Familial hyperglycerolemia.

A 70-yr-old mildly diabetic white male was discovered to have an elevated level of serum free glycerol in the range of 75 mg/dl and to excrete about 13 g of free glycerol in the urine per 24 h. During a 24-h fast the urine glycerol loss increased to 21.5 g per 24 h. Studies carried out in vitro using leukocytes prepared from the patient's blood which were incubated with [14C]glycerol demonstrated an almost complete absence of glycerol oxidation to 14CO2 and of glycerol phosphorylation, in contrast to control studies with leukocytes collected from normal subjects. Homogenates of the patient's leukocytes contained negligible activity of ATP:glycerol phosphotransferase (glycerokinase EC 2.7.1.30) as measured by a direct spectrophotometric method. Marked hyperglycerolemia has thus far been detected in one brother and in one son of the daughter of this patient. This evidence suggests an x-linked recessive inheritance pattern of the trait. There is a high prevalence of diabetes mellitus in this family.

Physical and functional interaction between wild-type p53 and mdm2 proteins.

The mdm2 oncogene, which is often amplified in mammalian tumors, produces a number of transcripts that encode distinct protein forms. Previous studies demonstrating that overexpression of the mdm2 gene can activate its transforming potential, and can inhibit the transcriptional activation function of p53, prompted us to begin to explore possible functional differences among the various mdm2 products. Utilizing a transient transfection assay, we have evaluated four naturally occurring murine mdm2 forms for their ability to inhibit p53-mediated transcriptional activation of reporter genes regulated by p53 response elements. Three of these mdm2 forms were found to physically associate with the wild-type p53 protein and to possess the ability to inhibit its transactivation function. A fourth form failed to exhibit either of these functions. This last mdm2 form lacks the N-terminal protein domain that is present in the other three splice forms examined, pointing to this region as one that is critical for complex formation with the p53 protein. Identifying such differences among mdm2 proteins provides important clues for dissecting their functional domains, and emphasizes that defining the individual properties of these products will be critical in elucidating the overall growth control function of the mdm2 gene.

MDM2 interacts with the C-terminus of the catalytic subunit of DNA polymerase epsilon.

MDM2 is induced by p53 in response to cellular insults such as DNA damage and can have effects upon the cell cycle that are independent or downstream of p53. We used a yeast two-hybrid screen to identify proteins that bind to MDM2 and which therefore might be involved in these effects. We found that MDM2 can bind to the C-terminus of the catalytic subunit of DNA polymerase epsilon (DNA pol epsilon), to a region that is known to be essential in yeast. In an in vitro system we confirmed that MDM2 could bind to the homologous regions of both mouse and human DNA pol epsilon and to full-length human DNA pol epsilon. DNA pol epsilon co-immunoprecipitated with MDM2 from transfected H1299 cells and also from a HeLa cell nuclear extract. We show here that the DNA pol epsilon-interacting domain of MDM2 is located between amino acids 50 and 166. Our studies provide evidence that MDM2 interacts with a region of DNA pol epsilon that plays a critical role in the function of DNA pol epsilon.

The pathway regulating MDM2 protein degradation can be altered in human leukemic cells.

The MDM2 protein regulates the functional activity of the p53 tumor suppressor through direct physical association. Signals that control MDM2 expression are poorly understood but are likely to play an important role in the regulation of p53 activity. We show here that the half-life of MDM2 protein is shorter in proliferating than in quiescent peripheral blood mononuclear cells. We also demonstrate that MDM2 protein half-life is extended in some, but not all, p53 mutant human leukemic cell lines. In at least one of these p53 mutant lines, increased MDM2 protein stability is associated with higher amounts of MDM2 protein. Moreover, we demonstrate that MDM2 protein accumulates to a much greater extent in proteasome inhibitor-treated cells containing unstable MDM2 than in cells possessing stable MDM2. These results demonstrate that MDM2 expression is regulated by events that control the stability of the protein and suggest that the normal regulation of MDM2 turnover can be altered in tumor cell lines.

Identification of a tumor-derived p53 mutant with novel transactivating selectivity.

MDM2 is a p53-responsive molecule that when overexpressed, can alter growth control pathways via p53-dependent and independent mechanisms. We have identified a mutant p53 containing line that expresses high levels of transcripts that are regulated by the p53-responsive promoter of the MDM2 gene. Analysis of cloned product obtained from these tumor cells revealed that they harbor a mutant p53 protein (possessing an Arg to Gln substitution at codon 213) that is a potent transactivator of MDM2 expression. Consistent with this activity, the R213Q mutant was found to have the ability to interact with DNA sequences located within the MDM2 promoter. In contrast to previously described tumor-derived p53 mutants which retain MDM2 transactivation function and possess partial growth suppressive activity, the R213Q mutant is severely compromised in its ability to induce p53-regulated transcripts that encode for proteins involved in cell-cycle arrest and apoptosis. The R213Q mutant can also be expressed at high levels in stably transfected cells and cells that harbor this mutant possess elevated levels of MDM2 protein. The R213Q mutant was also found to be able to up-regulate MDM2 during a genotoxic stress response. R213Q is the first described tumor-derived p53 mutant that is deficient at up-regulating both cell cycle arrest and apoptotic factors, but is highly proficient at inducing the growth-promoting molecule MDM2.

Enhanced translation: a novel mechanism of mdm2 oncogene overexpression identified in human tumor cells.

The cellular mdm2 gene, which has potential transforming activity that can be activated by overexpression, is amplified in a significant percentage of human sarcomas and in other mammalian tumors. Proteins encoded by the mdm2 gene can bind to, and inhibit the function of, the protein product of the p53 tumor suppressor gene. As reported here, we have identified human choriocarcinoma cell lines that express high levels of mdm2 proteins as well as the p53 protein. Several lines of evidence demonstrate that the p53 in these tumor cells has a wild-type nucleotide sequence, although the protein exhibits an extended half-life. Further, the more than 100-fold overexpression of mdm2 proteins in these cells cannot be explained by gene amplification, elevated RNA expression, or altered protein stability; rather our data indicate that elevated mdm2 protein levels in these choriocarcinoma cell lines result from enhanced translation. This mechanism has not previously been implicated in the regulation of mdm2 gene expression, and it represents a novel means by which the potential transforming activity of the mdm2 oncogene could be activated.

mdm2-P2 transcript levels predict the functional activity of the p53 tumor suppressor in primary leukemic cells.

The mdm2-P2 promoter is a transcriptional target of the p53 tumor suppressor. The aim of this study was to determine if there is an association between the level of these transcripts and the status of the p53 gene in human leukemic cells. A correlation between mdm2-P2 transcript levels and p53 gene status was observed in all cell lines examined. Primary malignant cells from 10 leukemic patients were also analysed for both mdm2-P2 levels and p53 gene status. All five patients with detectable mdm2-P2 transcripts possessed wild-type p53 alleles. However, only two of five patients with undetectable mdm2-P2 transcripts possessed mutant p53. mdm2-P2 levels were also determined in primary leukemic cells from 14 additional cases both before and after in vitro exposure to cisplatin. The p53 gene was found to be wild-type in all cases where mdm2-P2 levels were induced by cisplatin. There were four cases where no, or only modest, increases in mdm2-P2 levels were detected after cisplatin exposure. Two of these patients were found to harbor mutant p53 while one other possessed leukemic cells with elevated levels of mdm2 protein. These results show that induction of mdm2-P2 transcripts can be used to predict the presence of transcriptionally active p53 in primary leukemic cells.

Pulmonary phosphatidylcholine biosynthesis. Alterations in the pool sizes of choline and choline derivatives in rabbit fetal lung during development.

1. Progressive changes were noted in the pool sizes of choline in fetal rabbit lung between 25 and 30 days gestation (term, 31 days) and between 30 days gestation and adult lung. The level of choline in adult lung was double the level in the fetal lung at 25 days gestation. The pool size of choline phosphate decreased 10-fold during this period while the level of CDPcholine decreased by 30%. The phosphatidylcholine content increased 3-fold during development. The major change in the relative pool sizes was a marked decrease in the ratio of choline phosphate to CDPcholine from 26:1 at 25 days gestation to 3.4:1 in adult lung. 2. No differences were detected between the uptake of [14C]choline into slices from fetal lungs at 25 days gestation or slices from adult lung. However, the ability of the adult slices to convert [14C]choline into its derivatives was 30% lower than slices from fetal lung. In addition, whereas fetal slices contained significantly more radioactivity in choline phosphate and CDPcholine, adult slices incorporated significantly more [14C]choline into phosphatidylcholine. Experiments with [3H]choline and 32Pi revealed that the 3H/32P ratio of choline phosphate in fetal or adult slices was identical to the isotopic ratio in phosphatidylcholine, indicating that under the experimental conditions, negligible radioactivity was incorporated by base-exchange. Because of the marked decrease in the pool size of choline phosphate during development, it cannot be concluded that the increase in the incorporation of radioactive choline into phosphatidylcholine is indicative of increased production of phosphatidylcholine by the de novo pathway. The results suggest that if the de novo pathway is responsible for the increase in phosphatidylcholine content, this increase is due to a change in the parameter controlling the flux through the choline phosphate cytidylyltransferase step. The results also indicate that the metabolic flux through choline phosphotransferase is also enhanced during pulmonary development.

Altered expression of p53 and mdm-2 proteins at diagnosis is associated with early treatment failure in childhood acute lymphoblastic leukemia.

PURPOSE: To determine whether potential alteration in p53 function through p53 gene mutation or mdm-2 overexpression correlates with early treatment failure in childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Diagnostic marrow samples from 34 children were analyzed for p53 gene alterations by western blot and SSCP/DNA sequence analysis and for mdm-2 overexpression by western blot analysis. These samples were derived from two groups of children with ALL: 17 good outcome patients who are in long-term continuous complete remission and 17 poor outcome patients who did not achieve a complete remission or relapsed within 6 months of achieving remission. RESULTS: Two children within the poor outcome group were found to have p53 gene mutations. Furthermore, five poor outcome patients were shown to have greater than 10-fold overexpression of mdm-2 protein compared with the mean level of mdm-2 protein measured in the good outcome group. Aberrant p53 protein expression was found in only one good outcome patient, whereas no good outcome children were found to have elevated levels (> 10-fold) of mdm-2 protein. CONCLUSION: We show for the first time that potential alteration in p53 function in childhood ALL is more common (P = .036) in cases of early treatment failure than in children who remain in long-term continuous remission.

RNA abundance measured by a lysate RNase protection assay.

We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.

The mdm2 proto-oncogene.

The mouse double minute 2 (mdm2) proto-oncogene was originally discovered as one of three genes that was amplified in a tumorigenic cell line derived from non-transformed Balb/c cells. Consistent with the expression pattern of mdm2 in these cells, it was later shown that the transforming potential of the mdm2 proto-oncogene can be activated by experimental overexpression. Overexpression of mdm2 protein been detected in a number of diverse human malignancies, indicating that this oncogene plays a key role in human carcinogenesis. One mechanism by which mdm2 overexpression may lead to uncontrolled cellular proliferation is through its ability to physically associate with the p53 tumor suppressor and block p53's growth suppressive functions. Forced overexpression of mdm2 has been shown to block the transactivation, cell cycle arrest and apoptotic functions of p53. The mdm2 gene has also been shown to be a transcriptional target of p53 and the induction of p53 transcriptional activity leads to increases in mdm2 RNA and protein levels. Thus, it appears that an auto-regulatory feedback loop exists between these two proteins which keeps the growth suppressive functions of p53 in check during normal cell cycling. However, this block is thought to be overcome during certain cellular insults, including DNA damage, so that p53 can regulate the expression of genes involved in cell cycle arrest and/or apoptosis. Genetic lesions leading to elevated levels of mdm2 likely impair the ability of p53 to orchestrate the expression of genes controlling cell cycle progression during cellular insults. This may lead to the propagation of genetic errors, genomic instability and ultimately to an increase in the rate of tumor cell evolution. There is also recent evidence which suggests that mdm2 may play roles in p53-independent pathways regulating cellular proliferation. mdm2 has recently been shown to interact with the retinoblastoma tumor suppressor protein p(Rb), and the E2F-1 and DP1 transcription factors. These, and other clinical, cellular and biochemical studies relating to the mdm2 oncogene are reviewed here. In addition, a proposed role for mdm2 in pathways controlling cell cycle response to cellular perturbations is presented.

Clinical evaluation of serum thyroglobulin measurement using a commercial kit in the diagnosis of recurrent thyroid cancer.

The value of serum thyroglobulin assay employing a kit manufactured by Diagnostic Products Corporation in the detection of recurrence of thyroid carcinoma in patients treated by thyroidectomy and ablative therapy was assessed by clinical follow-up and radioiodine scanning of 122 patients over a 2-year period. A total of 204 specimens were analysed. The assay was found to be sensitive and highly specific for the detection of recurrent thyroid carcinoma provided that lipemic sera are clarified by ultracentrifugation prior to measurement and that results from those patients who demonstrate positive serum antithyroglobulin antibodies are excluded. The predictive value of a serum thyroglobulin level above 20 pmol/L was 96% for recurrence of thyroid carcinoma. The predictive value for non-recurrence was 98% for serum thyroglobulin levels below 10 pmol/L. The specificity and sensitivity of the assay were poor for serum thyroglobulin levels between 10 and 20 pmol/L. In recurrence-free patients, the serum thyroglobulin levels were not changed by withdrawal of thyroid replacement therapy 4-7 weeks prior to sampling.

Occupational lead exposure: studies in two brothers showing differential susceptibility to lead.

Unexpected differences in clinical and biochemical findings in two brothers occupationally exposed to the same source of lead for dissimilar lengths of time are presented. Only the brother with the shorter period of lead exposure was anemic and afflicted by nausea, vomiting, abdominal colic and arthralgia. His urinary PBG output yielded the high orders of magnitude found in acute intermittent porphyria in relapse. Prior to administration of a single dose of EDTA (1 g of the calcium disodium salt given intravenously in 325 mL 0.15 mol/L NaCl), his blood lead levels averaged 3.6 mumol/L. The amount of chelatable lead retrieved from his urine, 31 mumol/day, was more than twice that found in his asymptomatic counterpart who was exposed to lead for 13 months and whose pre-EDTA blood lead levels averaged 4.0 mumol/L. Not only the activity of delta-aminolaevulinic acid dehydratase, but also that of uroporphyrinogen I synthetase, was markedly inhibited by lead in red cells of both brothers. These activities were restored to normal levels in vitro by addition to the assay system of zinc and dithiothreitol. This ruled out a coexisting genetic deficiency of either enzyme. The anemia of the symptomatic brother with the shorter period of lead exposure was alleviated by folic acid, 15 mg/day. The differences in findings between the two brothers point to differential susceptibility to lead and illustrate the extent to which symptomatic lead poisoning may mimic biochemical and clinical features of the acute porphyrias.

Cholesterol granulomas in lymph nodes draining a benign ovarian neoplasm.

A 69-year-old woman underwent a hysterectomy and bilateral salpingo-oophorectomy for a 20-cm right ovarian tumor. Multiple peritoneal and lymph node biopsy specimens were obtained to determine the clinical stage. Despite the surgeon's concern of malignancy, pathologic examination demonstrated a benign mucinous cystadenoma. Periaortic and external iliac lymph nodes showed an unusual granulomatous reaction, with multinucleated giant cells surrounding cholesterol-like clefts. Analysis of cyst fluid from the ovarian tumor revealed high concentrations of protein and lipid; the lipid component was predominantly free cholesterol (0.61 mumole/mL) and phospholipid (0.225 mumole/mL). We speculate that fluid from the ovarian neoplasm drained into regional lymph nodes, causing this unusual granulomatous response.

A deficiency in Mdm2 binding protein inhibits Myc-induced B-cell proliferation and lymphomagenesis.

Mdm2 binding protein (MTBP) has been implicated in cell-cycle arrest and the Mdm2/p53 tumor suppressor pathway through its interaction with Mdm2. To determine the function of MTBP in tumorigenesis and its potential role in the Mdm2/p53 pathway, we crossed Mtbp-deficient mice to Emu-myc transgenic mice, in which overexpression of the oncogene c-Myc induces B-cell lymphomas primarily through inactivation of the Mdm2/p53 pathway. We report that Myc-induced B-cell lymphoma development in Mtbp heterozygous mice was profoundly delayed. Surprisingly, reduced levels of Mtbp did not lead to an increase in B-cell apoptosis or affect Mdm2. Instead, an Mtbp deficiency inhibited Myc-induced proliferation and the upregulation of Myc target genes necessary for cell growth. Consistent with a role in proliferation, Mtbp expression was induced by Myc and other factors that promote cell-cycle progression and was elevated in lymphomas from humans and mice. Therefore, Mtbp functioned independent of Mdm2 and was a limiting factor for the proliferative and transforming functions of Myc. Thus, Mtbp is a previously unrecognized regulator of Myc-induced tumorigenesis.

Actions of dietary orotic acid on liver synthesis of phosphatidylcholine and phosphatidylethanolamine in rats.

The in vivo rates of the reactions of the cytidine pathways of liver phosphatidylcholine and phosphatidylethanolamine synthesis were measured in rats after 1 day of feeding on a semisynthetic diet containing 1% orotic acid. The calculations were made from the specific and total radioactivity versus time curves of the precursors and products following intraportal injection of [1,2-14C]choline, [2-14C]ethanolamine, and [2-3H]glycerol. The liver CTP level was increased twofold and the rates of CDP-choline and phosphatidylcholine synthesis were stimulated 4.5-fold in the rats fed orotic acid. The rate of CDP-ethanolamine synthesis was increased but could not be accurately quantified because of its extreme rapidity. No change occurred in the rate of the ethanolaminephosphotransferase reaction and the overall rate of phosphatidylethanolamine synthesis was unchanged by orotic acid feeding. The catalytic activities of the enzymes of the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis were not affected by feeding orotic acid for 1 day. Similar findings were obtained 3 h following intragastric administration of 100 mg of orotic acid. The results suggest the possibility that changes in the levels of liver CTP may play a role in regulation of the cytidine pathway of liver phosphatidylcholine synthesis but not of phosphatidylethanolamine synthesis, because the latter pathway appears to be tightly controlled at the ethanolaminephosphotransferase step.

Changes in metabolism of ethanolamine and its derivatives in liver during fasting.

The time course of incorporation of radioactivity into liver phosphatidylethanolamine and its precursors was studied following intraportal injection of [1,2-14C]ethanolamine into rats that had been fasted for 24 h and into nonfasted rats. Marked diminution in the labelling of liver phosphatidylethanolamine and CDP-ethanolamine was present in the fasted groups while the radioactivity contained in the ethanolamine and phosphorylethanolamine pools of these livers was increased. The livers of the fasted rats contained significantly higher levels of ethanolamine, phosphorylethanolamine and CDP-ethanolamine while choline and phosphorylcholine were decreased. These changes are identical to those which we have described earlier in the livers of nonfasted rats fed a choline deficient diet. In a further experiment, the duration of the fasting period necessary to bring about these changes was studied. Incorporation of radioactivity into liver phosphatidylethanolamine at 2.5 min following intraportal injection of [1,2-14]ethanolamine declined progressively up to 7.5 h of fasting and did not decrease further after that time. There was a concomitant decrease in labelling of CDP-ethanolamine with retention of radioactivity in ethanolamine and phosphorylethanolamine. These results show that the liver metabolism of ethanolamine is altered quite early in the fasting period and suggest that it may be related in some way to the metabolic response to fasting.

Early effects of dietary orotic acid upon liver lipid synthesis and bile cholesterol secretion in rats.

Dietary orotic acid is known to cause impaired fatty acid synthesis and increased cholesterol synthesis in rats. We found that the impaired fatty acid synthesis occurs during the first day of orotic acid feeding and, in studies with albumin-bound [1-14C]palmitic acid, an associated decrease in the rate of esterification of this fatty acid into triacylglycerol, phospholipid, and cholesteryl ester was observed. These changes may result from the known decreases in liver levels of adenine nucleotides or, as reported here, from decreased liver CoASH levels in orotic acid-fed rats. The increase in hepatic cholesterol synthesis occurred during the second day of orotic acid feeding. It was detected by increased incorporation of [1,2-14C]acetate into cholesterol by liver slices and by a 7-fold increase in HMG-CoA reductase activity. At the same time the biliary output of cholesterol was increased 2-fold and studies using 3H2O revealed that the output of newly synthesized cholesterol in bile was increased 5-fold. The content of cholesteryl ester in hepatic microsomes decreased during orotic acid feeding but free cholesterol was unchanged. The findings are interpreted to suggest that the increased bile cholesterol secretion caused by orotic acid is a result of impaired hepatic cholesterol esterification and that the increase in HMG-CoA reductase activity is a result of diminished negative feedback due to the depleted content of cholesteryl ester in the hepatic microsomes.

A novel cellular protein (MTBP) binds to MDM2 and induces a G1 arrest that is suppressed by MDM2.

The MDM2 protein, through its interaction with p53, plays an important role in the regulation of the G(1) checkpoint of the cell cycle. In addition to binding to and inhibiting the transcriptional activation function of the p53 protein, MDM2 binds, inter alia, to RB and the E2F-1.DP-1 complex and in so doing may promote progression of cells into S phase. Mice transgenic for Mdm2 possess cells that have cell cycle regulation defects and develop an altered tumor profile independent of their p53 status. MDM2 also blocks the growth inhibitory effects of transforming growth factor-beta1 in a p53-independent manner. We show here that a novel growth regulatory molecule is also the target of MDM2-mediated inhibition. Using a yeast two-hybrid screen, we have identified a gene that encodes a novel cellular protein (MTBP) that binds to MDM2. MTBP can induce G(1) arrest, which in turn can be blocked by MDM2. Our results suggest the existence of another growth control pathway that may be regulated, at least in part, by MDM2.