Hydroxyl radical formation and lipid peroxidation enhancement by chromium. In vitro study. Chromium VI compounds have been shown to be carcinogenic in occupationally exposed humans, and to be genotoxic, mutagenic, and carcinogenic in a variety of experimental systems. In contrast, most chromium III compounds are relatively nontoxic, noncarcinogenic, and nonmutagenic. Reduction of Cr6+ leads to reactive intermediates, such as Cr5+, Cr4+, or other radical species. The molecular mechanism for the intracellular Cr6+ reduction has been the focus of recent studies, but the details are still not understood. Our study was initiated to compare the effect of Cr(6+)-hydroxyl radical formation and Cr(6+)-induced lipid peroxidation vs those of Cr3+. Electron spin resonance measurements provide evidence for the formation of long-lived Cr5+ intermediates in the reduction of Cr6+ by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr6+. Hydrogen peroxide suppresses Cr5+ and enhances the formation of hydroxyl radical. Thus, Cr5+ intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Comparative effects of Cr6+ and Cr3+ on the development of lipid peroxidation were studied by using rat heart homogenate. Heart homogenate was incubated with different concentrations of Cr6+ compounds at 22 degrees C for 60 min. Lipid peroxidation was determined as thiobarbituric acid reacting materiels (TBA-RM). The results confirm that Cr6+ induces lipid peroxidation in the rat heart homogenate. These observations might suggest a possible causative role of lipid peroxidation in Cr6+ toxicity. This enhancement of lipid peroxidation is modified by the addition of some metal chelators and antioxidants. Thus, strategies for combating Cr6+ toxicity should take into account the role of the hydroxy radicals, and hence, steps for blocking its chain propagation and preventing the formation of lipid peroxides.


Chromium, glucose tolerance, and diabetes. Chromium functions in maintaining normal glucose tolerance primarily by regulating insulin action. In the presence of optimal amounts of biologically active chromium, much lower amounts of insulin are required. Glucose intolerance, related to insufficient dietary chromium, appears to be widespread. Improved chromium nutrition leads to improved sugar metabolism in hypoglycemics, hyperglycemics, and diabetics.


Iodine deficiency, other trace elements, and goitrogenic factors in the etiopathogeny of iodine deficiency disorders (IDD). Severe goiter, cretinism, and the other iodine deficiency disorders (IDD) have their main cause in the lack of availability of iodine from the soil linked to a severe limitation of food exchanges. Apart from the degrees of severity of the iodine deficiency, the frequencies and symptomatologies of cretinism and the other IDD are influenced by other goitrogenic factors and trace elements. Thiocyanate overload originating from consumption of poorly detoxified cassava is such that this goitrogenic factor aggravates a relative or a severe iodine deficiency. Very recently, a severe selenium deficiency has also been associated with IDD in the human population, whereas in animals, it has been proven to play a role in thyroid function either through a thyroidal or extrathyroidal mechanism. The former involves oxidative damages mediated by free radicals, whereas the latter implies an inhibition of the deiodinase responsible for the utilization of T4 into T3. One concludes that: 1. Goiter has a multifactorial origin; 2. IDD are an important public health problem; and 3. IDD are a good model to study the effects of other trace elements whose actions in many human metabolisms have been somewhat underestimated.


Cultured human skin fibroblasts absorb 65Zn. Optimization of the method and study of the mechanisms involved. The radioactive isotope 65Zn was used to study the incorporation of zinc by cultured human skin fibroblasts. The development of the method for studying cell uptake of 65Zn in a minimal synthetic medium is presented. Kinetics carried out on control cultures up to 240 min indicated that zinc uptake occurred in three phases, the first being the most rapid. Temperature and pH affect zinc uptake, in favor of an active transport process. In addition, the rate of incorporation is considerably decreased during the first phases after adding potassium cyanide, during the last phases after adding sodium iodoacetate, and during all the phases if dithioerythritol is used. A hypothesis is therefore proposed according to which several types of mechanisms would be involved in zinc uptake by fibroblasts. At least a part of these mechanisms is energy-dependent.


The isolation of glucose tolerance factors from brewer's yeast and their relation to chromium. A new dietary factor, the glucose tolerance factor (GTF), was reported in 1957 that improved impaired glucose tolerance in rats. Most studies on GTF have used brewer's yeast as the starting material, and it has been postulated that the active material is a low-mol wt organic complex containing Cr3+. It seemed thus important to isolate an active GTF from chromium-rich yeast (228 ppm Cr) obtained by incubation with chromium and to compare each fraction with corresponding ones from untreated yeast (0.48 ppm Cr). We developed an isolation and purification procedure by fractionation of yeast extract on an anion and cation exchange resin, and tested the GTF activity (glucose oxidation) on rat adipocytes. PIXE (proton-induced X-ray emission) was used to measure the chromium content of the individual fraction. Individual fractions with GTF activity did not differ between Cr-rich and Cr-deficient yeast, and there was no relationship between Cr content and GTF activity. This does not support the hypothesis that chromium is an obligatory constituent of the GTF, assuming that GTF is a unique substance.


Absorption and metabolism of oral zinc gluconate in humans in fasting state, during, and after a meal. The absorption and metabolism of zinc in a commercial form for oral use (Rubozinc, 15 mg zinc as gluconate) were investigated in 10 subjects by a kinetic study of the serum zinc profile after administration of 45 mg zinc under three conditions: after an overnight fast, during a standardized breakfast, and 2 h after this meal. The pharmacokinetic parameters were calculated by a method suitable to the characterization of rebound effects (recycling of the element in the gastrointestinal tract). In fasting state, the parameters were comparable to those previously collected in the same subjects with oral 45 mg zinc as sulfate, except with very significantly higher Cmax and area under curve (AUC), showing a better bioavailability for zinc in the commercial form. The light meal perturbed the absorption process as evidenced by the significant increases in the lag time (+180%), the tmax (+57%), and the lag times for the first two cycles during the meal. However, the parameters returned to normal values 2 h after the meal. The Cmax only moderately decreased during the meal (31%) as did the AUC (-28%). An important delay in the absorption of zinc in the commercial form when taken during a meal was therefore demonstrated, but the effect on zinc bioavailability was only moderate.


Contribution of milk to iodine intake in France. A survey based on 838 samples of milk obtained from 537 dairies covering 70 of 95 districts in France was organized to assess iodine content of milk and its contribution to total intake. Iodine levels were significantly higher in winter than in summer. Very low iodine contents (less than 25 micrograms I/kg) were found in the eastern part of the country (the Vosges, Jura, and the Alpes) and the Massif Central. During milk processing, much of the iodine is lost in the whey. The other significant sources of dietary iodine are fish and eggs. Iodized salt is sold only to households and not to industry. Even if about 20% of the iodine is lost over the first 3 mo, salt remains the main source for this trace element. It is concluded that, if iodized salt is not provided systematically for both domestic and agro-industrial use, then milk may be the most important source of iodine. This key role may explain seasonal and geographical variations in the frequencies of goiter in France.


Selenium and rubidium changes in subjects with pathologically altered thyroid. Concentrations of selenium and rubidium in groups of subjects with hyperthyroidism, carcinomas, or adenomas and in controls were determined by neutron activation analysis with coirradiated inorganic standards and IAEA reference material. Se was decreased in all pathological groups with the greatest modification in thyroids with carcinomas. Rb was elevated in all pathological groups with the greatest increase in carcinomas as well. According to the literature, Se has a protective effect on carcinogenity as well as on biochemical pathways in thyroid cells. There are no data in the literature on the effects of Rb in those cells. On the grounds of the present results, it seems possible to use the trapping of Rb for diagnostic purposes in cases of pathologically altered thyroids.


Acute effects of various doses of iodide on thyroid iodine autoregulation. A study with 129I by means of analytical ion microscopy. Analytical ion microscopy (AIM) was used to determine alterations in the thyroid follicular lumen 127I stores of Wistar rats injected with different doses of 129I (low specific activity radionuclide). Animals fed a normal iodine diet (10 micrograms 127I/d) were divided into four groups: control group and three treated groups injected ip 24 h before sacrifice with 129I at doses of 10 micrograms (group 1), 30 micrograms (group 2), and 8500 micrograms (group 3). AIM was performed on thyroid semithin sections. The mean 129I concentration increased with the dose injected from group 1 (0.44 +/- 0.03 micrograms/mg, mean +/- SEM) to group 2 (2.05 +/- 0.14 micrograms/mg) and decreased in group 3 (0.57 +/- 0.08 microgram/mg). When compared to control group (4.14 +/- 0.17 micrograms/mg), the mean 127I concentration was not changed in group 1 (4.52 +/- 0.07 micrograms/mg), but altered in the other groups: It was significantly increased (7.14 +/- 0.41 micrograms/mg) in group 2 and slightly decreased (3.11 +/- 0.26 micrograms/mg) in group 3. These results underline the interest of AIM in the study of the effects of various doses of iodide on the thyroid autoregulation by iodide, a trace element actively trapped by this gland.


Iodine intakes assessed by urinary iodine concentrations in healthy children aged ten months, two years, and four years. Urinary iodine excretion was assessed in 642 healthy children aged 10 mo (n = 243), 2 yr (n = 183), and 4 yr (n = 216) living in the Paris area and originating from continental France (60.3%), North Africa (13.8%), the West Indies (9.1%), West Africa (8.3%), Southeast Asia (4.8%), and southern Europe (3.8%). Mild impairment of neurological (reflexes, tone, audiometry) and intellectual development (Brunet-Lézine scale) was assessed in relation to iodine status. Iodine excretions (median values) were 18.4, 11.9, and 10.9 micrograms/100 mL at 10 mo, 2 yr, and 4 yr, respectively, and risk of mild iodine deficiency (5-10 micrograms/100 mL) was 18.1%, 34.8%, and 38.3% for the same age groups. No relationship was found between anthropometry, global development quotient, and iodine status. High hearing thresholds were more commonly associated with lower iodine excretion, suggesting mild hearing defects. In spite of iodine prophylaxis, the risk of mild to moderate iodine deficiency still exists in France and in a number of European countries. Evaluation of neurological sequels of borderline iodine status is a major public health problem in European communities.


Selenium content of livers from sex-linked dwarf and normal broiler breeders. Influence of a thyrotropin-releasing hormone-induced growth hormone release. Plasma concentrations of cGH, T3, and T4 were not different between dwarf and normal broiler breeders. Normal hens had a liver selenium content of 710 +/- 35 ng/g, and dwarf hens 656 +/- nine ng/g (n = 8). Following injections into a wing vein of different doses (1.5, 3, 6, 12, and 24 micrograms/kg) of the hypothalamic hormone TRH, GH was increased after 15 min. This effect seemed to last longer in dwarf chickens. Plasma concentrations of T3 increased significantly 1 h after TRH in normal hens, but TRH was ineffective in raising T3 levels in dwarf animals. The selenium content of livers obtained following decapitation after 2 h was also increased in normal hens up to 902 +/- 42 ng/g using the highest dose of TRH (24 micrograms/kg). This seemed not to be the case for dwarf animals. A much smaller number of hepatic cGH receptors was also found in dwarf hens, whereas the affinity of the hepatic GH receptor was not influenced by the genotype. It is concluded that the sex-linked dwarf hens are unable to increase their hepatic T4 into T3 conversion following a TRH challenge probably because of a deficiency in hepatic GH receptors. The lower content of selenium in dwarfs and their inability to increase its uptake after TRH seem therefore to support the hypothesis that selenium has a direct role in the activity of the 5'-deiodinase complex.


Peptidylglycine alpha-amidating monooxygenase activity and TRH and CRF biosynthesis. Role of copper. Carboxy-terminal amidation of biologically active peptides, an important characteristic of more than half of these substances, occurs during the maturation process of peptide precursors. It is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme that is copper-dependent. We show here that alterations of copper stores in cultured cells from different origins (pancreas and hypothalamus) affect the immunoreactivity of thyrotropin-releasing hormone (TRH) and corticotropin-releasing factor (CRF) (two alpha-amidated peptides). This suggests that copper can affect neuropeptide biosynthesis and may play a role in the endocrine or central nervous system function.


Influence of thyroparathyroidectomy and thyroxine replacement on Cu and Zn cellular distribution and on the metallothionein level and induction in rats. Thyroid hormones are involved in copper and zinc distribution in rat tissues. We examined the influence of thyroparathyroidectomy (TPTY) and of a replacement therapy by T4 on Cu and Zn organ distribution. MT levels were also measured both in basal conditions and after induction by cadmium. The results confirm that a lack of T4 modified Cu and Zn in serum and tissues. In serum, TPTY increased Cu (+15%) and ceruloplasmin (+18%), and decreased Zn (-18%). In tissues, Cu was altered in liver (+13%), kidney (-24%), heart (-16%) duodenum (-18%), and Zn in liver (+25%) and kidney (-10%). The soluble fractions (100,000 g supernatant) were mainly affected in liver and kidney, and the subcellular fractions in heart and duodenum. MT levels were modified in basal conditions only in liver (+57%) and kidney (-36%). T4 administration partially prevented the effect of TPTY on both elements and MT concentrations. Therefore, no evidence is provided for a direct role of T4 in the metabolism of MT in a way comparable to the effects of glucocorticoids. However, MT could mediate the consequences of TPTY on metal distribution in certain organs, such as liver and kidney.


Effects of thyroparathyroidectomy on the distribution of bromine and iodine in rat tissues. The concentrations of iodine (I) and bromine (Br) were measured by inductively coupled plasma mass spectrometry in the plasma, kidney, heart, liver, and brain of control and thyroparathyroidectomized (TPTX) rats without and with an additional intake of either NaI or NaBr, 0.5 and 5 mumol/kg/d, respectively, for 21 d. In all groups, the highest concentrations of I and Br were found in the plasma. TPTX did not modify the concentrations of I in tissues, but slightly increased Br in plasma (+33%) and kidney (+24%). The additional intake of I with the drink induced an increase of I concentrations in the tissues tested (from 54 to 191%), except brain, both in control and TPTX rats. This additional intake of I also increased Br levels in the plasma of control (+24%) and TPTX rats (+53%). The additional intake of Br with the drink induced an increase of Br levels in all the tested tissues, brain included (from 85 to 284%). The augmentation was higher in the tissues, particularly brain, of TPTX rats than of controls. The increase of Br in brain after an additional intake contrasts with the absence of increase of I given in the same conditions. This difference between I and Br probably results from the smaller radius of Br ion in comparison with I ion radius. In conclusion, TPTX did not modify the distribution of I in the tested tissues, but slightly increased the concentrations of Br in plasma and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)


Zinc and insulin sensitivity.

Zinc and insulin sensitivity. Many studies have shown that zinc deficiency could decrease the response to insulin. In genetically diabetic animals, a low zinc status has been observed contrary to induced diabetic animals. The zinc status of human patients depends on the type of diabetes and the age. Zinc supplementation seems to have beneficial effects on glucose homeostasis. However, the mechanism of insulin resistance secondary to zinc depletion is yet unclear. More studies are therefore necessary to document better zinc metabolism in diabetes mellitus, and the antioxidant activity of zinc on the insulin receptor and the glucose transporter.


Urinary zinc and its relationships with microalbuminuria in type I diabetics. We investigated whether zincuria is associated with microalbuminuria in type I (insulin-dependent) diabetics (IDDM). In 169 IDDM, 215 overnight urine samples were collected for simultaneous assay of zinc and albumin. In 76 samples with excessive microalbuminuria (greater than 15 mg/L), zincuria was higher than in the 139 other samples (0.83 +/- 0.06 vs 0.58 +/- 0.03 mg/L p less than 0.001), though zincuria and microalbuminuria were not significantly correlated. An exercise provocation test was performed in 78 IDDM. Although microalbuminuria increased, zincuria did not change during the test. Another group of 83 IDDM underwent urinary zinc determination over a period of 1 h of recumbency. The 48 patients who had a zincuria higher than the mean + 2 SD of control values had higher microalbuminuria at rest (48 +/- 16 micrograms/min vs 12 +/- 2 p less than 0.01) and after exercise (111 +/- 33 vs 42 +/- 14 p less than 0.02) than the remaining 35 subjects. Both subgroups did not differ for zinc intake and zincemia. Thus, incipient nephropathy as detected by the measurement of microalbuminuria is associated with a highly significant increase in zinc excretion, which is not proportional to albumin leakage, nor is it amplified during exercise. Hyperzincuria is not explained by an increase in zinc intake and does not result in hypozincemia.


Zinc and copper in the serum of diabetic patients. The Zn/Cu ratio was examined in the serum of three groups of persons: healthy volunteers, diabetic patients on diabetic diet (NIDDM), and diabetic patients on diabetic diet and insulin (IDDM). Zinc, copper, the Zn/Cu serum ratio, and the blood glucose level were determined during fasting and 2 h after breakfast. Zn and Cu serum levels in NIDDM and IDDM patients were decreased. The Zn/Cu ratio was higher in both groups of diabetic patients. These changes in the Zn and Cu levels as well as in the Zn/Cu ratio were not related to chronic diabetic complications.


Effects of diabetes type and treatment on zinc status in diabetes mellitus. Zinc status was assessed in 53 diabetic patients: 18 insulin-dependent diabetic patients (IDDM), 22 noninsulin-dependent diabetic patients (NIDDM) treated with oral antidiabetic agents, and 13 insulin-treated, noninsulin-dependent diabetic patients (IRDM). Plasma zinc concentrations were in the usual range for healthy subjects in these three groups (15.3 +/- 0.9 mumol/L). Urinary zinc excretions were elevated in the IDDM group (18.3 +/- 4.1 mumol/24 h; p less than 0.01 vs normal) and in the NIDDM group (17.5 +/- 3.5 mumol/24 h; p less than 0.01 vs normal), but normal in the IRDM group (11.3 +/- 2.4 mumol/24 h). In 14 NIDDM patients treated with transient continuous sc insulin injections, urinary zinc decreased from 16.5 +/- 2.2 mumol/24 h before insulin treatment to 11.5 +/- 0.3 mumol/24 h after insulin treatment without any modification in plasma zinc concentrations.


Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes. Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37 degrees C (binding and internalization) and 16 degrees C (binding) using 125I insulin 0.05-20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.


Zinc bone loss in chronic renal failure and chronic metabolic acidosis. The effects of chronic metabolic acidosis (CMA) on zinc (Zn) bone content and urinary excretion were examined in the presence of normal or reduced renal function together with some aspects of calcium (Ca) metabolism. Four groups of rats were compared. All were fed a 30% protein and 9 mg Zn/100 g diet. Two were uremic (U): The first developed acidosis (UA), which was suppressed in the other (UNA) by NaHCO3 supplement. Two other groups had normal renal function: One was normal (CNA), and the other had NH4Cl in the drinking water and acidosis (CA). Femur total Zn and Ca content was markedly reduced by CMA and was not affected by uremia. Zn urinary excretion was increased by CMA and unaltered by uremia. Ca urinary excretion was markedly reduced in uremic rats, but was enhanced in both acidotic conditions. Urinary Ca and Zn showed a strong correlation in uremic and in control rats. Plasma parathormone and 1,25(OH)2D3 were unchanged by CMA. These data are in agreement with a direct primary effect of CMA on bone in releasing buffers. CMA induces bone resorption and a parallel decrease of mineral bone components, such as Ca and Zn, with little or no role of PTH, 1,25(OH)2D3 and of uremia itself.


Iron in granulocytes of nephrotic patients determined by Zeeman electrothermal atomic absorption spectrophotometry. One of the major threats to patients undergoing maintenance hemodialysis is an increased susceptibility to infections caused by microorganisms, among which Yersinia or Listeria are frequently recovered. In patients receiving hemodialysis, iron overload owing to multiple transfusions plays an important role in the mechanisms underlying the susceptibility to bacterial infections, partially mediated by impaired neutrophil function. In order to assess the true role of iron at the cellular level, an AAS method was developed to measure the iron content of granulocytes. Iron levels in the granulocytes were determined in an apparently healthy population and in a population of iron-overloaded renal hemodialysis patients. Granulocytes were isolated by a method modified from Böyum. The analyses were performed using pyrocoated graphite tubes, and in one of the char steps, oxygen was used to facilitate ashing. The mean iron level found in the granulocytes from apparently normal persons was 4.07 fg/cell (n = 17) with a CV of 44%; the mean value for the dialysis patient group was 4.59 fg/cell (n = 8) with a CV of 37%. There was no significant difference between the two groups, p = 0.70.


Effects of acute prednisolone administration on plasma and liver copper in rats with adjuvant arthritis. Several studies in animals and humans have shown that copper metabolism could be affected by inflammation or by corticosteroids. The relative importance of these two factors, often imbedded in clinical practice, was assessed by investigating the effects of acute prednisolone administration (30 mg/kg, ip) on healthy and adjuvant arthritis rats. Plasma copper levels were significantly higher in arthritic rats compared to healthy animals, whereas there was a slight, but nonsignificant increase in liver copper. Acute prednisolone administration in healthy rats resulted in a significant increase in plasma copper (10-15%) as early as 4 h after corticosteroid administration, which was maintained for 12 h. In arthritic rats, the response was much higher (25-40%), but somewhat delayed and shorter. Liver copper was not clearly modified by prednisolone treatment in both groups. This time-controlled study showed that acute prednisolone administration increased plasma copper in both healthy and arthritic rats, but in different ways, indicating that inflammation and corticosteroids may act synergistically.


The role of zinc in reproduction. Hormonal mechanisms. Zinc is a very important element in the reproductive cycle of species. In humans, it is necessary for the formation and maturation of spermatozoa, for ovulation, and for fertilization. During pregnancy, zinc deficiency causes a number of anomalies: spontaneous abortion, pregnancy-related toxemia, extended pregnancy or prematurity, malformations, and retarded growth. Delivery is adversely affected by deficiency. These different effects of zinc can be explained by its multiple action on the metabolism of androgen hormones, estrogen and progesterone, together with the prostaglandins. Nuclear receptors for steroids are all zinc finger proteins. Zinc supplementation has already proven beneficial in male sterility and in reducing complications during pregnancy. However, it would be worth conducting larger-scale trials to confirm these beneficial effects.


Hormonal effects of zinc on growth in children. Zinc deficiency impairs the metabolism of thyroid hormones, androgens, and above all growth hormones. In view of their important role in growth, it is not surprising to find growth disorders associated with zinc deficiency. Stunted growth linked to zinc deficiency is found during gestation, and also in the newborn and children up to adolescence. Depending on the country, 5-30% of children suffer from moderate zinc deficiency, responsible for small-for-age height. Zinc supplementation has proven effective in many studies, mainly in children where zinc deficiency has first been found. Finally, zinc supplementation makes it possible in certain cases to overcome resistance to growth hormone treatment.


Serum zinc and somatic growth in children with growth retardation. A possible role for zinc deficiency in some cases of growth retardation in southern France was investigated. Control values for zinc for 160 children (age = 12.5 +/- 2.4 yr) are 0.85 +/- 0.22 mg/L (mean +/- 2 SD). Twenty-five children with low serum zinc values (less than 0.63 mg/L) and 25 matched short children with normal serum zinc values (greater than 0.63 mg/L) were studied. Children in the two groups did not differ significantly in age, pubertal development, stature, and weight. For the 25 children whose serum values were low, we found significantly lower values for bone age delay, growth velocity in mm/month, as well as the ratio between calculated growth velocity and theoretical growth velocity for the bone age (so that zincemia was correlated to these parameters in the whole sample of 50 subjects). Nevertheless, no significant difference could be found between the two groups for serum somatomedin C, serum osteocalcin values, and GH responses to the GH stimulatory tests (exercise test, overnight sampling, insulin-induced hypoglycemia, arginine test). Therefore, low serum zinc is associated with a retardation in both somatic growth and pubertal maturation.


Comparative binding study of aluminum and chromium to human transferrin. Effect of iron. The characteristics of aluminum and chromium binding to apo-transferrin (apo-tf) have been investigated and compared. Both metal ions were taken up by human transferrin forming complexes with the maximum absorbances at 405 nm for chromium-transferrin (cr-tf) and 240 nm for aluminum-transferrin (Al-tf). In the presence of citric acid, chromium binding to transferrin is five times more than aluminum. The binding of aluminum or chromium to apo-transferrin was reduced by 18 and 22% in the presence of 200 ng/mL of iron. The binding of both metals to apo-tf appears to be pH dependent. In acidic pHs, less chromium and more aluminum binding occurred.


The effect of barium selenate injection on selenium concentration and glutathione peroxidase activity in blood of pregnant ewes fed selenium-deficient diet. Selenium (Se) levels in whole blood and plasma, and glutathione peroxidase (GSH-Px) activities in red cells and plasma were measured in ewes fed an Se-deficient diet injected with barium selenate before breeding season. Highly significant increases in Se levels and GSH-Px activities (P less than 0.001) were observed throughout the gestation period and during lactation. In the control group, Se levels and GSH-Px activities decreased significantly (P less than 0.001), and were at critically low levels during lambing and lactation periods.


Effects of a multivitamin mineral supplement on zinc and copper status during pregnancy. The effect of a multivitamin-mineral supplement was investigated during pregnancy according to a double-blind protocol by determining zinc and copper in maternal plasma, mononuclear and polynuclear zinc and copper at the third, sixth, eighth, and ninth months of gestation. The subjects were supplemented from the first trimester until delivery. A significant decrease was observed in plasma zinc that varied from 11.5 mumol/L to 10.8 mumol/L in the supplemented group (n = 29) and from 11 mumol/L to 10 mumol/L in the placebo group (n = 33) at 3 and 9 mo of gestation, respectively. In contrast, plasma copper levels increased in a way depending upon the stage of gestation in both groups: from 24.7 to 28.2 mumol/L in the treated group and from 24.9 to 30.9 mumol/L in the placebo group at 3 and 9 mo of gestation, respectively, but the difference was only significant in the placebo group. No difference between groups was observed in mononuclear and polynuclear zinc or copper levels. These trace elements were also determined in cord blood at delivery. There were no statistically significant differences in zinc and copper concentration found in placebo group and supplemented group. Finally, the beneficial effect of supplementation on muscular cramps and appearance of vergetures was noted.


Chromium status of full-term and preterm newborns. In order to obtain reference values from normal babies, Cr status of full-term newborns has been studied. Plasma and urine values were (mean +/- SEM) 0.7 +/- 0.1 micrograms/L and 0.9 +/- 0.3 micrograms/L, respectively, for the first month of life (n = 19), and 0.6 +/- 0.1 micrograms/L and 0.8 +/- 0.2 micrograms/L for the second-to-third-month period (n = 31). Premature newborns (gestational age 28-36 wk) were compared to these control values; concentrations were 0.9 +/- 0.1 micrograms/L and 1.1 +/- 0.2 micrograms/L for the first month (n = 47), and 1.0 +/- 0.2 micrograms/L and 1.5 +/- 0.3 micrograms/L for the second to third months (n = 27). For the whole group, there was a positive correlation between plasma and urine concentrations (p = 0.0001); multiple regression analysis was performed between plasma levels and gestational age at birth (p = -0.002) and postnatal age (NS). Plasma levels of prematures and full terms were statistically different (p = 0.03) only for the second- to third-month period. It is suggested that these high Cr levels result from high dietary intakes and/or high absorption rates.


Interleukin 2 production in iron-deficient children. The relationship between iron status and capacity for IL-2 production by lymphocytes was assessed in 81 children from 6 mo to 3 yr of age selected at random from a population with low socioeconomic status, undergoing free systematic examination in four children's health centers in the Paris area. Iron deficiency was defined by the existence of at least two abnormal values among the three indicators of iron status: serum ferritin level less than or equal to 12 micrograms/L, transferrin saturation less than 12%, and erythrocyte protoporphyrin concentration greater than 3 micrograms/g hemoglobin. According to this definition, 53 children were classified as iron deficient and 28 as iron sufficient. No differences were observed between the iron-deficient and iron-sufficient groups in terms of the IL-2 concentration without stimulation by PHA. IL-2 production by lymphocytes stimulated with PHA, as well as the stimulation index (ratio of IL-2 concentration following stimulation by PHA to that of IL-2 concentration without stimulation by PHA) were significantly lower in iron-deficient children. The reduction in IL-2 production by activated lymphocytes observed in our study of iron-deficient children may be responsible for impairments in immunity found by other authors, particularly in cell-mediated immunity.


Plasma chromium concentrations in normal infants and cystic fibrosis patients. Plasma Cr concentrations have been studied in normal children aged 0-14 yr. Levels ranged from 0.65 to 0.88 microgram/l and did not change with age. Plasma concentrations of CF patients given 0.5-0.75 microgram Cr/kg/d in addition to their diet were similar to normal values. There was no correlation between these plasma values and growth retardation.


The effect of chromium on parameters related to iron metabolism. The effect of chromium on some parameters related to iron metabolism was investigated. Preliminary experiments showed that this metal ion was taken up by serum proteins and was dependent on the amount of chromium present in the medium. It was also shown that the uptake of iron was reduced significantly in the presence of chromium. In vivo study showed that the serum levels of iron and total iron binding capacity (TIBC) were reduced by 28 and 11%, respectively, following daily administration of chromium (1 mg/kg) for 45 d. Serum ferritin was reduced by 22% under this condition. Hematocrit and hemoglobin levels were also affected in chromium-treated animals and were both reduced by 17%. Spectrophotometric titration of each individual amino acid located in the iron binding site of transferrin revealed that tyrosine might be the most suitable ligand for the binding of chromium to transferrin. These results suggest that chromium may compete with iron in binding to apo-transferrin, and influence iron metabolism and its related biochemical parameters.


Treatment and planning decisions in non-small cell carcinoma of the lung: an Australasian patterns of practice study. Fourteen practising radiation oncologists were surveyed to assess their treatment and planning habits utilizing six sample cases of non-small cell carcinoma of the lung. Respondents were first given a general questionnaire, designed to evaluate their theoretical treatment and planning recommendations based on various tumour and patient related variables. Respondents then undertook a practical planning exercise utilizing planning CT and simulator radiographs for each of the six sample cases. Each case was accompanied by a brief history and report outlining specific tumour stage and non-stage related variables. The practical planning exercise was repeated on the second day of the survey utilizing different non-stage related variables but identical radiology and stage-related information. This design enabled firstly, a comparison of clinicians' intended policy and planning methods with actual policy and planning decisions, and secondly, an assessment of intra-clinician variability in decision making and planning practice. Good agreement was evident among clinicians with respect to general, non-case specific treatment policies; however, very significant variation occurred at an inter- and intra-clinician level and involved the entire treatment and planning process for individual cases. Despite identical treatment intent across identical radiological case pairings, clinicians chose widely differing margins and target volumes in their planning exercise. Treatment intent appeared to be influenced more by non-stage related variables rather than stage related information and radiological appearances per se. We have shown that experienced radiation oncologists do not adhere to stated case selection criteria and show inconsistencies in their treatment planning for non-small cell carcinoma of the lung.


Chromium metabolism. A literature review. The trivalent state of chromium (Cr3+) is that encountered in biological milieus and is responsible for its nutritional activity. The principal route by which trivalent chromium enters the body is the digestive system. Chromium in foods is present both in the inorganic form and as organic complexes. Intestinal absorption of chromium is low (0.5-2%), and the mechanism has not yet been fully elucidated. Absorbed chromium circulates as free Cr3+, as Cr3+ bound to transferrin or other plasma proteins, or as complexes, such as glucose tolerance factor (GTF)-Cr. Circulating trivalent chromium can be taken up by tissues, and its distribution in the body depends on the species, age, and chemical form. It is excreted primarily in the urine by glomerular filtration or bound to a low-mol-wt organic transporter. Chromium metabolism is still imperfectly understood. The use of 51Cr has nevertheless furnished valuable data concerning its transport and excretion.


Chromium content in the hair of children and students in southern Poland. Chromium concentrations in hair were compared in groups of children and students from Southern Poland. There were no statistically significant differences (at p greater than 0.05) between girls and boys. The results are similar to those found by other authors in different countries.


Samarium-153-labelled EDTMP for bone metastases from cancer of the prostate. A Phase I dose-escalation trial of a bone seeking phosphonate labelled with Samarium-153 was conducted on heavily pretreated patients with widespread bony metastases from cancer of the prostate. The bone marrow dose was calculated initially on a 4:1 uptake of cortical to cancellous bone, but subsequent information suggested the distribution was approximately equal so that the marrow received about three times the dose that has been prescribed. As a result, what were at first thought to be doses of 0.5 Gy, 1.0 Gy and 1.5 Gy were probably 1.5 Gy, 3.0 Gy and 4.5 Gy respectively. Three patients were treated at each dose level. The dose was repeated in the first three. Pain relief was delayed for 2 weeks, and was maximal by 4 weeks. All but one patient gained some benefit but this was transitory, lasting only 4-6 weeks in most. A recalculated dose of 3 Gy proved the most effective. The major toxicity was to platelets. Thrombocytopenia was fatal in four cases (two with repeat doses). All patients died but three survived more than 6 months with the help of third line hormonal measures and local radiotherapy to maintain comfort. A second group of five patients, not previously irradiated, were given 153Sm-EDTMP to a marrow dose of 3 Gy and all have survived for more than 4 months and achieved minimal to excellent relief of pain. The technique is recommended as initial therapy in unirradiated patients with good bone marrow function and with significant pain above and below the diaphragm, making half-body irradiation less likely to be a useful palliative procedure.


Segregated thalamocortical pathways to inferior parietal and inferotemporal cortex in macaque monkey. Inferior parietal and inferotemporal cortex, which process different aspects of visual information through largely segregated pathways from the visual cortex, both receive thalamic afferents from the pulvinar complex. We examined the topography of pulvinar projections to these two cortical regions by placing multiple injections of different tracers (fluorescent dyes, horseradish peroxidase) in the inferotemporal and inferior parietal cortex of macaque monkeys. The patterns of label observed after injections in inferotemporal gyrus indicate that area TEO and the ventral part of area V4 receive a major input from the ventral part of the lateral pulvinar (PuLv) while area TE has strong connections with the caudal pole of the medial pulvinar (PuM) and only minor connections with PuLv. In contrast, injections in the caudal inferior parietal cortex demonstrate that area PGc, on the lateral surface of the inferior parietal gyrus, and area POa, in the ventral bank of intraparietal sulcus, receive strong projections from PuM and the adjacent fringe of the dorsal part of the lateral pulvinar (PuLd). Paired injections of two different tracers in the inferotemporal and inferior parietal cortex of the same hemisphere revealed a nearly complete segregation of the two populations of labeled neurons in the pulvinar, with only a small region of overlap in PuM, close to the PuM/PuLd border. These results demonstrate a clear separation of the thalamic afferents to the inferior parietal and inferotemporal cortex which parallels the separation of prestriate afferents to these two cortical territories (Morel & Bullier, 1990).


Physiological chromium determination in serum by Zeeman graphite furnace atomic absorption spectrometry. A serious challenge. Several authors have already underlined chromium implication in glucose and lipids metabolism of humans. In this field, physiological chromium determination in serum could be helpful, but the discrepancies reported in numerous papers are confusing. Here we report some results obtained by Zeeman correction Graphite Furnace Atomic Absorption Spectrometry. This technique includes a dilution of serum with 12.5 mM ultrapure nitric acid and 0.25% Triton X-100 (final concentrations). Some main features can be outlined: (1) the contamination constitutes a serious drawback and (2) the sensitivity of the technique is critical (characteristic mass found: 1.76 pg/0.0044 A.s). Our results obtained from 27 healthy subjects (2.01 +/- 0.77 nmol/L) agree with most recent studies and indicate that serum chromium level does not seem to be sex-related.


Ocular dominance columns in area 17 of Old World macaque and talapoin monkeys: complete reconstructions and quantitative analyses. The effects of monocular deprivation on cytochrome-oxidase (CO) expression were used to reveal ocular dominance columns in flatmounts of the striate cortex in macaque (Macaca fascicularis) and talapoin (Miopithecus talapoin) monkeys. This procedure allowed the first direct visualization of the complete pattern of ocular dominance bands in macaque monkeys, and less complete reconstructions in talapoin monkeys. In a second macaque monkey, the ocular dominance organization was revealed by injecting wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into one eye. The organization of ocular dominance columns in the macaque monkeys conforms to previous descriptions, but the flat-mounted hemispheres provide accurate details on (1) the arrangement of columns, (2) the orientation of the representation of the optic disc, and (3) the breakdown of the bands in the cortex between the optic disc and monocular representations into a pattern of dots activated by the ipsilateral eye and larger surrounds related to the contralateral eye. Talapoin monkeys, the smallest of Old World monkeys, have sharply segregated ocular dominance bands. The columns in talapoins are narrower than those in macaques, so that even with less striate cortex than macaques, talapoins have more ocular dominance hypercolumns.


Substance P modulates calcium current in retinal bipolar neurons. Retinal bipolar cells are non-spiking interneurons that relay information from photoreceptors to amacrine and ganglion cells. In turn, bipolar cells receive extensive synaptic feedback from amacrine cells, some of which contain neuropeptides, including substance P. We have examined the effect of substance P on single bipolar neurons isolated from goldfish retina and find that substance P (0.1-1 nM) produced a voltage-dependent inhibition of calcium current in these cells. The inhibition was strongest at negative potentials, with the peak suppression occurring at -20 to -30 mV; at potentials positive to 0 mV, there was little effect on calcium current. Thus, the net effect was to shift the voltage range of activation of calcium current toward more positive potentials. The inhibition of calcium current by substance P required GTP in the patch pipette and was blocked by internal GDP-beta-S. Similar effects on calcium current were observed with somatostatin and metenkephalin, which are also found in amacrine cells.


Chromium content of foods and diets.

Chromium content of foods and diets. Comparatively few valid data are available on the chromium content of foods and on the dietary chromium (Cr) intake of various populations. This is chiefly because of the difficulties encountered in contamination control during sampling, sample pretreatment, and analysis. Moreover, there are several analytical problems involved that are mostly owing to the low concentration level of Cr in foods. However, with the recent establishment of food reference materials with certified low concentrations of Cr, the analytical validity of studies on Cr content of foods and on its dietary intake by various populations can be ascertained. With the exception of herbs and condiments, and certain other special food items with a relatively low average consumption rate, such as tea, coffee, and some candies, most foods contain Cr below 100 micrograms/kg. Staple foods, particularly cereals and milk, are very low (less than or equal to 10 micrograms/kg) in Cr, showing little or no geographic variation. Food processing may increase food Cr content depending on the process. Processes, such as meat grinding and homogenization using stainless-steel equipment, very strongly increase the Cr content of foods. Also, acidic fruit juices in contact with steel cans are high in Cr, whereas cooking in aluminium vessels reduces the Cr content of foods. Average dietary Cr intake seems to fluctuate considerably among countries. In many developing countries, such as Brazil, the Sudan, and Iran, the dietary intake is high, from 50-100 micrograms/d, whereas in certain developed countries, such as Finland, Sweden, Switzerland, and the US, the intake is 50 micrograms/d or lower and, consequently, at or below the estimated safe and adequate daily dietary intake range of 50-200 micrograms/d established by the US National Academy of Sciences. The average Cr content of human milk is below 0.5 micrograms/L, thus resulting in a very low average intake of 0.3 microgram Cr/d by exclusively breast-fed infants in the US and Finland.


Theoretical study of the intake of trace elements (nutrients and contaminants) via total diet in some geographical areas of Spain. The aim of this study was to monitor exposure to heavy metal contaminants from habitual diets and to estimate the health risk for the consumer by comparing the analyzed content with the acceptable daily intake (ADI). This study is based on the household consumption as assessed by the National Institute of Statistics (INE) and our department. The number of food groups (237) consumed by the population (national and the 17 Autonomy Communities) is considered. A list of the metal content of foods in included. The data were collected from reports issued by the Ministry of Health, other official organizations, and from specialized literature. The results show that the intakes of arsenic (0.019 mg/person/d), mercury (0.004 mg/person/d), and lead (0.082 mg/person/d) do not reach the ADI, but that of cadmium exceeds the ADI in some geographical areas. The intake of zinc and chromium does not exceed the dietetic recommendations.


[3H]iloprost and prostaglandin E2 compete for the same receptor site on cardiac sarcolemmal membranes. We have previously demonstrated that high-affinity PGE receptors are present on purified cardiac sarcolemmal (SL) membrane from bovine heart (Lopaschuk et al. (1989) Circ. Res. 65, 538-545). In this study we determined whether PGI2 receptors are also present on the cardiac SL membrane. Due to the extreme lability of prostacyclin (PGI2) under physiological conditions, the PGI2 analogue, Iloprost was substituted for PGI2. 3H-Iloprost specifically bound to two sites on the SL membrane; one of high affinity (Kd = 0.3 nM, Bmax = 97.0 fmol/mg SL), and one of lower affinity (Kd = 20.6 nM, Bmax = 1589 fmol/mg SL). Competition studies demonstrated that the concentrations of PGE2 and PGE1 necessary to displace 50% of the specific binding of 20 nM [3H]Iloprost on cardiac SL were 15-fold lower than the concentrations of unlabelled Iloprost necessary to displace 50% of binding. In contrast, a 15-fold higher concentration of unlabelled Iloprost was needed to displaced 50% of specific binding of 2 nM [3H]PGE2 compared to the concentrations of PGE1 or PGE2 required to displace 50% of [3H]PGE2 binding. In summary, our results indicate that a prostacyclin receptor is present on the cardiac sarcolemmal membrane, and that PGI2 competes for the same receptor site as PGE2.


Effect of cell exposure to top or bottom phase prior to cell partitioning in dextran-poly(ethylene glycol) aqueous phase systems: erythrocytes as a model. Cells exposed to dextran (Dx)-rich bottom phase prior to cell partitioning in Dx-poly(ethylene glycol) (PEG) aqueous two-phase systems have lower partition ratios than cells exposed to PEG-rich top phase. Aspects of this previously observed phenomenon were explored. In the present work charge-sensitive phases made with Dx T500 and PEG 8000 were used exclusively. It was found that: (1) even on countercurrent distribution (CCD) red cells (RBC) loaded in bottom phase have a lower apparent partition ratio, G, than the same cells loaded in top phase; (2) when part of the same cell population is loaded into top phase and part into bottom phase of the same load cavities for CCD, with the cells loaded into top or bottom bearing an isotopic tracer (51Cr), the cells loaded into top phase have a higher G value than the cells loaded into bottom phase; (3) the shift in the CCD curves of human or of rat RBC between cells loaded in top or bottom phase using systems having the same polymer concentration (though different salt compositions) shows no striking difference and is, for the number of experiments run, not statistically significant; (4) when the quantity of cells loaded for CCD is reduced from 10(9) to 10(8), the G value of cells loaded in top phase is reduced slightly while that of cells loaded in bottom phase is diminished more appreciably; (5) increasing polymer concentrations yield larger differences in G values between (rat) RBC loaded in top or bottom phase; (6) when cells exposed to top or bottom phase, respectively, are centrifuged and suspended in bottom or top phase, respectively, their CCD patterns are qualitatively similar to cells exposed to these latter respective phases initially; (7) rat RBC populations containing 59Fe-labeled cells of different but distinct age are fractionated on CCD irrespective of whether loaded in top or bottom phase. An exception are populations containing very young mature labeled cells (e.g., 4-d old) which are resolved when loaded in top phase but not in bottom phase. Thus cell populations exist which can be resolved by CCD when loaded in one of the phases but not when loaded in the other. Glutaraldehyde-fixed rat RBC containing 4-d old labeled cells are fractionated by CCD irrespective of whether loaded in top or bottom phase.


Effects of temperature variation and phenethyl alcohol addition on acyl chain order and lipid organization in Escherichia coli derived membrane systems. A 2H- and 31P-NMR study. Using 2H- and 31P-NMR techniques the effects of temperature variation and phenethyl alcohol addition were investigated on lipid acyl chain order and on the macroscopic lipid organization of membrane systems derived from cells of the Escherichia coli fatty acid auxotrophic strain K1059, which was grown in the presence of [11,11-2H2]oleic acid. Membranes of intact cells showed a gel to liquid-crystalline phase transition in the range of 4-20 degrees C, which was similar to that observed for the total lipid extract and for the dominant lipid species phosphatidylethanolamine (PE). Phosphatidylglycerol (PG) remained in a fluid bilayer throughout the whole temperature range (4-70 degrees C). At 30 degrees C acyl chain order was highest in PE, followed by the total lipid extract, PG, intact cells, and isolated inner membrane vesicles. Acyl chain order in E. coli PE and PG was much higher than in the corresponding dioleoylphospholipids. E. coli PE was found to maintain a bilayer organization up to about 60 degrees C, whereas in the total lipid extract as well as in intact E. coli cells bilayer destabilization occurred already at about 42 degrees C. It is proposed that the regulation of temperature at which the bilayer-to-non-bilayer transition occurs may be important for membrane functioning in E. coli. Addition of phenethyl alcohol did not affect the macroscopic lipid organization in E. coli cells or in the total lipid extract, but caused a large reduction in chain order of about 70% at 1 mol% of the alcohol in both membrane systems. It is concluded that while both increasing temperature and addition of phenethyl alcohol can affect membrane integrity, in the former case this is due to the induction of non-bilayer lipid structures, whereas in the latter case this is caused by an increase in membrane fluidity.


Permeabilizing action of filipin III on model membranes through a filipin-phospholipid binding. The binding of the pentaene antibiotic filipin to egg-yolk phosphatidylcholine (EPC) and dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles, has been studied by ultraviolet (UV) absorption and circular dichroism (CD). A stoichiometry of one molecule of filipin for five molecules of phospholipid was demonstrated by CD when phospholipids were in fluid phase. The similarity of the CD spectra with EPC and DMPC established a similar filipin-phospholipid assemblage in both membranes. We therefore postulated that filipin incorporation leads to the formation of gel-like domains in fluid EPC membranes as previously demonstrated for fluid DMPC membranes (Milhaud, J., Mazerski J., Bolard, J. and Dufoure, E.J. (1989) Eur. Biophys. J. 17, 151-158). The release of fluorescent probes (carboxyfluorescein (CF) or calcein (CC)), entrapped in EPC small unilamellar vesicles (SUV), due to the action of filipin, was followed by fluorescence and CD measurements concomitantly. The following observations were made. (1) The percentage of released probe, as a function of the filipin/phospholipid molar ratios, was the same whether or not membranes contained cholesterol. (2) The permeabilization of vesicles proceeded concomitantly with filipin-phospholipid binding while filipin-cholesterol binding leveled off. (3) The release of the content of vesicles occurred by an all-or-none mechanism leaving the depleted vesicles intact. From these observations and from the previous structural findings, a new interpretation of the action of filipin is proposed. Precluding any disruptive effect, inducement of permeability would result from the high intrinsic permeability of the interfacial region at the boundaries of the gel-like domains corresponding to the filipin-phospholipid aggregates. Additionally, we obtained the permeability coefficients for the anionic forms of CC and CF across EPC SUV, 0.6.10(-10) cm s-1 and 2.10(-10) cm s-1, respectively, as compared to 2.5.10(-14) cm s-1 for the counterion Na+ (Hauser, H, Oldani, D. and Phillips, M.C. (1973) Biochemistry 12, 4507-4517).


Abnormal responsiveness of granulocyte-committed progenitor cells in cyclic neutropenia. The mechanism(s) driving cyclic hematopoiesis in human cyclic neutropenia remains unknown. Clinical trials suggest that an abnormal responsiveness of bone marrow progenitor cells to hematopoietic growth factors might cause oscillatory blood counts. Studies were performed to determine whether an abnormal responsiveness to multiple growth factors exists in this disorder and whether the defect could be shown in highly enriched populations of marrow progenitor cells. Bone marrow mononuclear cells from patients with congenital cyclic neutropenia required higher concentrations of added granulocyte-colony-stimulating factor (G-CSF) to achieve half-maximal colony growth than cells from normal subjects (478 +/- 90 pmol/L v 53 +/- 12 pmol/L, P less than .01). Patients also differed in requirement for granulocyte-macrophage-CSF (P less than .05), but not for interleukin-3 (P greater than .30). CD34+ bone marrow cells from three patients also showed this difference in G-CSF responsiveness (P less than .05). These data suggest that the defect in congenital cyclic hematopoiesis lies in growth factor receptor binding or the postreceptor signal transduction system that drives granulocytopoiesis.


Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia. Patients with sickle cell anemia were treated with daily doses of hydroxyurea, to assess pharmacokinetics, toxicity, and increase in fetal hemoglobin (Hb) production in response to the drug. Plasma hydroxyurea clearances were not a useful guide to maximum tolerated doses of the drug. The mean daily single oral dose that could be maintained for at least 16 weeks was 21 mg/kg (range, 10 to 35 mg/kg). Among 32 patients, last HbF levels were 1.9% to 26.3% (mean, 14.9%) with increases in HbF over initial values of 1.4% to 20.2% (mean, 11.2%). The most significant predictors of last HbF were last plasma hydroxyurea level, initial white blood count and initial HbF concentration. Last HbF was not related to beta globin haplotype or alpha globin gene number. No serious toxicity was encountered. Clinically significant bone marrow depression was avoided, and chromosome abnormalities after 2 years of treatment were no greater than those observed before treatment. The period of observation has been too short to evaluate the risk of carcinogenesis. Patient's red cells developed striking macrocytosis. Median red cell Hb concentrations did not change. Hb concentrations increased, on average 1.2 g/dL, but serum erythropoietin levels increased. Patients' body weights increased, and some returned to work or school, but no conclusions regarding therapeutic efficacy could be drawn from this uncontrolled open-label study.


Decreased expression of eosinophil peroxidase and major basic protein messenger RNAs during eosinophil maturation. We evaluated the levels of mRNAs encoding cationic proteins in peripheral blood eosinophils (PBE) purified from patients with eosinophilia and in eosinophils differentiated from cord blood cells (CBC) by culture with recombinant human interleukin-3 (rhIL-3), rhGM-CSF, and rhIL-5. Messenger RNAs encoding eosinophil peroxidase (EPO), major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP) were detected by Northern blot hybridization with the respective specific oligonucleotide probes. In mature PBE, MBP mRNA appeared to be absent, whereas EPO mRNA was barely detectable in only 5 of the 19 patients. In contrast, EDN and ECP mRNAs were observed in the PBE of all patients. In CE, EPO, and MBP, mRNAs were abundant in immature eosinophils and their amounts decreased after differentiation toward eosinophils. ECP and EDN mRNAs followed the same patterns, but mRNAs were less abundant at all timepoints studied. Study of mRNA t1/2 during the time course of differentiation indicated that changes in the stability of the different mRNAs were not responsible for the variations observed in the steady-state levels. Together, these results suggest that regulation of expression differs among EPO, MBP, EDN, and ECP mRNAs during the time course of eosinophil differentiation.


Long-term generation of colony-forming cells in liquid culture of CD34+ cord blood cells in the presence of recombinant human stem cell factor. Human cord blood was used as a source of progenitor and stem cells to evaluate the effect of recombinant human stem-cell factor (SCF) on colony formation and the generation of colony-forming cells (CFC) under highly defined, serum-deprived conditions. SCF interacted with a number of hematopoietic growth factors to stimulate colony growth and was particularly effective in stimulating the formation of mixed-cell colonies from CD34+ soybean agglutinin negative (SBA-) cells. In suspension culture of CD34+, SBA- cells, SCF alone was unable to maintain cell numbers or CFC but, in combination with interleukin-3 (IL-3), increased input numbers of cells by 10-fold and increased CFC of all kinds by nearly 20-fold. This included erythroid burst-forming cells (BFU-E), granulocyte/macrophage (GM) CFC, and mixed-cell CFC. In contrast, CD34- SBA- cells neither gave rise to CFC nor were maintained by combinations of growth factors including SCF. SCF interacted with erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF) to maintain large numbers of cells as well as to generate a twofold to threefold increase in CFC in the case of Epo, and a 10-fold increase in CFC in the case of G-CSF. With Epo, the predominant CFC generated were BFU-E and erythroid CFC and many of the cells in suspension were erythroblasts. In contrast, SCF plus G-CSF resulted in large numbers of granulocytes at various stages of maturation and the CFC generated were almost exclusively granulocytic-CFC. IL-1 and IL-6, alone or in combination with SCF, showed little or no ability to increase cell numbers or generate CFC. In summary, SCF interacts with a variety of hematopoietic growth factors to promote colony formation, particularly mixed-cell colony formation, and also, in suspension culture, SCF interacts with IL-3, G-CSF, and Epo to generate large numbers of differentiated cells as well as a variety of CFC for up to 1 month.


Interferon-gamma enhances factor-dependent myeloid proliferation of human CD34+ hematopoietic progenitor cells. Numerous studies have shown that interferon-gamma (IFN gamma) inhibits the proliferative effects of colony-stimulating factors (CSFs) on human bone marrow cells. In the present study we investigated the effects of IFN gamma and other described inhibitory factors on the proliferation of highly purified CD34+ human hematopoietic progenitor cells (HPC) in response to recombinant CSFs. While transforming growth factor-beta (TGF beta) and IFN alpha were highly inhibitory, IFN gamma strongly potentiated interleukin-3 (IL-3) and, to a lesser extent, granulocyte-macrophage-CSF (GM-CSF) induced growth of CD34+ HPC. IFN gamma had no significant proliferative effect per se, and did not affect granulocyte-CSF (G-CSF)-dependent cell proliferation. Within 10 days the number of viable cells generated in the presence of IL-3 + IFN gamma was two times higher than in the presence of IL-3 alone. Limiting dilution analysis showed that IFN gamma acts directly on its target cell to increase the frequency of IL-3-responding cells without affecting the average size of the IL-3-dependent clones. Enhanced frequency of IL-3- and GM-CSF-responding cells was also observed in colony assays where the addition of IFN gamma increased by twofold to threefold the number of granulocyte colony-forming units (CFU-G), macrophage CFUs (CFU-M), granulocyte-macrophage CFUs (CFU-GM), and mixed erythroid (E-MIX). In contrast, IFN gamma did not affect the generation of erythroid burst-forming units (BFU-e) in such cultures. In longer-term culture, the combination of IFN gamma and IL-3 did not alter the lineage distribution of the cells when compared with IL-3 alone. However, after 15 days, when mature cells were present in the cultures, IFN gamma displayed cell concentration-related growth-inhibitory effects. Thus, IFN gamma appears to stimulate the early stage of myelopoiesis by enhancing the frequency of growth factor-responding cells but, unlike tumor necrosis factor alpha (TNF alpha), does not alter cell differentiation.


Plasminogen activator inhibitor-1 messenger RNA expression is induced in rat hepatocytes in vivo by dexamethasone. Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tissue plasminogen activator (tPA), plays a crucial role in the regulation of fibrinolysis. Both hepatocytes and endothelial cells have been implicated as major sources of plasma PAI-1. To study the relative contribution of these cell types to hepatic PAI-1 production, we have separated hepatocytes and hepatic sinusoidal endothelial cells by fractionation of freshly isolated rat livers using metrizamide density gradients and centrifugal elutriation. In untreated animals, PAI-1 messenger RNA (mRNA) was detected only in the purified endothelial cell fraction, and not in the hepatocyte fraction or in unfractionated liver. However, when the animals were treated with dexamethasone, PAI-1 mRNA expression was transiently induced in the liver. This induction paralleled the appearance of PAI-1 mRNA in purified hepatocytes, while PAI-1 expression in sinusoidal endothelial cells was unchanged. Four hours after dexamethasone treatment, plasma PAI-1 levels were increased approximately twofold over levels measured in animals treated with the diluent alone. These data suggest that PAI-1 production by hepatocytes may contribute to elevated plasma PAI-1 levels in the setting of acute injury and stress.


In vivo efficacy of B43 (anti-CD19)-pokeweed antiviral protein immunotoxin against BCL-1 murine B-cell leukemia. We show that a highly aggressive subclone of murine BCL-1 B-lineage leukemia expresses a single 2.4-kb transcript hybridizing to the human CD19 cDNA probe and reacts strongly with the anti-human CD19 monoclonal antibodies (MoAb) B43, B4, Leu-12, and J3-119. In contrast to their strong reactivity with anti-human CD19 MoAb, BCL-1 cells show no reactivity with MoAb directed against human CD22, CD72, HLA-DR, IgD, or IgM. Western blot analysis of BCL-1 whole cell lysates with the anti-human CD19 MoAb J3-119 showed a single 69-Kd protein band, which was not detected by the negative control MoAb G19.4 (anti-CD3). In contrast to BCL-1 cells, normal BALB/c splenocytes or mouse splenocyte/myeloma hybridoma cell lines did not (1) express any transcripts that hybridized to the human CD19 cDNA probe, (2) react with B43/anti-CD19 MoAb, or (3) express the 69-Kd protein that reacts with the anti-human CD19 MoAb J3-119. Murine BCL-1 B-cell leukemia thus provides a unique model of disseminated B-lineage leukemia to evaluate the antileukemic efficacy of anti-CD19 immunotoxins. This model was subsequently used to evaluate the in vivo homing ability, pharmacokinetics, and antileukemic efficacy of B43 MoAb conjugated to the plant hemitoxin pokeweed antiviral protein (PAP). B43-PAP immunotoxin (1) showed strong and antigen-specific reactivity with BCL-1 cells, (2) promptly penetrated the spleens of leukemic mice, (3) rapidly reduced the BCL-1 leukemia burden of leukemic mice and, most importantly, (4) improved survival. Finally, B43-PAP immunotoxin was more effective against BCL-1 leukemia than 700 cGy (LD100/30) total body irradiation (TBI) followed by syngeneic bone marrow transplantation (BMT).


Impaired in vitro growth of purified (CD34+) hematopoietic progenitors in human immunodeficiency virus-1 seropositive thrombocytopenic individuals. In this report the role played by human immunodeficiency virus type-1 (HIV-1) in the pathogenesis of HIV-1-related thrombocytopenia was investigated. CD34+ hematopoietic stem/progenitor cells were purified from the bone marrow (BM) of HIV-1(+) thrombocytopenic patients, HIV-1(+) nonthrombocytopenic individuals, HIV-1(-) patients with immune thrombocytopenic purpura, and HIV-1(-) normal donors. CD34+ cells from HIV-1(+) thrombocytopenic individuals alone showed a reduced capacity to give rise to megakaryocytic colonies (CFU-Meg) and also a progressive and significant decline in cell number when placed in liquid culture containing recombinant human interleukin-3 (rIL-3). This decline involved not only megakaryocyte but also erythroid and granulocyte/macrophage progenitors. The defects in megakaryocyte colony formation and CD34+ cell growth did not result from a productive HIV-1 infection of CD34+ cells. Moreover, HIV-1 DNA was absent from CD34+ cells in 10 of 12 thrombocytopenic patients examined. On the other hand, the decreased survival/proliferation of CD34+ cells in liquid culture, within the HIV-1(+) thrombocytopenic patients, was correlated with the presence of HIV-1 p24 antigen in BM plasma. These results demonstrate an impairment of CD34+ cells in HIV-1(+) individuals presenting thrombocytopenia as the only hematologic manifestation. Furthermore, these findings suggest that increased viral replication in the BM microenvironment may cause this impairment and possibly contributes to HIV-induced thrombocytopenia.


Mutations of p53 gene and their relation to disease progression in B-cell lymphoma. The alteration of p53 tumor suppressor gene was studied in 48 patients with B-cell lymphoma. A sequential combined technique of polymerase chain reaction-mediated single-strand conformational polymorphism (PCR-SSCP) or reverse transcription (RT)-PCR-SSCP and direct sequencing were used as a simple and sensitive approach to analyze nucleotide changes. By these methods, we identified 8 missense point mutations and 2 codon deletions in 9 of the 48 patients. These mutations were located in or close to the evolutionally highly conserved regions of the p53 gene. Eight of nine patients having p53 gene alterations were in advanced clinical stage (IV). It is the first report of p53 gene mutations in follicular and diffuse lymphoma. These observations suggest that the p53 gene alteration may play an important role in lymphomagenesis and/or disease progression in some types of B-cell lymphoma.


Cloning and analysis of a recombinant antigen containing an epitope specific for human T-cell lymphotropic virus type II. An immunodominant HTLV-I-specific epitope in the HTLV-I envelope glycoprotein (GP) 46 has been described. To determine if the analogous region of HTLV-II contains a similarly immunogenic and specific epitope, the polymerase chain reaction (PCR) was used to amplify HTLV-II DNA fragments encoding various portions of the putative epitope. The synthesized DNAs were cloned into lambda-phage gt11 and screened for production of immunoreactive fusion protein using sera from HTLV-II- or HTLV-I-infected individuals. Antisera from HTLV-II-infected individuals identified three of four recombinant clones when tested in a plaque immunoassay. Fusion protein from one of the clones, GH2-K15, was purified and analyzed by Western blot against a panel of HTLV-I and HTLV-II antisera. Twenty-one of 22 HTLV-II-infected sera were reactive with the GH2-K15 epitope. Sera from HTLV-I-infected and HTLV-I-uninfected individuals did not cross-react with GH2-K15. Western blot analysis of recombinant proteins encoding portions of the HTLV-II sequences in the Gh2-K15 antigen localized the HTLV-II-specific epitope to a 17-amino acid sequence. Recombinant antigens containing this epitope should be useful for type-specific serologic diagnosis of HTLV-II infection.


Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis. Long-term bone marrow cultures support both differentiation and conservation of primitive human hematopoietic progenitors in the absence of exogenous cytokines. It is believed that hematopoiesis in such cultures requires direct contact between hematopoietic progenitors and stroma. In the present study, we demonstrate that primitive progenitors physically separated from the stromal layer by a 0.45-microns microporous membrane continue to generate differentiated progenitors for at least 8 weeks. Moreover, primitive progenitors are conserved to a greater extent under these conditions, as when cultured in direct contact with the stroma. However, excessive production of granulocyte-macrophage progenitors occurs when primitive progenitors are not allowed to interact directly with the stroma. Thus, direct contact between hematopoietic and stromal cells is not required for either differentiation or conservation of primitive hematopoietic progenitors but is essential for the regulated production of mature blood elements. These findings can now be used to define the role of diffusible factors and cell-cell or cell-extracellular matrix adhesion events in the regulation of conservation, proliferation, and differentiation of primitive human hematopoietic progenitors in vitro.


A diverged homeobox gene is involved in the proliferation and lineage commitment of human hematopoietic progenitors and highly expressed in acute myelogenous leukemia. HB24 is a diverged homeobox gene known to be expressed in hematopoietic progenitor cells. We show here that the inhibition of HB24 expression in CD34+ bone marrow cells via antisense (AS) oligonucleotides impaired the proliferation of these cells in response to interleukin-3 and granulocyte-macrophage colony-stimulating factor. The treatment of CD34+ cells with HB24 AS oligonucleotides also reduced the levels of c-fos, c-myc, c-myb, cyclin B, and p34cdc2 messenger RNAs compared with cells treated with control oligonucleotides. Conversely, the transient transfection of HB24 into a subpopulation of CD34 cells inhibited their differentiation into mature hematopoietic cell types. In addition, HB24 messenger RNA transcripts were elevated in bone marrow and peripheral blood mononuclear cells isolated from patients with acute myelogenous leukemia compared with normal controls. These data suggest that HB24 is an important transcription factor during hematopoietic progenitor proliferation and that differentiation to specific cell types requires its downregulation. Furthermore, dysregulated expression of HB24 impairs the normal differentiation of hematopoietic progenitors and may contribute to leukemogenesis.


Pharmacokinetics of subcutaneous recombinant human granulocyte colony-stimulating factor in children. Fifteen children (age 1.2 to 9.4 years) with advanced neuroblastoma were treated with myelosuppressive chemotherapy (cyclophosphamide, cisplatin, doxorubicin) followed by 5 (n = 5), 10 (n = 5), or 15 (n = 5) micrograms/kg recombinant granulocyte colony-stimulating factor (rG-CSF) subcutaneously (SC) once daily for 10 days, starting the day after chemotherapy. Serial serum samples obtained on days 1 and 10 were analyzed for G-CSF activity by a specific proliferation assay using NFS-60 cells. G-CSF serum concentration-time data were best described by a one-compartment model, with zero-order absorption and first-order elimination. After SC injection, absorption was prolonged, with peak concentrations of G-CSF (3 to 117 ng/mL) being reached after 4 to 12 hours. The relatively slow absorption, with a mean elimination half-life of 5.8 hours on day 1 and 4.5 hours on day 10, provided measurable G-CSF concentrations for the entire 24-hour dosing interval in all patients at each dosage level. The median apparent clearance of G-CSF on day 10 was significantly higher than on day 1 (0.57 v 0.31 mL/min/kg, P = .02), and was positively correlated with the absolute neutrophil count (ANC) (r2 = .33, P = .003). Systemic exposure to G-CSF was dose-related, but interpatient pharmacokinetic variability yielded overlap in area under the concentration-time curve (AUC) at all three dosage levels. Stepwise regression analysis showed that G-CSF AUC could be predicted by a model that includes rG-CSF dosage and ANC on the day of administration (r2 = .82, P = .0001).


Enhancement of murine hematopoiesis by synergistic interactions between steel factor (ligand for c-kit), interleukin-11, and other early acting factors in culture. Entry into the cell cycle of dormant hematopoietic progenitors appears to be regulated by multiple synergistic factors, including interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), IL-11, and the ligand for c-kit, which is also known as steel factor (SF). We have tested the effects of these and other hematopoietic factors on the proliferation of partially enriched dormant murine progenitors in the presence and absence of serum. In serum-containing cultures, SF and IL-11 interacted to support the formation of multilineage colonies; the level of colony formation was comparable with the colony formation supported by other effective two-factor combinations. In serum-free cultures, colony formation supported by two factors was significantly less than that in serum-containing culture and the most effective two-factor combination in serum-free culture was SF plus IL-3. In serum-free cultures, three-factor combinations consisting of SF, IL-3, and one of IL-6, G-CSF, or IL-11 yielded colony formation that was comparable with that seen in serum-containing cultures. These studies indicate that IL-11 belongs to a group of early-acting hematopoietic synergistic factors that now includes IL-6, G-CSF, and IL-11. In contrast, SF is unique among the synergistic factors in that it interacts either with growth factors such as IL-3 or GM-CSF or with synergistic factors such as IL-6, IL-11, or G-CSF.


The clonal proliferation of normal mouse hematopoietic cells: enhancement and suppression by colony-stimulating factor combinations. Combinations of relatively high concentrations of the four colony-stimulating factors (CSFs) in cultures of normal mouse bone marrow cells stimulated subadditive responses in the number of colonies developing but, with some combinations, superadditive increases in mean cell numbers per colony. This latter effect was due largely to the induced development of small numbers of giant colonies containing macrophages with or without granulocytes. However, in cultures including a combination of granulocyte-macrophage-CSF (GM-CSF) with macrophage-CSF (M-CSF), a selective reduction in the number of pure macrophage colonies was observed together with a change in the morphology of those colonies that did develop. Recloning studies on macrophage colonies showed that the inhibitory action of the GM-CSF plus M-CSF combination was a direct one on the colony cells. The example of inhibition observed suggests that combined stimulation by two positive growth factors can sometimes result in a selective reduction of the production of certain cells, a possibility needing further exploration.


Granulocyte-macrophage colony-stimulating factor, interleukin-3, and steel factor induce rapid tyrosine phosphorylation of p42 and p44 MAP kinase. Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin-3 (IL-3), and Steel Factor (SF) induce proliferation of hematopoietic cells through binding to specific, high-affinity, cell surface receptors. However, little is known about postreceptor signal transduction pathways. In previous studies, we noted that each of these three factors could independently support proliferation of the human MO7 cell line, and also that each factor induced a rapid increase in protein-tyrosyl phosphorylation. Although the proteins phosphorylated on tyrosine by GM-CSF and IL-3 are similar or identical in MO7 cells, many of the proteins that are phosphorylated on tyrosine after SF are different. However, two proteins, p42 and p44, were prominently phosphorylated in response to all three of the factors. In MO7 cells, the tyrosyl phosphorylation of p42 and p44 was transient, peaking at 5 to 15 minutes. In contrast to many of the other proteins which are tyrosyl phosphorylated in response to these factors, phosphorylation of p42 and p44 was temperature-dependent, occurring at 37 degrees C, but not at 4 degrees C. We identified the p42 protein as p42 Mitogen-Activated Protein Kinase (p42mapk, ERK-2) and the p44 as a p42mapk-related protein using monospecific antisera to MAP kinase. GM-CSF, IL-3, and SF were each found to induce MAP kinase activity when assayed in vitro using myelin basic protein (MBP) as a substrate. Remarkably, we found that GM-CSF-induced tyrosyl phosphorylation of p42 and p44 even in nonproliferative cells (neutrophils) that respond to this CSF, and that p42 and p44 were two of the most prominently tyrosyl phosphorylated proteins following GM-CSF stimulation of these cells. These results implicate p42mapk and p44 as important signal transducing molecules in myeloid cells, and it is likely that these kinases play a role as part of a sequential "kinase cascade" linking growth factor receptors to mitogenesis and other cellular responses.


Regulation of complement functional efficiency by histidine-rich glycoprotein. The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.


Growth- and differentiation-associated expression of bcl-2 in B-chronic lymphocytic leukemia cells. The bcl-2 gene is translocated into the Ig loci in about 80% of human follicular lymphomas and in 10% of B-type chronic lymphocytic leukemias (B-CLL), resulting in a high level of expression. We have compared the expression of bcl-2 transcripts and protein in B-CLL cells in their normal equivalent CD5+ B cells and in normal B-cell populations representative of different in vivo and in vitro stages of activation and proliferation. We report here that bcl-2 was expressed in 11 of 11 cases of CD5+ B-CLL clones, contrasting with the absent expression in normal CD5+ B cells. Activation of 173 and 183 B-CLL cells by phorbol esters (12-O-tetradecanoylphorbol-13-acetate [TPA]) to IgM secretion without concomitant DNA synthesis resulted in a rapid but transient downregulation of bcl-2 expression. In contrast, the reduction of bcl-2 at both the messenger RNA and protein levels was sustained after mitogenic stimulation, suggesting that bcl-2 expression and proliferation are inversely related in these cells. This notion was further supported by immunocytochemical analysis showing that bcl-2 was primarily expressed in small resting lymphocytes and in cells differentiating to the plasma cell stage, but less expressed in Ki67-positive proliferating B blasts. Moreover, it was also supported by the low level of bcl-2 in exponentially growing Epstein-Barr virus-carrying lymphoblastoid and B-CLL cell lines. The regulation of bcl-2 expression in B-CLL resembled that of normal tonsillar follicular B cells, in which a high level of expression was found in resting mantle zone B cells but not in the proliferating germinal center B cells. Based on these findings and the role of bcl-2 in maintaining B-cell memory, we propose that the phenotype of B-CLL cells corresponds to a mantle zone memory-type B cell.


Complement activation during storage of blood under normal blood bank conditions. Effects of proteinase inhibitors and leukocyte depletion. During storage of CPD-A1 preserved whole blood factors of the complement cascade become activated, as evidenced by a rapid increase in the concentrations of C3a-desArg and C4a-desArg. After 10 to 14 days of whole blood storage, the elevations of C3a and C4a levels were highly significant. This increase was paralleled by an increase in the concentration of the lysosomal proteinase elastase from polymorphonuclear (PMN) granulocytes. By contrast, the concentration of the C3 activator complex C4b2b remained unchanged even after 3 weeks of storage. The supplementation of the anticoagulant CPD-A1 with the polyvalent-proteinase-inhibitor aprotinin and the specific elastase-inhibitor eglin C failed to inhibit complement activation, whereas leukocyte depletion could partially abolish the increase of the concentration of C4a, but had no effect on C3a concentrations. These observations support the notion that cleavage of C4 during storage of whole blood is partially leukocyte dependent, whereas the activation of C3 is possibly caused by the activation of the alternate pathway of the complement system by contact of plasma with plastic surfaces.


Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF): receptor biology, signal transduction, and neutrophil activation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are two of the growing number of recognized cytokines involved in the regulation of hematopoiesis. The purification of these factors and the subsequent cloning of the cDNAs which encode these proteins have led to their widespread clinical use in the setting of therapy or disease-induced myelosuppression. Although originally purified on the basis of their colony-stimulating properties, GM-CSF and G-CSF may also play important roles in the regulation of effector cell function. The mechanisms underlying progenitor cell proliferation and effector cell stimulation remain poorly understood. However, the characterization of the GM-CSF and G-CSF receptors and recent work in signal transduction are helping to elucidate these mechanisms. This paper will review the biology of the GM-CSF and G-CSF receptors, the mechanisms of post-receptor signal transduction, and the resultant effects on neutrophil function. In addition, the current and potential clinical uses of these factors will be examined in light of their ability to activate and perhaps enhance the function of neutrophils.


Diagnostic value of skin and laboratory tests in cow's milk allergy/intolerance. The applicability of a panel of five skin and laboratory tests in the diagnosis of cow's milk allergy/intolerance (CMAI) was investigated. The tests used were prick and patch tests, RAST, basophil histamine release test (BHRT) and lymphocyte proliferation test (LPT). Twenty-two atopic children who had experienced either immediate or delayed cutaneous symptoms upon challenge with cow's milk (CM), and 12 non-milk-allergic controls with atopic dermatitis (AD) were included in the study. RAST, prick test, BHRT and LPT to CM and patch test to alpha-casein were positive in the CMAI group and non-milk-allergic atopic controls as follows: 59% and 33%, 57% and 0%, 55% and 17%, 77% and 17%, 33% and 0%. RAST, prick test and BHRT were more often positive in children exhibiting immediate reactions, and patch test and LPT more often positive in those having delayed reactions to CM. The panel of five tests detected 21/22 children with CMAI and gave false-positive results in 5/12 of non-milk allergic controls. The sensitivity and specificity of the panel in the diagnosis of CMAI were 95% and 58%, respectively.


Specificity mapping of patients IgE response towards the tree pollen major allergens Aln g I, Bet v I and Cor a I. The specificity pattern of IgE from non-treated tree pollen allergic patients (n = 38) were evaluated by solid phase absorption of serum samples followed by CRIE on alder, birch and hazel CIE precipitation profiles. The majority of the serum samples seemed to contain IgE antibodies with the following characteristics: specific towards Bet v I alone and common between Aln g I, Bet v I and/or Cor a I, 'II'. The IgE specificity profiles observed for 95% of the sera tested are compatible with birch pollen allergens being the only sensitizing allergens, indicating that the patients react to allergens from other tree pollens of the Fagales order due to IgE cross-reaction with the major allergens of birch and alder and/or hazel pollens.


Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine. We have studied an aspect of the functional heterogeneity of human mast cells, namely responsiveness to the inhibitory effects of sodium cromoglycate and nedocromil sodium. The effects of these drugs were examined on the release of histamine and PGD2 from mast cells of human skin, lung, tonsils, adenoids and intestine. A high concentration, 1000 microM, of sodium cromoglycate was required to significantly inhibit histamine release from lung and tonsillar mast cells. Nedocromil sodium, 1000 microM, was more effective than sodium cromoglycate against histamine release from lung, tonsillar and adenoidal cells. Both compounds showed tachyphylaxis in lung and tonsillar mast cells but not in adenoidal and intestinal mast cells. In contrast, in intestinal mast cells, the effect of nedocromil sodium was weaker and more variable than sodium cromoglycate. Skin mast cells differed from mast cells of the other anatomical sites in being unresponsive to sodium cromoglycate and nedocromil sodium. Our results confirm that high concentrations of sodium cromoglycate and nedocromil sodium are required to achieve even modest inhibition of mediator release from human mast cells under in vitro conditions. Notwithstanding this, the results also indicate that differences exist among skin, lung, tonsillar, adenoidal and intestinal mast cells with respect to their sensitivity to sodium cromoglycate and nedocromil sodium, thus extending our knowledge of functional heterogeneity within the human mast cell populations.


Signal peptides open protein-conducting channels in E. coli. Plasma membrane vesicles and protoplasts of Escherichia coli were fused to planar lipid bilayers and studied with electrophysiological techniques. Large transmembrane aqueous channels were opened when 0.2 nM LamB signal peptide was added to the cytoplasmic side of the membrane. These aqueous pores are similar in conductance to those previously observed in mammalian endoplasmic reticulum when puromycin is used to release and thus unplug nascent translocating chains. Signal sequences have been previously shown to be necessary and sufficient for targeting proteins to cellular membranes. These results demonstrate that signal peptides are sufficient for opening the protein-conducting channels. We suggest that they are the physiological ligands that open protein-conducting channels at the initiation of protein translocation across prokaryotic plasma membrane and mammalian endoplasmic reticulum.


Signaling from LFA-1 contributes signal transduction through CD2 alternative pathway in T cell activation. LFA-1, a member of the integrin family of molecules, is involved in mediating cellular adhesion in all phases of the immune response, playing a role in the interaction of helper T cells as well as in killing of target cells by both cytotoxic T cells and natural killer cells. We have developed a monoclonal antibody, anti-HVS6B6, which recognizes a functionally unique epitope of the LFA-1 molecule. Although this mAb itself was not mitogenic against T cells, it induced a strong proliferative response when added to T cells with submitogenic concentrations of anti-CD2 (anti-T11(2) and anti-T11(3)) mAbs. In contrast, other anti-LFA-1 mAbs (CD11a and CD18) suppressed this anti-CD2 mAb-induced T cell proliferation. Kinetic studies showed that anti-HVS6B6 acts on an early event in CD2-mediated T cell activation. Although T11(3)-epitope expression induced by anti-T11(2) mAb was not affected by treatment of cells with anti-HVS6B6, both Ca2+ influx and phosphatidylinositol turnover induced by anti-CD2 mAbs were markedly enhanced by the pretreatment of T cells with anti-HVS6B6 mAb. These results indicate that the LFA-1 mediating signal contributes to a very early phase of signal transduction during CD2-mediated T cell activation.


The substitution of cysteine 17 of recombinant human G-CSF with alanine greatly enhanced its stability. Human recombinant granulocyte-colony stimulating factor (rhG-CSF) has one free cysteine at position 17 and has two disulfide bridges (Cys36-Cys42 and Cys64-Cys74). The Cys17 of rhG-CSF was substituted with Gly, Ala, Ser, Ile, Tyr, Arg, and Pro, or deleted using site-directed mutagenesis in order to improve its thermostability. With the exception of Pro17-rhG-CSF, all mutant proteins retained biological activity which promotes the growth of mouse bone marrow cells in vitro. Among these mutant proteins, Ala17-rhG-CSF had more than 5 times higher stability than rhG-CSF. But Ser17-rhG-CSF had almost same stability as rhG-CSF and other mutant proteins had only lower stability.


Studies on the mechanism of the synergistic interaction between 2'-deoxy-5-azacytidine and cisplatin. 2'-Deoxy-5-azacytidine (5-aza-CdR) and cisplatin interact to produce synergistic cytotoxicity against many human tumor cell lines. Preliminary experiments designed to explore the mechanism of this synergy suggested a poor correlation between synergy and the degree of genomic hypomethylation measured following exposure to 5-aza-CdR. Subsequent studies using plasmid DNA suggested that rather than DNA hypomethylation, incorporation of 5-aza-CdR into DNA mediated increased cisplatin binding to DNA and could therefore be essential to the synergistic interaction between these two agents. In this series of experiments, we evaluated the degree of synergy with cisplatin produced against two human melanoma cell lines by two additional antimetabolites that were chosen on the basis of their biochemical properties. In addition, we investigated the synergy between 5-aza-CdR and cisplatin in parental and 5-aza-CdR-resistant murine cell lines, which differed in their sensitivity to 5-aza-CdR and DNA methylation status but incorporated similar amounts of 5-aza-CdR into DNA when exposed to this antimetabolite. In the studies testing additional antimetabolites, cytosine arabinoside, which is incorporated into DNA but does not hypomethylate it, produced synergy with cisplatin that was similar or superior to that obtained using 5-aza-CdR. With 3-deaza-adenosine, which is not incorporated into DNA but produces DNA hypomethylation through inhibition of S-adenosylhomocysteine hydrolase, a primarily antagonistic interaction was observed in the two cell lines studied. In the 5-aza-CdR-sensitive and -resistant cell lines, a very similar synergistic interaction was documented for 5-aza-CdR and cisplatin despite the significant difference observed in DNA methylation levels. Taken as a whole, these data suggest that DNA hypomethylation was not critical to the synergistic cytotoxicity produced by 5-aza-CdR and cisplatin. This finding suggests additional strategies that could further modulate this interaction.


Potent inhibitors for the deamination of cytosine arabinoside and 5-aza-2'-deoxycytidine by human cytidine deaminase. Deamination of the nucleoside analogues ARA-C and 5-AZA-CdR by CR deaminase results in a loss of antileukemic activity. To prevent the inactivation of these analogues, inhibitors of CR deaminase may prove to be useful agents. In the present study we investigated the effects of the deaminase inhibitors Zebularine, 5-F-Zebularine, and diazepinone riboside on the deamination of CR, ARA-C, and 5-AZA-CdR using highly purified human CR deaminase (EC 3.5.4.5). These inhibitors produced a competitive type of inhibition with each substrate, the potency of which followed the patterns diazepinone riboside greater than 5-F-Zebularine and THU greater than Zebularine. 5-AZA-CdR was more sensitive than ARA-C to the inhibition produced by these deaminase inhibitors. The inhibition constants for diazepinone riboside lay in the range of 5-15 nM, suggesting that this inhibitor could be an excellent candidate for use in combination chemotherapy with either ARA-C or 5-AZA-CdR in patients with leukemia.


Radiotherapy in the treatment of middle ear and mastoid carcinoma. The treatment of temporal bone carcinoma is a widely discussed topic with marked variation in published results. Most conclude that a combination of radical surgery and radiotherapy is the optimum treatment. The present study reviews the results of radiotherapy used as the main primary treatment for this condition. Five-year survival in 56 patients was 32% for radical and palliative therapy, with an excellent response in 'early' cases. It is concluded that improvement in survival could be attained by defining those groups which would benefit from a combination of treatment methods.