Heterogeneity of protein phosphorylation in metastatic variants of B16 melanoma.

The polypeptide and phosphoprotein profiles of a spectrum of B16 melanoma clones of defined metastatic potential have been analyzed by two-dimensional gel electrophoresis. To accommodate the documented instability of metastatic properties in B16 clones, in vitro biochemical assays were always accompanied by in vivo assays of the metastatic behavior using replicate samples of the same clonal populations harvested on the same day. To exclude differences in polypeptide and phosphoprotein profiles resulting from inherent variation in electrophoretic measurements made at different times, polypeptides and phosphoproteins were analyzed in unison for every clone, and a series of clones was examined in parallel in each experiment. Also, samples were electrophoresed simultaneously using a custom-designed apparatus capable of accommodating 20 two-dimensional samples. When tested under these stringent conditions, the polypeptide profiles of B16 clones were indistinguishable. Significant qualitative and quantitative differences in phosphoprotein expression were detected in each clone, but no correlations were found between alterations in protein phosphorylation and metastatic potential. Over 200 discrete phosphoproteins were detected in each clone, but interclonal variation was confined to approximately 10 to 15 phosphoproteins. Expression of three phosphoproteins with the following molecular weights (in kilodaltons) and isoelectric points was strictly qualitative: pp96 (7.9); pp30 (8.2); and pp30 (8.8). In any given clone, they were present individually at equal intensities or were completely absent, but their expression was not coordinate. The data indicate that expression of polypeptide gene products is similar in B16 melanoma clones with widely differing metastatic abilities, but considerable clonal variability exists in posttranslational covalent modification of cell proteins. The possible contribution of protein phosphorylation and other posttranslational pathways in generating the extensive phenotypic heterogeneity observed in tumor cell subpopulations within the same tumor and in the rapid generation of new clonal variants with altered metastatic properties are discussed.

The presence of dialkylphosphates in fresh fruit juices: implication for organophosphorus pesticide exposure and risk assessments.

This study was designed to determine whether dialkylphosphates (DAPs) are present in fresh fruit juices, as a result of organophosphorus (OP) pesticides degradation. Fresh conventional and organic fruit (apple and orange) juices were purchased from local grocery stores. DAPs were found in both conventional and organic juices, and the original levels were higher, for both apple and orange juices, in conventional than in organic juices. Additional DAPs were found in OP pesticide fortified juices after 72 h of storage at 4 degrees C, suggesting a degradation of OP pesticides in juices. Overall, 12% and 36.2% of fortified azinphosmethyl, a dimethyl OP pesticide, and the combination of fortified diazinon and chlorpyrifos, both diethyl OP pesticides, were degraded to dimethyl and diethyl DAPs, respectively. Although the exact mechanism of the degradation is unknown, hydrolysis is likely the cause of OP pesticide degradation in juice. The presence of DAPs in fresh fruit juices clouds the validity of using urinary DAP measurements for estimating OP pesticide exposures in humans, particularly in children. The overestimated OP pesticide exposures based on urinary DAPs reported in other studies is likely due to the coexistence of preformed DAPs and DAPs resulting from OP pesticide exposures. Thus, before urinary DAP concentrations can be reliably used in exposure and risk assessment, the proportion of the concentration attributable to environmental DAP exposure, particularly through the diet, must be ascertained. In conclusion, urinary DAPs have many limitations when being used as biomarkers for OP pesticides in exposure and risk assessment, and caution should be exercised when interpreting DAPs results.

Evaluation of transdermal theophylline pharmacokinetics in neonates.

Theophylline may be administered by several routes, but problems are associated with neonatal dosing. The transdermal route may provide a safer and noninvasive method of administration, yet produce therapeutic concentrations in a consistent and reliable manner. To study the feasibility of this in the apnea of prematurity, stable neonates were administered a subtherapeutic transdermal dose for 24 hours in order to assess pharmacokinetics and bioavailability. This was followed with routine intravenous theophylline therapy according to institutional policy. Six of nine neonates had detectable serum theophylline concentrations that increased slowly after patch application. Mean (+/- SD) maximum serum concentration was 2.4 +/- 1.3 micrograms/ml, mean time to maximum serum concentration was 22 +/- 8.2 hours, and mean latency period was 8.0 +/- 4.9 hours. Mean total amount of theophylline delivered to the skin was 18.6 +/- 4.1 mg. Mean fractional absorption at 30 hours was 0.25 +/- 0.12. These data demonstrate that it is possible to produce systemic theophylline concentrations with a transdermal patch in preterm infants sufficient to study pharmacokinetics and bioavailability, and that transdermal delivery of therapeutic doses is technologically feasible.

[Normal levels of collagen-type-I telopeptide in the first 90 days of life]. / Livelli normali di telopeptide del collagene di tipo I nei primi 90 giorni di vita.

Serum levels of carboxyterminal telopeptide of type I collagen (ICTP), a marker of matrix degradation, were measured by RIA test, on 184 samples of healthy newborns and children aging from 1 (cord blood) to 90 days of life. We found ICTP values about tenfold higher than the adults', with highly significant variations (P < 0.001) in the whole period studied. During the first three months of life serum levels of the bone marker show a progressive increase from 0 to 7 days, they remain unchanged until the 30th day and then decrease until the 45th day, maintaining similar values from the 45th to the 90th day of life. The authors think that the pattern of ICTP in the first week of life is under the influence of the adapting phenomena following delivery, in which catabolic processes are predominant, while in the second period ICTP modifications are related to growing processes and then to bone turnover.

[Platelet factor 4 levels in full-term newborns undergoing phototherapy]. / Livelli di fattore piastrinico 4 (FP4) in neonati a termine sottoposti a fototerapia.

PF4 levels and platelets counts were studied in a group of 15 term newborn infants before treatment and after 24-48-72 and 96 hours of phototherapy and in a control group of 10 babies. Unlike data found by other AA. in vitro and in preterm infants, our values show only minimal, not statistically significant, differences in PF4 levels and platelets counts between the two groups. The AA. believe that in term infants, with adequate weight for gestational age, proper phototherapy treatment does not cause any change in platelet function, owing to the thicker and more mature skin and to the better bone marrow compensation typical of term versus preterm infants.

[The relationships between the degrees of oxygenation and the serum calcitonin levels in the term newborn]. / Relazioni tra gradi di ossigenazione e livelli di calcitonina sierica nel neonato a termine.

The authors have studied the correlations between serum levels of calcitonin and the degree of oxygenation assessed by means of transcutaneous pO2 and pCO2 and capillary pH in 40 term newborns of adequate birth weight. Highly significant correlations (P < 0.001) were found at the 24th hour of life between calcitonin levels and the asphyxia parametres and between the latter measured at the 12th or the 24th hour and calcitonin levels found respectively at the 24th or the 48th hour. Similar correlations were found subdividing the studied newborns with regard to the type of delivery. We conclude that the severity of neonatal asphyxia is indeed the main determining factor of the magnitude of the calcitonin hyperincretion.

[Normal levels of carboxyterminal propeptide of type I procollagen in the first three months of life]. / Livelli normali del propeptide carbossiterminale del procollagene di tipo I nei primi tre mesi di vita.

Serum levels of type I procollagen were measured on 118 samples from cord blood or from healthy infants aging from 1 to 90 days of life. Significant variations (P = 0.001) were noticed in the values of the marker in the whole period under investigation. We observed a decrease of PICP from cord blood to the end of the first day of life with a sharp rise on the 5th day lasting until the 30th day which then became stable till the end of the third month. Our results show a peculiar pattern of PICP levels during the first month of life which has to be taken into account to evaluate normal values of the marker in this period of life.

[Calcium phosphate metabolism in thalassemia]. / Metabolismo calciofosforico nella talassemia.

The AA. performed a screening test on 113 patients affected by beta thalassemia major ranging between 2 and 40 years of age, randomized among those who come to the Microcitemic Center of our Institute, and on a control group. In all of them serum levels of calcium, phosphate, parathyroid hormone (PTH), calcitonin and 25-OH vitamin D were measured. Average serum levels of PTH were significantly (P < 0.001) lower in our patients than in control group and 12.4% of the former were clearly under normal range, especially in the group over 16 years of age. Also serum levels of 25-OH vitamin D were lower in thalassemic patients than in controls, because of the presence of 32 patients with average values under normal limit. Our results are in agreement with current literature and underline the increasing incidence of endocrine complications in thalassemic patients who undergo high transfusion regimens, because of to the increase of hemosiderosis due to the low compliance to iron chelation therapy. Controversial is the pathogenesis of the absence of hypocalcemia in many patients with hypoparathyroidism and the cause of the deficit of vitamin D.

[Bone metabolism markers in thalassemia]. / Markers del metabolismo osseo nella talassemia.

The A.A. performed a screening on 113 patients affected by beta-thalassemia major ranging in age between 2 and 40 years, randomized among those which come to the Microcitemic Center of our Institute, and in a control group. In everybody, serum levels of calcium, phosphate, parathyroid hormone (PTH), calcitonin and 25-OH vitamin D were measured. Average serum levels of PTH were significantly (P < 0.001) lower in patients than controls and 12.4% of them were clearly under normal range, especially in the group above 16 years of age. Also serum levels of 25-OH vitamin D were lower in thalassemic subjects than in controls, because of the presence of 32 patients with values under normal limit. Our results are in agreement with current literature that underline the increasing incidence of endocrine complications in thalassemic patients which undergo to high transfusion regimens, owing to the increase of emosiderosis due to the low compliance to iron chelation therapy. Controversial is the pathogenesis of the absence of hypocalcemia in many patients with hypoparathyroidism and the determinism of the deficit of vitamin D.

[Calcium-phosphorus metabolism in celiac disease in children]. / Metabolismo calcio-fosforico nella malattia celiaca del bambino.

The Authors studied the changes of the main parameters of calcium-phosphate metabolism in twenty four untreated celiac children (mean age: 23.7 +/- 14.4 months) and in eleven control subjects (mean age: 28.5 +/- 21.2 months). 16 patients were checked again one and three months after treatment was begun. Compared with controls patients show at diagnosis a significant increase of serum phosphate (P = 0.025) and a decrease of calcitonin levels (P = 0.02), whereas serum calcium is slightly lower and parathyroid hormone higher with serum levels above normal range in 5 of the coeliac patients (20.8%). During the three months of gluten free diet we find a significant increase of calcemia values (ANOVA: P = 0.025) together with a decrease of serum phosphate (ANOVA: P = 0.009) and of parathyroid hormone levels (ANOVA: P = 0.042). No significant change was found in vitamin D metabolites levels. The Authors conclude that coeliac disease affect clearly mineral metabolism. Actually the tendency to hypocalcemia, due to abnormalities of the intestinal mucosa, and the comparative iperphosphatemia, cause in some patients a compensatory increase of PTH levels. This increase seems to be the cause of the osteoporotic lesions described in current literature. Rickets due to the lack of vitamin D does not occur.

Serum levels of osteocalcin and type I procollagen in children with celiac disease.

BACKGROUND: Bone metabolism may be disturbed in children with celiac disease. METHODS: Two markers of bone turnover were used: the level of osteocalcin (BGP) and the level of carboxylterminal peptide of type I procollagen (PICP). BGP and PICP were measured by radioimmunoassays in 18 untreated children with celiac disease (mean age: 22.9 +/- 15.6 months) and in 15 control subjects (mean age 28.5 +/- 21 months). All the patients were rechecked after 1 month and again after 3 months from beginning of a gluten-free diet (GFD). RESULTS: Compared with controls at diagnosis our patients had significantly lower serum levels of BGP and PICP (p = 0.003 and p = 0.018 by Student's t test, respectively). These levels increased markedly during the 1st 3 months of GFD. CONCLUSIONS: The alteration in calcium phosphate homeostasis caused by celiac disease directly affects the synthesis of both components of the connective matrix of bone. Measurements of BGP and PICP provide a reliable and rapidly obtainable index of normalization of the processes of bone growth which can be achieved with a GFD.

Alterations in the phosphoprotein composition of tumor cell clones induced by cell culture techniques used routinely in assessing metastatic behavior in vivo.

To investigate whether the cell dispersion techniques commonly employed to harvest monolayer cultures of tumor cells for injection into experimental animals might induce alterations in cellular biochemistry, we compared the phosphoprotein profiles of 6 B16 melanoma clones of distinct metastatic potential using 2-D gel electrophoresis after growth in monolayer culture, after suspension by treatment, with trypsin/EDTA and after injection of suspended cells into syngeneic mouse plasma. Trypsin/EDTA treatment and subsequent exposure to syngeneic mouse plasma induced significant alterations in phosphoprotein composition in all clones. Most alterations were quantitative, involving either enhanced or diminished expression of specific phosphoproteins, but qualitative changes involving expression of novel phosphoproteins were also observed. None of the changes in phosphoprotein composition correlated with metastatic potential. The principle alteration induced in all clones by trypsin/EDTA involved enhanced phosphorylation of an NP-40-soluble component with a molecular weight of 79,000 and an isoelectric point of 6.3 [pp79 (6.3)]. This determinant was detected in extracts of B16 monolayer cultures but its level of phosphorylation was enhanced significantly by trypsin/EDTA treatment and by exposure of the harvested cells to syngeneic mouse plasma. These data indicate that procedures commonly employed to harvest tumor cells for assay of tumorigenic and metastatic potential may provide extensive alterations in phosphoprotein composition and that biochemical investigations of tumor cells grown in monolayer culture may not accurately reflect the metabolic status of the same cells immediately prior to and following i.v. injection into experimental animals.

Familial Jarcho-Levin syndrome.

Jarcho-Levin syndrome is a variety of autosomal recessive spondylocostal dysostosis characterized by severe deformity of the thoracic cage, leading to respiratory failure and early death. There are often associated dysmorphic features. The disease is more frequent in Puerto Ricans and rare in Europe. A Sicilian family with four affected individuals in two interrelated sibships is reported.