low density lipoproteins (LDL) and their unrestricted scavenger receptor-dependent uptake is believed to account for cholesterol deposition in macrophage-derived foam cells. It has been suggested that vitamin E that is transported by LDL plays a critical role in protecting against LDL oxidation. We hypothesize that the maintenance of sufficiently high vitamin E concentrations in LDL can be achieved by reducing its chromanoxyl radicals, i.e., by vitamin E recycling. In this study we demonstrate that: i) chromanoxyl radicals of endogenous vitamin E and of exogenously added alpha-tocotrienol, alpha-tocopherol or its synthetic homologue with a 6-carbon side-chain, chromanol-alpha-C6, can be directly generated in human LDL by ultraviolet (UV) light, or by interaction with peroxyl radicals produced either by an enzymic oxidation system (lipoxygenase + linolenic acid) or by an azo-initiator, 2,2'-azo-bis(2,4-dimethylvaleronitrile) (AMVN; ii) ascorbate can recycle endogenous vitamin E and exogenously added chromanols by direct reduction of chromanoxyl radicals in LDL; iii) dihydrolipoic acid is not efficient in direct reduction of chromanoxyl radicals but recycles vitamin E by synergistically interacting with ascorbate (reduces dehydroascorbate thus maintaining the steady-state concentration of ascorbate); and iv) beta-carotene is not active in vitamin E recycling but may itself be protected against oxidative destruction by the reductants of chromanoxyl radicals. We suggest that the recycling of vitamin E and other phenolic antioxidants by plasma reductants may be an important mechanism for the enhanced antioxidant protection of LDL.


B-less: a strain of profoundly B cell-deficient mice expressing a human lambda transgene. We have created several transgenic mouse strains that bear the human lambda light chain gene driven by its own promoter and a mouse immunoglobulin heavy chain enhancer. The transgene is expressed in many tissues, with particularly high levels of expression in the bone marrow, thymus, spleen, and lymph nodes. One of these transgenic lines, B-less, displays a dramatic phenotype characterized by an acute susceptibility to bacterial and viral infections. Analysis of this strain shows it to be profoundly deficient in both immature (pre-B) and mature B cells, as well as in circulating immunoglobulin. The pre-B and B cell defects are cell autonomous, as judged by cell culture and bone marrow graft chimeras. Despite this B cell deficiency, the T cell lineage appears grossly normal as assessed by flow cytometric analysis and by its response to mitogen stimulation. Since an independently derived transgenic strain bearing the same human lambda construct displays a partial B-less phenotype, it is likely that the B lineage deficiency is due to a dominant effect of transgene expression rather than to the insertional perturbation of an endogenous mouse gene. It is interesting that the deficiency phenotype is fully expressed in the FVB/N genetic background, but is suppressed in F1 hybrids formed between the FVB/N and C57BL/6 inbred strains. Evidently, there are one or more dominant genetic suppressors of B-less in the C57BL/6 genome.


Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. Dithiocarbamates and iron chelators were recently considered for the treatment of AIDS and neurodegenerative diseases. In this study, we show that dithiocarbamates and metal chelators can potently block the activation of nuclear factor kappa B (NF-kappa B), a transcription factor involved in human immunodeficiency virus type 1 (HIV-1) expression, signaling, and immediate early gene activation during inflammatory processes. Using cell cultures, the pyrrolidine derivative of dithiocarbamate (PDTC) was investigated in detail. Micromolar amounts of PDTC reversibly suppressed the release of the inhibitory subunit I kappa B from the latent cytoplasmic form of NF-kappa B in cells treated with phorbol ester, interleukin 1, and tumor necrosis factor alpha. Other DNA binding activities and the induction of AP-1 by phorbol ester were not affected. The antioxidant PDTC also blocked the activation of NF-kappa B by bacterial lipopolysaccharide (LPS), suggesting a role of oxygen radicals in the intracellular signaling of LPS. This idea was supported by demonstrating that treatment of pre-B and B cells with LPS induced the production of O2- and H2O2. PDTC prevented specifically the kappa B-dependent transactivation of reporter genes under the control of the HIV-1 long terminal repeat and simian virus 40 enhancer. The results from this study lend further support to the idea that oxygen radicals play an important role in the activation of NF-kappa B and HIV-1.


Extracellular processing of peptide antigens that bind class I major histocompatibility molecules. One problem associated with the use of synthetic peptides as antigens in vivo is their susceptibility to inactivation by proteolytic degradation. A situation is described in which a serum protease, angiotensin-converting enzyme (ACE), is actually responsible for the class I binding activity of a commonly used influenza antigen, nucleoprotein (NP)(147-158R-). This peptide has been reported to be a highly efficient class I antigen. Evidence is presented that demonstrates that the peptide is inactive until cleaved by ACE, which is a normal constituent of serum. The enzyme removes a COOH-terminal dipeptide resulting in the sequence NP(147-155), which is identical to the naturally processed peptide. Such extracellular processing of peptides and proteins may occur for a variety of antigens both in vitro and in vivo, and could have important implications for the design of proteolytically resistant vaccines.


Selective upregulation of platelet-derived growth factor alpha receptors by transforming growth factor beta in scleroderma fibroblasts. Transforming growth factor beta (TGF-beta), a multifunctional cytokine, is an indirect mitogen for human fibroblasts through platelet-derived growth factor (PDGF), particularly the A ligand-alpha receptor arm of that system. TGF-beta effects on PDGF alpha receptor expression were studied in vitro using ligand binding techniques in three human dermal fibroblast strains: newborn foreskin, adult skin, and scleroderma (systemic sclerosis, SSc). Each cell strain responded differently to TGF-beta. In newborn foreskin fibroblasts, PDGF alpha receptor number decreased in a dose-dependent manner after exposure to low concentrations of TGF-beta (0.1-1 ng/ml). Responses of normal skin fibroblasts were varied, and mean net receptor number was unchanged. Increases in PDGF alpha receptor number by TGF-beta occurred consistently with SSc fibroblasts and low concentrations of TGF-beta (0.1-1 ng/ml) were particularly stimulatory. Increased surface expression of alpha receptor subunit by TGF-beta in SSc fibroblasts correlated with increased new PDGF alpha receptor synthesis as demonstrated by radioimmunoprecipitation analysis of metabolically labeled cells and with increased steady-state levels of corresponding mRNAs. In normal adult skin fibroblasts, TGF-beta had no effect on either synthesis or mRNA expression of alpha receptor subunits. Proliferative responses to PDGF-AA after pretreatment with TGF-beta correlated positively with effects of TGF-beta on expression of alpha receptor subunit. Decreased mitogenic responses to PDGF-AA were observed in foreskin fibroblasts, small changes in responses in adult fibroblasts, and significant increases in SSc fibroblasts. Thus, costimulation with PDGF-AA and TGF-beta selectively enhanced proliferation of fibroblasts with the SSc phenotype. Immunohistochemical examination of SSc and control skin biopsies revealed the presence of PDGF-AA in SSc skin. Data obtained by ligand binding, immunoprecipitation, mRNA, and mitogenic techniques are consistent with the hypothesis that activation of the PDGF-AA ligand/alpha receptor pathway is a characteristic of the SSc fibroblast and may contribute to the expansion of fibroblasts in SSc.


In vivo and in vitro clonal deletion of double-positive thymocytes. To study the processes of thymic development, we have established transgenic mice expressing and alpha/beta T cell antigen receptor (TCR) specific for cytochrome c associated with class II major histocompatibility complex (MHC) molecules. The transgenic TCR chains are expressed by most of the thymocytes in these mice, and these cells have been shown to efficiently mature in association with Ek- and Ab-encoded class II MHC molecules. This report describes a characterization of the negative selection of these transgenic thymocytes in vivo that is associated with the expression of As molecules. Negative selection by As molecules appears to result in the deletion of a late stage of CD4/CD8 double-positive thymocytes in that there is a virtual absence of transgenic TCR bearing CD4 single-positive thymocytes. This phenotype is accompanied by the appearance of CD4/CD8 double-negative thymocytes and peripheral T cells that are functionally antigen reactive. The process of negative selection has also been investigated using an in vitro culture system. Upon presentation of cytochrome c by Eb-expressing nonthymic antigen-presenting cells, there occurs an antigen dose-dependent deletion of the majority of CD4/CD8 double-positive thymocytes. In contrast, presentation of Staphylococcal enterotoxin A by Eb in vitro results in minimal deletion of double-positive thymocytes. In addition, we use this in vitro model to examine the effects of cyclosporin A on negative selection. In contrast to its effects on mature T cells, and the findings of others in vivo, cyclosporin A does not inhibit antigen-induced deletion of double-positive thymocytes. Finally, a comparison of the antigen dose responses for thymocyte deletion and for peripheral T cell activation indicates that double-positive thymocyte recognition is more sensitive than mature T cells to antigen recognition.


Anti-CD8 impairs clearance of herpes simplex virus from the nervous system: implications for the fate of virally infected neurons. The role of CD8+ T cells in resistance to herpes simplex virus (HSV) was examined. After cutaneous inoculation, HSV spreads to the peripheral nervous system (PNS) where it replicates in ganglionic neurons. In normal mice, replication of virus in the PNS was rapidly terminated and evidence of neuronal destruction, assessed by a quantitative histological assay, was sparse. Clearance of infectious virus was impaired, and a strikingly high proportion of ganglionic neurons was killed, in mice treated with an antibody that depleted them of CD8+ T cells. These results suggest that CD8+ T cells play an important role in maintaining the integrity of the sensory nervous system during primary infection with HSV. Therefore, viral epitopes recognized by CD8+ T cells and restricting class I major histocompatibility complex genes are, in principle, implicated as interacting genetic determinants of neurovirulence.


Signal transduction by Fc gamma RIII (CD16) is mediated through the gamma chain. To determine the functional role of the two isoforms of Fc gamma RIII (CD16) (IIIA, IIIB), the signal transduction capabilities of wild-type and mutant forms of these receptors were analyzed in transfected lymphoid, myeloid, and fibroblastic cell lines. Functional reconstitution of receptor signalling was observed in hematopoietic T and mast cells, and was absent in nonhematopoietic (CHO) cells. Fc gamma RIIIA, a hetero-oligomeric receptor composed of a ligand-binding subunit alpha and dimeric gamma chains, generated both proximal and distal responses in Jurkat and P815 cells, typical of what is seen in natural killer cells and macrophages upon receptor activation. In contrast, Fc gamma RIIIB, which is normally attached to the cell surface via a glycosyl-phosphatidylinositol anchor, was incapable of transducing signals. After crosslinking, Fc gamma RIIIA signalling was dependent only upon the gamma chain. Fc gamma RIIIA chimeras in which the alpha subunit transmembrane and cytoplasmic domains were substituted with the corresponding gamma chain sequences functioned as well as wild-type hetero-oligomeric receptors. These data indicate that the ability of the Fc gamma RIIIA complex to activate the appropriate pathways for cell activation is cell-type restricted and independent of the transmembrane and cytoplasmic domains of the alpha subunit. The presence of the gamma chain is responsible for the assembly of, as well as the signal transduction by, the functional cell surface complex.


Validating screening instruments for neuroepidemiologic surveys: experience in Sicily. Sicilian Neuro-Epidemiologic Study (SNES) Group. In a hospital setting in Sicily, we assessed a screening instrument developed for a prevalence survey of parkinsonism, peripheral neuropathies, stroke, and epilepsy. The subjects consisted of (1) hospital patients with any of the above-mentioned diseases, to investigate sensitivity; and (2) hospital visitors free of all these diseases, to investigate specificity. The standard for comparison was a clinical evaluation based on specified criteria. Trained interviewers administered the screening instrument, asking subjects to answer symptom questions and to perform simple physical tasks. For the questions and tasks together, the sensitivity estimates were 100% for parkinsonism (n = 21), 96% for peripheral neuropathies (n = 22), 96% for stroke (n = 22), and 96% for epilepsy (n = 22), while the specificity estimate was 86% (n = 21). Analogous estimates were computed for the set of questions, for the set of tasks, and for each question and task individually. Despite limitations in our approach, we concluded that the screening instrument would be adequate for its intended use.


Treatment of recurrent gliomas and metastatic brain tumors with a polydrug protocol designed to combat nitrosourea resistance. The study was undertaken to evaluate a chemotherapy protocol against recurrent malignant gliomas that was designed to combat presumed chloroethyl-nitrosourea (NU) resistance. All patients had malignant gliomas and had failed prior therapy. Patients were stratified as having either glioblastoma multiforme (GM) or anaplastic gliomas (AG) and as having failed radiotherapy (RT) only or both RT and chemotherapy. Chemotherapy consisted of six drugs: before lomustine (CCNU), thioguanine (TG), dibromodulcitol (mitolactol; DBD), and procarbazine (PCB) were given to enhance CCNU-induced tumor-cell kill and to reduce alkyltransferase repair of ethylated DNA. A fluorouracil-hydroxyurea (FUHU) combination was given 2 weeks later to kill cells that began to cycle after the challenge of the first four drugs (TPDC-FUHU chemotherapy). Of the 88 assessable patients, 37 had GM, 38 had AG, and 13 had other primary and metastatic brain tumors. For GM patients, 61% had a partial response (PR) or stable disease (SD) for a median of 9.3 months if RT only failed, and 58% had a PR or SD for a median of 5.1 months if they had previously been treated with an NU. For AG patients, 92% had a PR or SD for a median of 15 months if RT only had failed, but only 38% had a PR or SD for a median of 10.6 months if they had been previously treated with a NU. Activity was also seen against other recurrent or progressive primary and metastatic brain tumors. TDPC-FUHU chemotherapy is a highly effective form of chemotherapy for both recurrent GM and AG patients. This study suggests but does not prove that this combination may be superior to other NU-based treatments for recurrent malignant glioma patients who fail RT. Because of the activity of this chemotherapy, we intend to evaluate more fully this approach in a randomized study.


Combination chemotherapy with carboplatin, etoposide, and vincristine as first-line treatment in small-cell lung cancer. The antineoplastic activity of carboplatin and etoposide may be improved by the addition of vincristine (CEV) because of its low myelosuppressive potential and its activity in small-cell lung cancer (SCLC). A phase II study with CEV was carried out. One hundred twenty-one untreated patients with SCLC (63 with limited disease [LD], 58 with extensive disease [ED]) were treated with a combination of 300 mg/m2 intravenous (IV) on day 1, etoposide 140 mg/m2 IV daily on days 1 to 3, and vincristine 1.4 mg/m2 IV on days 1, 8, and 15 every 4 weeks. A 90% rate overall response rate including 56% complete responses (CRs) was achieved in LD and an 83% overall response rate including 35% CRs was observed in ED. Median survival time was 13 months in limited disease and 9.5 months in extensive disease. The 24 and 36 months survival rates were 29% in LD and 9% in ED. Myelosuppression was the main form of toxicity. The combination of CEV is a new active and well-tolerated regimen in the treatment of SCLC. Prospective randomized studies of CEV with conventional chemotherapy are warranted.


Multimodality treatment of stage IIIA non-small-cell lung carcinoma: a critical review of the literature and strategies for future research. Stage IIIA non-small-cell lung carcinoma (NSCLC) is composed of regionally advanced yet potentially resectable disease. Many trials have evaluated a variety of therapeutic strategies. Most have been single-arm phase II trials, although a number of comparative phase III trials have also been performed. This review critically evaluates the existing literature on the treatment of stage IIIA NSCLC, particularly the various multimodality approaches that have been used. A review of the existing literature does not establish the optimal treatment of stage IIIA NSCLC. Although radiation therapy alone remains the most commonly administered therapy for apparently unresectable disease, the status of radiation as a "standard" therapy can be seriously challenged based on existing data. Similarly, although a number of studies have established that surgical resection is clearly feasible in selected patients with stage IIIA disease, the efficacy of surgery also remains to be definitively established. Many studies have explored neoadjuvant chemotherapy, either in conjunction with radiation or surgery, with results that are best described as conflicting and controversial. Without question, randomized phase III trials are required at this time to define what is to be considered optimal treatment. An attempt is made to define the most important questions that should be addressed in future phase III trials. Additionally, a number of study designs are suggested to best answer the therapeutic questions posed.


Paraventricular neuron chemosensitivity and activity related to blood pressure control in emotional behavior. 1. Unit activity in the paraventricular nucleus (PVN) and blood pressure (BP) of the rat were recorded during discrimination learning of cue tone (CTS+) predicting reward [glucose and intracranial self stimulation (ICSS)], and cue tone (CTS-) predicting aversion (electric shock or tail pinch). Adrenergic systems in the PVN were investigated by electrophoretic application of norepinephrine (NE), and by electrophoretic application or direct microinjection of its antagonists (prazosin, yohimbine, and propranolol). 2. Of 65 PVN neurons analyzed, 35 responded during CTS learning in one or more phases of an operant task. Of these, activity of 20 neurons was increased nondiscriminatively by CTS+ and CTS-, and reward and aversion. In parallel with these neuronal activity increases, BP (systolic, diastolic, and mean) was also increased by both CTS+ and CTS- (10-35 mmHg), and reward and aversion (20-47 mmHg). Both neuronal activity and BP increases during CTS learning were suppressed during extinction. 3. Paraventricular neurons, the activity of which correlated with BP increase during CTS learning, were excited by electrophoretic application of NE and Na+ and fired phasically during inter-trial intervals. Activity increases of these neurons during CTS learning, which was related to BP increase, were blocked by electrophoretically applied prazosin (alpha 1-antagonist), but not by yohimbine (alpha 2-antagonist) or propranolol (beta-antagonist). 4. Direct microinjection of prazosin into the PVN suppressed BP increase during CTS learning. Operant licking for reward was not suppressed by prazosin injection. Microinjection of yohimbine or propranolol into the PVN did not suppress either BP increase during CTS learning or operant licking to obtain reward. 5. These results suggest that afferent alpha 1-adrenergic input to the PVN is important to the central BP control during CTS learning in emotional behavior.


Gastric uptake of gallium-67 in AIDS.

Gastric uptake of gallium-67 in AIDS. We describe gastric localization of 67Ga in 13 patients with acquired immunodeficiency syndrome (AIDS) among 148 referred primarily to rule out Pneumocystis carinii pneumonia (PCP). Endoscopic biopsies in five of the patients indicated cytomegalovirus (CMV) infection in one, gastritis in two, and normal tissue in two. Other associated, but nongastric, infections in these 13 patients included esophageal candidiasis, PCP, Mycobacterium avium-intracellulare (MAI) complex, coccidioidomycosis, toxoplasmosis and Isospora belli. Only six of the patients exhibited gastric symptoms, and even fewer proved to have gastric pathology. Although gastric 67Ga uptake in a patient with AIDS may not require specific treatment, opportunistic infections as a possible cause of gastritis should be considered.


Treatment of proteoglycan aggregates with physeal enzymes reduces their ability to inhibit hydroxyapatite proliferation in a gelatin gel. In vitro, cartilage proteoglycans (PGs) are effective inhibitors of hydroxyapatite formation and growth. Their inhibitory ability decreases with decreasing PG size and charge density. It has been suggested that the enzyme-mediated alteration in the size and conformation of PGs in the growth plate may similarly facilitate the calcification process. In this study, a gelatin gel system was used to monitor hydroxyapatite formation and growth in the presence of proteoglycan aggregates, before and after enzyme treatment. To reproduce the physeal degradation cascade, an enzyme preparation was used that contained all of the growth plate enzymes. At a concentration of 500 micrograms/ml, the untreated proteoglycan aggregates reduced the amount of mineral formed by 30%. When the aggregates were treated with the heat-inactivated enzyme, the same extent of inhibition was found. In contrast, treating the aggregates with the crude growth plate enzyme preparation removed all the inhibitory ability, such that 500 micrograms/ml of proteoglycan preparation yielded 10% more mineral than the controls. Treatment of the aggregates with chondroitinase ABC and trypsin, similarly removed all the inhibitory ability. These data, suggest that enzymatic degradation of proteoglycans may contribute to the regulation of growth plate calcification.


Slow release of anticancer drugs from porous calcium hydroxyapatite ceramic. We have developed a new delivery system for sustained release of an anticancer drug (cis-platinum) by enclosure into blocks of porous calcium hydroxyapatite ceramic. The slow release of this drug from this system was confirmed in in vitro experiments. When this system was implanted into normal back muscle, or the tibia, sustained release of cis-platinum was observed during a 12-week period after implantation. The diffusion rate of cis-platinum into blood and other organs (liver, kidney, brain) was less than 10% of that at the implanted site. This delivery system placed into experimental tumors of mice also showed a uniform release of anticancer drug for more than 3 months. Inhibition of tumor growth was more marked after local implantation of this system than after intraperitoneal administration of cis-platinum. These results indicate that this new approach to a drug delivery system may well have an important role in cancer chemotherapy. In bone tumors it is attractive because the mechanical strength of calcium hydroxyapatite ceramic permits partial surgical excision and replacement of the bone defect at the same time.


[Adenoid cystic carcinoma of the head and neck: a clinical review of 29 cases]. Pathological data and the outcomes of 29 patients with adenoid cystic carcinoma of the head and neck treated between 1975 and 1990 were reviewed. Patients consisted of 21 females and 8 males, with a median age of 59 years. The overall 5-year and 10-year survival rates determined by Kaplan-Meier analysis were 84.2% and 52.9%, respectively. Patients in whom pathological data indicated a frequent solid pattern of carcinoma had high incidences of metastasis and poor prognoses. The effectiveness of local control was found to have no relation to the pathological data of the tumor, but rather depended upon whether anatomical limitations restricted complete resection of the tumor. All 7 patients who had cervical lymph node metastasis underwent neck dissection, which was useful in controlling metastatic spread. Distant metastases were present in 6 patients, and the lung was the most frequent site of metastasis.


Strand-specific LINE-1 transcription in mouse F9 cells originates from the youngest phylogenetic subgroup of LINE-1 elements. LINE-1 (L1) is a mammalian family of highly repeated DNA sequences that are members of a class of transposable elements whose movement involves an RNA intermediate. Both structural and evolutionary data indicate that the L1 family consists of a small number of active transposable elements interspersed with a large number of L1 pseudogenes. In the mouse, the longest, characterized L1 sequences span about 7000 base-pairs and contain two long open reading frames. Two subfamilies of mouse L1 elements, A and F, have been defined on the basis of the type of putative transcriptional regulatory sequence found at the 5' end. In order to identify a transcribed subset of L1 elements in mouse F9 teratocarcinoma cells, we have examined the strand-specificity of L1 transcription by Northern analysis and compared the open reading frame-1 sequences of ten A-type cDNAs with fifteen genomic A-type L1 elements. Transcripts containing A-type sequence are far more abundant than those containing F-type sequence. Although the majority of L1 RNA in F9 cells appears to be transcribed non-specifically from both strands, our results provide evidence for a subpopulation of variable length, strand-specific transcripts arising from A-type transcriptional regulatory sequences. F9 cell cDNA sequences, which share greater than 99.5% sequence identity with one another, represent a homogeneous subset of the genomic L1 population. Examination of genomic mouse L1 sequences reveals three types of length polymorphism in a defined segment of the first open reading frame. Phylogenetic analysis shows a correlation between the type of length polymorphism in the first open reading frame and the relative age of an individual A-type genomic L1 element. Comparison of the cDNA and genomic sequences indicates that the youngest subgroup of A-type L1 elements is preferentially transcribed in F9 cells. This subgroup may be currently dominating the L1 dispersal process in mice.


NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae. The yeast nuclear gene NAM7 was previously isolated within a genomic fragment of 7.7 kb (1 kb = 10(3) bases or base-pairs), having the ability to suppress mitochondrial intronic mutations defective in RNA splicing. We have identified and sequenced the region on the insert corresponding to the NAM7 gene. A long open reading frame has been revealed which could code for a protein of 971 amino acids. Comparison of the NAM7 putative protein with data libraries did not reveal any strong similarity with known proteins. However, the NAM7 protein contains several motifs typical for proteins interacting with nucleic acids: (1) five motifs diagnostic for a superfamily of helicases appear in the same order and with similar distances; (2) the N-terminal portion possesses potential Zn-ligand structures belonging to the C chi superfamily. Deletion of the chromosomal copy of NAM7 gene leads to a partial impairment in respiratory growth that is particularly striking at low temperature. Southern blot analysis of DNA extracted from a nam7 :: URA3 deleted strain revealed the presence of a second gene whose sequence is related to that of the NAM7 gene and which could participate in the same process.


Effect of the Asn52----Ile mutation on the redox potential of yeast cytochrome c. Theory and experiment. Theoretical methods for correlation of sequence changes and redox potential of electron transport proteins are examined using the Asn52----Ile mutation in cytochrome c as a test case. The first approach uses the protein dipoles Langevin dipoles (PDLD) method and the high resolution X-ray structures of the native and the mutant proteins. This approach is found to give reliable results where all the solvent molecules are represented by Langevin dipoles and also when some bound water molecules are represented explicitly. A free energy perturbation method is also found to give reasonable results but at the expense of much more computer time. Finally, an approach that generates mutant structures from the native structure by molecular dynamics simulation and then uses these configurations in PDLD calculations is found to give a reasonable estimate of the effect of the mutation on the corresponding redox potential. The encouraging results obtained here and in a preliminary test case of the Phe82----Ser mutation indicates that the present strategies can provide a useful tool for structure-redox and sequence-redox correlation in proteins.


Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering. Soluble spin labels, which "bleach" the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed.


His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study. In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)


Contribution of hydration and non-covalent interactions to the heat capacity effect on protein unfolding. The heat capacity change upon protein unfolding has been analysed using the heat capacity data for the model compounds' transfer into water, corrected for volume effects. It has been shown that in the unfolding, the heat capacity increment is contributed to by the effect of hydration of the non-polar groups, which is positive and decreases with temperature increase, and by the effect of hydration of the polar groups, which is negative and decreases in magnitude as temperature increases. The sum of these two effects is very close to the total heat capacity increment of protein unfolding at room temperature but is likely to deviate from it at higher temperatures. Therefore, the expected heat capacity effect caused by the increase of configurational freedom of the polypeptide chain upon unfolding seems to be compensated for by some other effect, perhaps associated with fluctuation of the native protein structure.


Internal motional averaging and three-dimensional structure determination by nuclear magnetic resonance. Dynamic averaging effects from internal motions on interproton distances estimated from nuclear Overhauser effects (NOE) are determined by using a molecular dynamics simulation of lysozyme. Generalized order parameters measuring angular averaging and radial averaging parameters are calculated. The product of these two parameters describes the full averaging effects on cross-relaxation. Analysis of 2778 non-methyl NOE interactions from the protein interior and surface indicates that distances estimated by assuming a rigid molecule have less than 10% error for 89% of the NOE interactions. However, analysis of 1854 methyl interactions found that only 68% of the distances estimated from cross-relaxation rates would have less than 10% error. Qualitative evaluation of distances according to strong, medium and weak NOE intensities, when used to define only the upper bound for interproton separation, would misassign less than 1% of the distance constraints because of motional averaging. Internal motions do not obscure the identification of secondary structure, although some instances of significant averaging effects were found for interactions in alpha-helical regions. Interresidue NOEs for amino acids more than three residues apart in the primary sequence are more extensively averaged than intraresidue or short-range interresidue NOEs. Intraresidue interactions exhibit a greater degree of angular averaging than those involving interresidue proton pairs. An internal motion does not equally affect all NOE interactions for a particular proton. Thus, incorporation of averaging parameters in nuclear magnetic resonance structure determination procedures must be made on a proton-pair-wise basis. On the basis of the motional averaging results, particular fixed-distance proton pairs in proteins are suggested for use as distance references. A small percentage of NOE pairs localized to three regions of the protein exhibit extreme averaging effects from internal motions. The regions and types of motions involved are described.


Refined structure of the complex between guanylate kinase and its substrate GMP at 2.0 A resolution. The crystal structure of guanylate kinase from Saccharomyces cerevisiae complexed with its substrate GMP has been refined at a resolution of 2.0 A. The final crystallographic R-factor is 17.3% in the resolution range 7.0 A to 2.0 A for all reflections of the 100% complete data set. The final model has standard geometry with root-mean-square deviations of 0.016 A in bond lengths and 3.0 in bond angles. It consists of all 186 amino acid residues, the N-terminal acetyl group, the substrate GMP, one sulfate ion and 174 water molecules. Guanylate kinase is structurally related to adenylate kinases and G-proteins with respect to its central beta-sheet with connecting helices and the giant anion hole that binds nucleoside triphosphates. These nucleotides are ATP and GTP for the kinases and GTP for the G-proteins. The chain segment binding the substrate GMP of guanylate kinase differs grossly from the respective part of the adenylate kinases; it has no counterpart in the G-proteins. The binding mode of GMP is described in detail. Probably, the observed structure represents one of several structurally quite different intermediate states of the catalytic cycle.


X-ray diffraction by crystals of beef heart ubiquinol: cytochrome c oxidoreductase. Beef heart mitochondrial ubiquinol:cytochrome c oxidoreductase has been crystallized in the shape of hexagonal bipyramids. At present the crystals diffract X-rays to 4.7 A. From preliminary analysis the diffraction pattern appears to be consistent with space group P6(1)22 or P6(5)22 and with unit cell parameters a = b = 212 A and c = 352 A.


Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference. Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.


Planarian mitochondria. I. Heterogeneity of cytochrome c oxidase subunit I gene sequences in the freshwater planarian, Dugesia japonica. We have detected sequence heterogeneity in the cytochrome c oxidase subunit I (COI) gene of freshwater planarian, Dugesia japonica, collected in one locality. A part of the COI gene was amplified via the polymerase chain reaction (PCR) using template DNA prepared from a mixture of 500 individuals or from each of 18 individuals. Analyses of DNA sequences by standard strategies for cloning and sequencing or by direct sequencing clearly show that (1) considerable sequence heterogeneity exists in DNA prepared from the mixed individuals, (2) 11 individuals have almost identical sequences (type A), and (3) 7 individuals have sequences different from one another (Seq-D1 to Seq-D7; collectively called type D). Each of the Seq-D1-D7 sequences except for Seq-D5 shows some heterogeneity even in a single individual (heteroplasmy). A possible cause of the sequence heterogeneities is discussed.


Planarian mitochondria. II. The unique genetic code as deduced from cytochrome c oxidase subunit I gene sequences. The cytochrome c oxidase subunit I (COI) gene sequences from planarian (Dugesia japonica) DNA, most probably of mitochondrial origin, are heterogeneous. Taking advantage of the heterogeneity that occurs primarily in silent sites of the COI DNA sequences, amino acid assignments of several codons have been deduced as nonuniversal: UGA = Trp, AAA = Asp, and AGR (R: A or G) = Ser. In addition, UAA, a stop codon in the universal genetic code, is tentatively assumed to be a tyrosine codon, because three of the sequences examined have UAA at the well-conserved tyrosine site of UAY (Y: U or C) in other planarian sequences as well as in the mitochondria of human, Xenopus, sea urchin, Drosophila, Trypanosoma, and Saccharomyces cerevisiae. AUA would most probably be an isoleucine codon in these mitochondria, whereas it is a methionine codon in the majority of nonplant mitochondria.