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The authors' impression is that the CBC provides much more information than is routinely used. When anemia is present, the CBC contains considerable information regarding its cause, which can assist in formulating a differential diagnosis and directing further evaluation. White blood cell and platelet count levels may similarly direct practitioners to consider or dismiss underlying conditions. This article assists the pediatrician in optimizing use of this familiar diagnostic tool.

A comprehensive discussion of professionalism in medicine must include its impact on successive generations of physicians. Fifty years ago, doctors acting professionally emphasized medicine as a calling and an ability to act as the authority for patients in crisis at home and in hospitals. Therapeutic options were limited relative to the modern era, and the laying on of hands was practiced as science and art. Today, doctors balance increasing demands on time and efficiency with the sense of primacy of patient care. Technological innovation and patients' increasing access to medical knowledge through varying media of inconsistent quality challenge physicians in novel ways. Fifty years in the future, doctors will have access to vast amounts of information through a multitude of noninvasive diagnostics. Progressively more personalized medicine should inspire doctors to become even more adept at communicating effectively with patients. Professionalism in medicine throughout these generations embodies similar fundamental behaviors, such as demonstrating compassion, respect, and humility; adhering to high ethical and moral standards; subordinating personal interest to that of others; and reflecting on actions and decisions. Despite the dynamic nature of the profession itself, the omnipresent need for such traits will define medical professionalism for decades to come.

1. Chasnoff 1J, Burns W J, Schnoll SH, Burns KA. Cocaine use in pregnancy. N Engl J Meal 1985;313:666-9. 2. Fulroth R, Phillips B, Durand DJ. Perinatal outcome of infants exposed to cocaine and/or heroin in utero. Am J Dis Child 1989;143:905-10. 3. Madden JD, Payne TF, Miller S. Maternal cocaine abuse and effect on the newborn. Pediatrics 1986;77:209~l 1. 4. Bingol N, Fuchs M, Diaz V, Stone RK, Gromisch DS. Teratogenicity of cocaine in humans. J PEDIATR 1987;110: 93-6. 5. Oro AS, Dixon SD. Perinatal cocaine and methamphetamine exposure: maternal and neonatat correlates. J PEDtATR 1987; 111:571-8. 6. Chasnoff I J, Hunt CE, Kletter R, Kaplan D. Prenatal cocaine exposure is associated with respiratory pattern abnormalities. Am J Dis Child 1989;143:583-7. 7. Bauchner H, Zuckerman B, McClain M, Frank D, Fried LE, Kayne H. Risk of sudden infant death syndrome among infants with in utero exposure to cocaine. J PED1ATR 1988;112:831-4. 8. Lubchenco LO, Hansman C, Boyd E. Intrauterine growth in length and head circumference as estimated from live births at gestational ages from 26 to 42 weeks. Pediatrics 1966;37: 403-8. 9. Chavez C J, Ostrea EM, Stryker JC, Smialek Z. Sudden infant death syndrome among infants of drug,dependent mothers. J PEDIATR 1979;95:407-9. 10. Ward SLD, Schuetz S, Krishna V, et al. Abnormal sleeping ventilatory pattern in infants of substance-abusing mothers. Am J Dis Child 1986;140:1015-20.

Leadership has emerged as a key theme in the rapidly growing movement to improve patient safety. Leading an organization that is committed to providing safer care requires overcoming the common traps in thinking about error, such as blaming individuals, ignoring the underlying systems factors, and blaming the bureaucracy of the organization. Leaders must address the system issues that are at work within their organizations to allow individual and organizational learning to occur.

We describe two children who had breath-holding spells that were accelerating in frequency and severity and in one case had recrudesced. Both patients had signs, symptoms, and laboratory findings of severe anemia. With correction of anemia the breath-holding spells promptly and completely resolved in each case. We conclude that in some patients anemia may be a factor contributing to breath-holding spells and that correction of concomitant anemia may produce amelioration or remission of the spells. (J P EDIATR 1995;126:395-7)

After having made decisions about college, medical school, and specialty training, some residents may delay consideration of their future career in pediatrics. Others, having decided what path they will pursue, are not clear as to what steps need to be taken to ensure that they approach their choice armed with the most appropriate and accurate information. Residents seek career choice information from various sources. For many, the resources may not be able to provide as complete information in all facets of career decision-making. An Internet accessible site for career planning for pediatric residents has been developed (www.ucch.org/residency/CareerPlanning.html). The advantages, beyond ease of access by all residents, are many, particularly the power of links to other Internet resources. In addition, a website provides the ability to rapidly update and add information, which is not feasible in printed form. career planning, pediatric residents, jobs, private practice, pediatric specialties.

IN 1974, companion papers in the Journal discussed the improved disease-free survival among patients with osteosarcoma who had received adjuvant chemotherapy after surgery for the primary tumor. On...

Presentation of Case A 10-year-old girl was admitted to the hospital because of pain in the shoulder. She was well until two months earlier, when right mastoiditis was treated with amoxicillin; a r...

Normal human platelets were shown to contain the enzyme dihydropteridine reductase. The enzyme was not found in a variety of other cells of hematogenous origin. Partial purification and kinetic and physical data indicated that the platelet enzyme is similar to that previously characterized from liver. Dihydropteridine reductase is important for the regeneration of tetrahydrobiopterin, a required cofactor in hydroxylation reactions involved in biogenic amine formation. The presence of the enzyme may indicate that some synthesis de novo of serotonin and/or catecholamines occurs in platelets, as opposed to a purely storage and transport function. In addition, screening for hyperphenylalaninemia due to dihydropteridine reductase deficiency may become feasible by assaying platelets for enzyme activity.

A thin-layer chromatographic method for the separation of thymidine and deoxyuridine nucleotides and nucleosides is described. This procedure involves the following sequence of steps: (i) Ion-exchange thin-layer chromatography to afford separation into fractions of increasing degree of phosphorylation, (ii) conversion of each fraction into an equivalent mixture of thymine and uracil through the combined actions of alkaline phosphatase and thymidine phosphorylase, and (iii) partition thin-layer chromatographic separation of thymine and uracil. A key feature of the method is the specificity afforded by the second step which converts only thymidine and deoxyuridine nucleotides and nucleosides to the corresponding pyrimidine bases. An application of the method to the study of [3H]deoxyuridine metabolism in L1210 cells, as well as the effect of methotrexate on this metabolism is also described.

The addition of whole serum to G0-arrested, confluent Balb c -3 T 3 cells induces them to progress through G1 and synthesize DNA after a 12-h lag period. Prior to the onset of DNA synthesis, RNA is synthesized and RNA content increases. Serum has been fractionated into two sets of growth factors: a platelet-derived growth factor present in heat-treated (100°C) platelet extracts and platelet-poor plasma. Addition of whole serum, platelet-derived growth factor or platelet-poor plasma induces quiescent cells to increase their cytoplasmic RNA content, but the cells treated with platelet-poor plasma do not synthesize DNA. Messenger RNA content increases within 2 h after stimulation with whole serum or platelet-poor plasma, and after 18 h, mRNA has accumulated to a greater degree than rRNA.

The proportion of hnRNA processed into cytoplasmic mRNA in resting and growing 3T3 and 3T6 cells has been investigated by measuring the efficiency of transfer of nuclear poly(A) to the cytoplasm. When nuclear poly(A) is either labeled continuously with precursor or pulse labeled and then chased with cordycepin, growing cells transfer approximately twice as much of the poly(A) from the nucleus to the cytoplasm as do resting cells. When cells undergo a serum-induced transition from the resting to growing state, the efficiency of poly(A) transfer is increased to that characteristic of growing cells by 3 hr after the addition of the serum. On the other hand, the proportion of hnRNA which is polyadenylated and the total nuclear poly(A) content are the same in resting and growing cells. It appears that the principal means by which the cell regulates its poly(A)+ mRNA content in the growing and resting states is by regulating the efficiency with which nuclear poly(A)+ RNA is converted to cytoplasmic mRNA.

The authors report their experience with adjuvant chemotherapy for osteosarcoma at Children’s Hospital Center and Sidney Farber Cancer Institute. Results of three studies showed a significant improvement in disease-free and overall survival rates over those of historical controls. Some aspects of the tumor that may affect the results of adjuvant therapy trials are also discussed, and experimental design factors that may resolve the controversy over the role of adjuvant chemotherapy are suggested.

We conducted a randomized controlled trial to determine whether intensive multi-agent adjuvant chemotherapy improves the chances of relapse-free survival in patients with nonmetastatic high-grade osteosarcoma of the extremity, as compared with concurrent controls. After undergoing definitive surgery, 36 patients were randomly assigned to adjuvant chemotherapy or to observation without adjuvant treatment. At two years the actuarial relapse-free survival was 17 percent in the control group, similar to that found in studies before 1970, and 66 percent in the adjuvant-chemotherapy group (P less than 0.001). Similar results were observed among 77 additional patients who declined to undergo randomization but who elected observation or chemotherapy. We conclude that the natural history of osteosarcoma of the extremity has remained stable over the past two decades, that adjuvant chemotherapy increases the chances of relapse-free survival of patients with high-grade osteosarcoma, and that it should be given to all such patients.

In previous reports, it was shown that both the concentration and rate of production of rRNA and mRNA were greater in growing than in resting 3T6 fibroblasts. Studies on isolated nuclei indicated that ribosomal RNA production is apparently controlled at the level of transcription. In contrast, hnRNA, the putative precursor of mRNA, appeared to be synthesized at the same rate in resting and growing cells. This finding was unexpected and has been tested in several ways. In this report, we show by an independent method that the relative rate of production of mRNA compared to hnRNA is several-fold higher in growing than in resting cells. However, the kinetics of processing of mRNA appear unchanged. This result suggests either that mRNA arises from a small subfraction of hnRNA or that the efficiency of processing of the hnRNA precursor is an important control mechanism which determines mRNA production ingrowing and resting states. Comparison of the initial rates of labeling of hnRNA and cytoplasmic message gives the efficiency with which the cytoplasmic mRNA is produced from nucleoplasmic RNA.The very low efficiency (3-4% in growing and 1-2% in resting cells) suggests that not every hnRNA molecule gives rise to a cytoplasmic message. In contrast to the similar kinetics of mRNA production in resting and growing states, processing of ribosomal RNA is much slower in the resting state and the emergence time for 28S RNA from nucleolus is greatly lengthened.

Mammalian cells selected for resistance to certain cytotoxic drugs frequently develop cross-resistance to a broad spectrum of other drugs unrelated in structure to the original selective agent. This phenomenon constitutes a major problem in cancer chemotherapy. Multi-drug resistance arises from decreased intracellular drug accumulation, apparently due to an alteration of the plasma membrane. The observation of double minute chromosomes or homogeneously staining regions in some of the multi-drug-resistant cell lines suggests that gene amplification underlies this phenomenon. We have used the technique of DNA renaturation in agarose gels to detect, compare and clone amplified DNA sequences in Adriamycin- and colchicine-resistant sublines of Chinese hamster cells. We show that both Adriamycin- and colchicine-resistant cells contain amplified DNA fragments, some of which are amplified in both of these independently derived cell lines. Furthermore, loss of the multi-drug resistance phenotype on growth in the absence of drugs correlates with the loss of amplified DNA. These results strongly suggest that the DNA sequences which are amplified in common in multi-drug-resistant cell lines include the gene(s) responsible for a common mechanism of multi-drug resistance in these cells. We have cloned one of the commonly amplified DNA fragments and show that the degree of amplification of this fragment in the cells correlates with the degree of their drug resistance.

Human melanomas are naturally resistant to methotrexate (MTX). The mechanism of intrinsic drug resistance has been explored in 3 melanoma cell lines not previously exposed to this agent. All 3 lines exhibited relative MTX resistance with ID50 values of greater than 1 microM. Drug uptake studies were performed over an extracellular concentration range of 0.1 to 10 microM MTX. The uptake was linear over the initial 10 min at all concentrations and subsequently reached plateau levels only at the 10 microM concentration. Lineweaver-Burke transformations yielded apparent Km (uptake) values of 1.4 to 5 microM, similar to data obtained from other human cell lines. The level of dihydrofolate reductase (DHFR) in the human melanoma cells ranged between 8.42 to 11.98 pmoles/mg protein. The melanoma DHFR levels are several fold higher than in MTX-sensitive human tumor lines and up to a hundred-fold higher than that measured in human brain tumor cells by our assay. The intrinsic resistance of these melanoma lines has therefore been attributed to elevated intracellular levels of DHFR.

The number and time to appearance of pulmonary metastases were evaluated in 15 patients with osteogenic sarcoma receiving adjuvant chemotherapy with high-dose methotrexate and doxorubicin (adjuvant group). The results were compared to 33 age- and sex-matched controls (control group). The adjuvant group demonstrated a reduction in the number and a delay in the appearance of the metastases. The median time to development of metastases was 17 mo in the adjuvant group and 7 mo in the control group, and the median number of metastases was 2 and 12, respectively.

The pharmacokinetics and metabolism of dibutyl methotrexate (DBMTX) and gamma-monobutyl methotrexate (gamma-MBMTX) were studied in Rhesus monkeys. When a bolus IV dose of either [3H]DBMTX or [3H] gamma-MBMTX was given, the principal species in serum for up to 1 h was the monoester, with MTX accounting for less than 10% of the total radioactivity. Products other than gamma-MBMTX and MTX were formed in substantial amounts with DBMTX, but not with gamma-MBMTX. Total radioactivity recovered in the bile 5 h after [3H]DBMTX injection accounted for 32% of the administered dose, indicating high hepatic extraction for this lipophilic compound. Serum and CSF levels of unchanged gamma-MBMTX, as well as of MTX arising via esterase cleavage, were measured by HPLC after IV infusion of gamma-MBMTX (10 g/m2). Efflux of monoester from CSF was slower than disappearance from serum. However, gamma-MBMTX levels in CSF were no higher than could be attained by infusing MTX itself at the same dose rate. While CSF/serum ratios were ca. 10-fold higher for gamma-MBMTX than for MTX, this difference could be explained on the basis of the very different affinities of the two compounds for serum proteins. HPLC analysis of serum processed by methanol precipitation as opposed to ultrafiltration of the proteins showed gamma-MBMTX to be greater than 99% bound, whereas for MTX this value was 50% or less. When gamma-MBMTX and MTX levels measured after ultrafiltration were corrected for this difference in serum protein binding the total amount of the two drugs in serum became almost equivalent.

After partial purification, rabbit brain extracts were assayed for dihydrofolate reductase by spectrophometric, radiochemical and methotrexate binding assays. By these assays, the specific activity of rabbit brain dihydrofolate reductase was about 15 per cent that of liver. Both rabbit liver and brain dihydrofolate reductase activities were abolished by methotrexate and had comparable K m (3 μM), pH maxima (4.8), and cofactor requirements (NADPH). In vivo , a small percentage of [ 3 H]folic acid was reduced to [ 3 H]methyltetrahydrofolate when injected into the left lateral ventricle. Thus, both in vitro and in vivo , the central nervous system has the ability to reduce oxidized folates.

Product identification and kinetic data are presented for the conversion of 7,8-dihydrobiopterin into tetrahydrobiopterin by purified rabbit brain dihydrofolate reductase.

Dihydrofolate reductase (DHFR), the main target for methotrexate and other antifolate compounds was found to be present in 100-200 times higher concentration in human cell lines grown in vitro than in human tumors or cells obtained in situ. The DHFR content of human cell lines in vitro however were equivalent to rodent tumor lines also measured in vitro. The enzyme was quantitated by [3H]methotrexate binding, [3H]dihydrofolate reduction to [3H]tetrahydrofolate, and immunoprecipitation with a monospecific anti-serum to DHFR. Additional studies revealed only a liver sample to contain significant amounts of an inhibitor of DHFR activity. It is postulated either that low levels of DHFR in fresh human tissue reflect low cell turnover or conversely that high levels in vitro and in animal tissues reflect high levels of enzyme due to selection because of high levels of folic acid in culture medium and prepared feeds.

Four separate groups of patients have been studied: (1) The effect of high-dose methotrexate (MTX) administration on glomerular filtration rate was determined by pre- and posttreatment inulin and creatinine clearances in nine patients. Measurements were made prior to and 24-40 hr after drug administration. Inulin and creatinine clearances both decreased a mean of 43%. No signs of systemic toxicity occurred. (2) Three other patients given high-dose courses of MTX developed MTX toxicity. Their creatinine clearance decreased an average of 61%. (3) In a separate group of five patients undergoing weekly MTX treatment, comparison of serum MTX pharmacokinetics with and without alkalinization of the urine demonstrated no significant difference in peak serum MTX levels or serum MTX decay. (4) Eight additional patients with severe renal dysfunction secondary to MTX were treated with increased doses of leucovorin and a continuous infusion of thymidine (8 g/m2/day) once renal failure was recognized. When high-dose leucovorin and thymidine were begun 48-72 hr after the MTX infusion, severe toxicity in the form of leukopenia, thrombocytopenia, diffuse mucositis, stomatitis, or skin rash was averted. We concluded the following: (1) high-dose MTX causes a subclinical decrease in glomerular filtration rate with each administration, even in nontoxic courses; (2) alkalinization of the urine with sodium bicarbonate does not alter plasma MTX decay, while volume expansion (hydration) is maintained constant; and (3) rigorous monitoring of serum creatinine and serum MTX levels 24-48 hr after MTX administration allows for the institution of rescue measures, including leucovorin and thymidine, which will abort the systemic toxicity that accompanies MTX-induced renal failure.

We have examined the relative quantities of 18S and 28S rRNA, 4S RNA and poly (A) + mRNA in the following cultured cells: the mouse fibroblast lines 3T3 and 3T6 in the resting (contact inhibited) and growing (sparse) states, 3T3 clones transformed with SV40 (SV3T3) and with both SV40 and polyoma SV-Py 3T3), hamster lung fibrobalsts (v79), human cervical carcinoma cells (HeLa), and human diploid fibroblasts at early and late passage. The relative quantities of the RNA species were determined by labeling the cells to equilibrium with 32PO4 and measuring the amount of label in each RNA species. The ratio of mRNA to rRNA varied form 1.1% to 2.7% in the different cell lines, the more rapidly growing cell lines usually giving a higher ratio. In cells experiencing growth limitation either by contact inhibition or due to senescence, the ratio of mRNA to rRNA was about 30% lower than in the corresponding cells in the growing state. In most cell lines the ratio of 4S RNA to 18S rRNA was between 0.8 and 1.2, but in seescent fibroblasts, this ratio increased to greater than 1.7. Senescent fibroblasts also contained much more total RNA per unit of DNA than the same cells at early passage or than 3T6 or 3T3 cells.

Chemotherapeutic agents have generally been considered to exert their cytotoxic effects by directly interacting with DNA metabolism or by inhibiting enzymatic steps within the multiple pathways of purine and pyrimidine nucleotide metabolism (for review, see Roy-Burman, 1970). It has now become increasingly clear that most agents have multiple effective sites or targets within the cell, and that their effect on macromolecular metabolism may be of prime importance. This chapter will deal only with ribonucleic acid (RNA) metabolism and how various RNA species can be selectively inhibited by drugs. In addition, where possible, suggestions will be given on how this selectivity may be strategically utilized in designing a rational basis for chemotherapy.

The effects of methotrexate di-n-butyl ester (DBMTX) on thymidine (dThd) metabolism in cultured L1210 cells have been studied. In contrast to methotrexate. DBMTX markedly suppresses the incorporation of exogenous radiolabeled dThd into DNA. Uptake of dThd into the pools of thymidine nucleotides is decreased markedly. The DNA polymerases and thymidine kinase were unaffected by DBMTX in vitro. There was no net efflux of soluble dThd DNA precursors from cells. No specific block at any of the phosphorylation steps was found. dThd transport, however, was inhibited significantly, as were deoxyuridine (dUrd) and uridine (Urd) transport. These results suggest that DBMTX inhibits dThd incorporation by inhibiting initial transport of dThd across the cell membrane.

Ten primary and two metastatic central-nervous-system (C.N.S.) tumours were assayed. They all contained dihydrofolate reductase. This finding provides a biochemical rationale for antifolate therapy of C.N.S. tumours by methotrexate.

A major obstacle in the successful treatment of malignancies is the emergence of drug resistance. We have chosen a Chinese hamster model system to study the gene(s) responsible for multiple drug resistance and the mechanisms by which they arise. To analyze DNA rearrangements, we used adriamycin(ADR)-resistant subclones of V79 cells, 77A and LZ which are resistant to a 5-fold or 3000-fold concentration respectively of ADR relative to V79, and C5, a colchicine-resistant subline of CHO cells which is cross-resistant to a 150-fold concentration of ADR. DNA from these lines was assayed for the presence of amplified sequences by a novel in-gel renaturation technique (I.B. Roninson, Nuc. Acids Res. 11:5413, 1983). Both LZ and C5 cells contain amplified DNA fragments, a subset of which were commonly amplified in these two independently selected lines, thereby suggesting that they contain the gene(s) responsible for a common mechanism of drug-resistance. We have cloned one of the commonly amplified fragments and shown that the degree of amplification of this fragment correlated with the degree of drug resistance. We have also used this fragment as a probe to isolate several phage clones from a genomic library of LZ DNA. Transcription of these clones is being analyzed. We are currently extending these studies to include an analysis of DNA from newly diagnosed and relapsed patients with leukemia.

170 DE-COUPLING OF RNA AND DNA SYNTHESIS IN PLATLET-POOR SERUM STIMULATED G o ARRESTED BALB/C 3T3 FIBROBLASTS