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Multiple endocrine neoplasia type 1 is an autosomal dominant tumor syndrome. Manifestations include neoplasms of the parathyroid glands, enteropancreatic neuroendocrine cells, and the anterior pituitary gland. The MEN1 tumor suppressor gene encodes menin, a 610 amino acid nuclear protein without sequence homology to other proteins. To elucidate menin function, we used immunoprecipitation to identify interacting proteins. The NF-kappaB proteins p50, p52 and p65 were found to interact specifically and directly with menin in vitro and in vivo. The region of NF-kappaB proteins sufficient for binding to menin is the N-terminus. Furthermore, amino acids 305-381 of menin are essential for this binding. Menin represses p65-mediated transcriptional activation on NF-kappaB sites in a dose-dependent and specific manner. Also, PMA (phorbol 12-myristate 13-acetate)-stimulated NF-kappaB activation is suppressed by menin. These observations suggest that menin's ability to interact with NF-kappaB proteins and its modulation of NF-kappaB transactivation contribute to menin's tumor suppressor function.

A process was developed for producing human menin from transformed Drosophila Schneider 2 cells. Protein expression was achieved after inducing the metallothionein promoter by adding copper sulfate to cells growing in suspension in a stirred-tank reactor. Experiments in shake flasks showed that the production of menin was improved when the induction was conducted late in the exponential phase of cell growth at a concentration of 1–2 × 107 cells ml-1, with a copper concentration of 0.2 mM for no more than 24 h. This observation was confirmed by experiments in bench-scale fermentors. Subsequently, a pilot-scale fermentation yielded 1 mg l-1 culture of purified menin.

Primary hyperparathyroidism is characterized by hypercalcemia and elevated parathyroid hormone levels. It can be caused by overactivity of one (adenoma or carcinoma) or more (hyperplasia or multiple adenoma) parathyroid glands. Parathyroid adenoma and hyperplasia are usually mono- or oligoclonal neoplasms. To establish whether parathyroid cancer has a genetic composition distinct from parathyroid adenoma, we analyzed 10 adenoma and 10 carcinoma cases by comparative genomic hybridization (CGH). Results show clear differences between the constitution of adenoma and carcinoma genomic DNA. The most frequent genomic alterations in adenoma included deletions on chromosomes 11, 17 (5 of 10 cases), and 22 (7 of 10 cases). In parathyroid carcinoma, frequent chromosomal deletions were on chromosome arm 1p (4 of 10 cases) and chromosome 17 (3 of 10 cases), and gains were on chromosome 5 (3 of 10 cases). Our data indicate that different genetic changes could contribute to the development of parathyroid adenoma and carcinoma; genomic losses predominate in adenoma, and gains along with some losses are found in carcinoma. Furthermore, the CGH results implicate several chromosomal regions that may harbor genes that could be potentially involved in the development of parathyroid adenoma and carcinoma. Published by Elsevier Science Inc.

Dideoxyfingerprinting was used to screen for germline and somatic MEN1 mutations. This method, applied to a panel of germline DNA from 15 probands with multiple endocrine neoplasia type 1 (MEN-1), allowed confident discovery of the MEN1 gene. Germline MEN1 mutation has been found in 47 out of 50 probands with familial MEN-1, in 7 out of 8 cases with sporadic MEN-1, and in 1 out of 3 cases with atypical sporadic MEN-1. Germline MEN1 mutation was not found in any of five probands with familial hyperparathyroidism. Somatic MEN1 mutations were found in 7 out of 33 parathyroid tumours not associated with MEN-1. Allowing for repeating mutations, a total of 47 different germline or somatic MEN1 mutations have been identified. Most predict inactivation of the encoded 'menin' protein. supporting expectations that MEN1 is a tumour suppressor gene. The 16 observed missense mutations were distributed across the gene, suggesting that many domains are important to its as yet unknown functions.

Familial multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder with affected individuals developing parathyroid, gastrointestinal (GI) endocrine, and anterior pituitary tumors. Four large kindreds from the Burin peninsula/Fortune Bay area of Newfoundland with prominent features of prolactinomas, carcinoids, and parathyroid tumors (referred to as MEN1Burin) have been described, and they show linkage to 11q13, the same locus as that of MEN1. Haplotype analysis with 16 polymorphic markers now reveals that representative affected individuals from all four families share a common haplotype over a 2.5 Mb region. A nonsense mutation in the MEN1 gene has been found to be responsible for the disease in the affected members in all four of the MEN1Burin families, providing convincing evidence of a common founder. Hum Mutat 11:264–269, 1998. Published 1998 Wiley-Liss, Inc.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder that manifests as varying combinations of tumors of endocrine and other tissues (parathyroids, pancreatic islets, duodenal endocrine cells, the anterior pituitary and others). The MEN1 gene is on chromosome 11q13; it was recently identified by positional cloning. We previously reported 32 different germline mutations in 47 of the 50 familial MEN1 probands studied at the NIH. Eight different germline MEN1 mutations were encountered repeatedly in two or more apparently unrelated families. We analyzed the haplotypes of families with recurrent MEN1 mutations with seven polymorphic markers in the 11q13 region surrounding the MEN1 gene (from D11S1883 to D11S4908). Disease haplotypes were inferred from germline DNA and also from tumors with 11q13 loss of heterozygosity. Two different disease haplotype cores were shared by apparently unrelated families for two mutations in exon 2 (five families with 416delC and six families with 512delC). These two repeat mutations were associated with the two founder effects that we reported in a prior haplotype analysis. The disease haplotypes for each of the other six repeat mutations (seen twice each) were discordant, suggesting independent origins of these recurrent mutations. Most of the MEN1 germline mutations including all of those recurring independently occur in regions of CpG/CpNpG, short DNA repeats or single nucleotide repeat motifs. In conclusion, recurring germline mutations account for about half of the mutations in North American MEN1 families. They result from either founder effects or independent occurrence of one mutation more than one time. Hum Mutat 12:75–82, 1998. Published 1998 Wiley-Liss, Inc.1

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder in which affected individuals develop tumors primarily in the parathyroids, anterior pituitary, endocrine pancreas, and duodenum. The locus for MEN1 is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis has previously placed the MEN1 gene within a 2-Mb interval flanked by markers D11S1883 and D11S449. Loss of heterozygosity (LOH) studies in MEN1 and sporadic tumors have helped narrow the location of the gene to a 600-kb interval between PYGM and D11S449. Eighteen new polymerase chain reaction (PCR)-based polymorphic markers were generated for the MEN1 region, with ten mapping to the PYGM-D11S449 interval. These new markers, along with 14 previously known polymorphic markers, were precisely mapped on a 2.8-Mb (D11S480-D11S913) high-density clone contig-based, physical map generated for the MEN1 region.

Multiple endocrine neoplasia type 1 (MEN 1) is an inherited cancer syndrome in which affected individuals develop multiple parathyroid, enteropancreatic, and pituitary tumors. The locus for MEN1 is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis places the MEN1 gene within a 2-Mb interval flanked by the markers D11S1883 and D11S449. Loss of heterozygosity studies in MEN 1 and sporadic tumors suggest that theMEN1 gene encodes a tumor suppressor and have helped to narrow the location of the gene to a 600-kb interval between PYGM andD11S449. Focusing on this smaller MEN1 interval, we have identified and mapped 12 transcripts to this 600-kb region. A precise ordered map of 33 transcripts, including 12 genes known to map to this region, was generated for the 2.8-Mb D11S480–D11S913interval. Fifteen candidate genes (of which 10 were examined exhaustively) were evaluated by Southern blot and/or dideoxy fingerprinting analysis to identify the gene harboring disease-causing mutations.[The sequence data described in this paper have been submitted to GenBank under accession nos. EST06996, U93236,AF001540–AF001547, AF001433–AF001436, AF001891–AF001893,N55476, R19205, and W37647 (see Table 1 for listing of transcripts). The BAC clone sequences have been submitted to GenBank under accession nos. AC000134, AC000159, and AC000353.]

Adriamycin (AdM) aglycones have dramatic effects on isolated heart mitochondria, oxidizing pyridine nucleotides, modifying sulfhydryl groups, and triggering a permeability transition of the inner membrane that results in free passage of solutes smaller than 1500 Da. In this investigation, the role of glutathione (GSH) peroxidase in these actions of the aglycones was evaluated, by comparing mitochondria from selenium-deficient and selenium-supplemented rats, with the following results. Selenium deficiency was without effect on the permeability transition of heart mitochondria, followed via Ca2+ release and triggered by AdM aglycone or by t-butyl hydroperoxide (TBH) or H2O2, both of which are authentic substrates of the peroxidase. The permeability transition of liver mitochondria was delayed by selenium deficiency regardless of the triggering agent; however, substantial triggering by the aglycone and TBH persisted in mitochondria from selenium-deficient animals. Selenium deficiency inhibited thiol modification elicited by AdM aglycone and H2O2 in heart mitochondria and by the aglycone, TBH, and possibly H2O2 in liver mitochondria. It would thus appear that AdM aglycone, TBH, and H2O2 can induce the permeability transition of isolated heart mitochondria via a process (or processes) distinct from the catalytic activity of the peroxidase. Furthermore, even in liver, where involvement of the peroxidase is observed, mechanisms other than the GSH cycle can contribute to transition induction by the aglycone and by TBH. Finally, mitochondrial-SH group modification by the aglycones appeared not to be causally linked to induction of the permeability transition. This laboratory has suggested that the effects of aglycone metabolites of AdM on mitochondria mediate the cardiotoxicity that limits use of the parent drug. The data presented in this paper argue against the involvement of GSH peroxidase in that process. They are in agreement with in vivo studies, which have generally failed to find evidence for amelioration of AdM cardiotoxicity in selenium-deficient animals.

Transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25D3), when added simultaneously to a human osteosarcoma cell line, MG-63, induce alkaline phosphatase activity 40-70-fold over basal levels, 6-7-fold over 1,25D3 treatment alone, and 15-20-fold over TGF beta treatment alone. TGF beta and 1,25D3 synergistically increased alkaline phosphatase specific activity in both matrix vesicles and plasma membrane isolated from the cultures, but the specific activity was greater in and targeted to the matrix vesicle fraction. Inhibitor and cleavage studies proved that the enzymatic activity was liver/bone/kidney alkaline phosphatase. Preincubation of MG-63 cells with TGF beta for 30 min before addition of 1,25D3 was sufficient for maximal induction of enzyme activity. Messenger RNA for liver/bone/kidney alkaline phosphatase was increased 2.1-fold with TGF beta, 1.7-fold with 1,25D3, and 4.8-fold with the combination at 72 h. Human alkaline phosphatase protein as detected by radioimmunoassay was stimulated only 6.3-fold over control levels with the combination. This combination of factors was tested for their effect on production of three other osteoblast cell proteins: collagen type I, osteocalcin, and fibronectin. TGF beta inhibited 1,25D3-induced osteocalcin production, whereas both factors were additive for fibronectin and collagen type I production. TGF beta appears to modulate the differentiation effects of 1,25D3 on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state.

The polypeptide antibiotic duramycin has been reported to interact selectively with phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol (Navarro et al., 1985, Biochemistry 24, 4645-4650). PE is a major component of mitochondrial membranes. Duramycin was used to probe the role of PE in mitochondrial energy conversion reactions with the following results: (i) Duramycin uncoupled mitochondrial respiration, decreasing the respiratory control ratio to 1 at 5 microM. At concentrations of duramycin in excess of 10 microM, ADP addition inhibited electron transport. (ii) Duramycin inhibited oxidative phosphorylation (C50 less than 2 microM). (iii) Duramycin stimulated mitochondrial ATP hydrolysis modestly. The antibiotic was 7- to 16-fold less effective in this regard than concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (F-CCP) which produced comparable uncoupling. (iv) Duramycin inhibited uncoupled ATPase activity (C50 = 8 microM). Inhibition of the ATPase activity of intact mitochondria was blocked by 1 mM MgCl2 and 5 mM CaCl2; inhibition persisted in sub-mitochondrial particles assayed in the presence of 3 mM MgCl2. The effects on mitochondrial function of free fatty acids (FFA) and duramycin are similar in many respects. It is suggested that duramycin, like FFA, uncouples via a nonclassical mechanism, possibly by disrupting intramembrane H+ transfer between redox and ATPase complexes. In addition, interaction of duramycin, either direct or indirect, with the F0 moiety of the mitochondrial ATPase and with one or more components of the respiratory electron transport chain is proposed.

The pharmacokinetics of theophylline and its metabolites were studied in 13 patients with cystic fibrosis (CF) after an intravenous bolus of aminophylline (6 mg/kg) followed by multiple dosing with sustained release theophylline (Theo-Dur®) (7.7-14.8 mg/kg/day). An agematched group of 18 healthy subjects, taking a single oral dose of theophylline (300 mg), was used as a control group for comparing theophyline pharmacokinetic parameters. Literature values were used for the pharmacokinetics of the theophylline metabolites in normal subjects. Serum and urine concentrations of theophylline and its three main metabolites [3 methylxanthine (3MX), 1 methyluric acid (1MU), and 1,3 dimethyluric acid (1,3DMU)] were assayed by HPLC. The total body clearance and volume of distribution were significantly greater (p

While much of diagnostic radiology is practiced in a private setting, many of the papers and presentations describing picture archiving and communications systems (PACS) implementation to date have been sponsored by university and government health facilities. We will present a 4-year retrospective review of a private practice PACS project. The challenges, benefits, and cost analysis of the PACS project will be presented.

Multiple endocrine neoplasia type 1 (MENI) is a promising model to understand endocrine and other tumors. Its most common endocrine expressions are tumors of parathyroids, entero-pancreatic neuro-endocrine tissue, and anterior pituitary. Recently, collagenomas and multiple angiofibromas of the dermis also have been recognized as very common. MEN1 can be characterized from different perspectives: (a) as a hormone (parathyroid hormone, gastrin, prolactin, etc.) excess syndrome with excellent therapeutic options; (b) as a syndrome with sometimes lethal outcomes from malignancy of entero-pancreatic neuro-endocrine or foregut carcinoid tissues; or (c) as a disorder than can give insight about cell regulation in the endocrine, the dermal, and perhaps other tissue systems. The MEN1 gene was identified recently by positional cloning, a comprehensive strategy of narrowing the candidate interval and evaluating all or most genes in that interval. This discovery has opened new approaches to basic and clinical issues. Germline MEN1 mutations have been identified in most MEN1 families. Germline MENI mutations were generally not found in families with isolated hyperparathyroidism or with isolated pituitary tumor. Thus, studies with the MENI gene helped establish that mutation of other gene(s) is likely causative of these two MEN1 phenocopies. MEN1 proved to be the gene most frequent L4 mutated in common-variety, nonhereditary parathyroid tumor, gastrinoma, insulinoma, or bronchial carcinoid. For example, in common-variety parathyroid tumors, mutation of several other genes (such as cyclin D1 and P53) has been found, but much less frequently than MEN1 mutation. The majority of germline and somatic MEN1 mutations predicted truncation of the encoded protein (menin). Such inactivating mutations strongly supported prior predictions that MEN1 is a tumor suppressor gene insofar as stepwise mutational inactivation of both copies can release a cell from normal growth suppression. Menin is principally a nuclear protein; menin interacts with junD. Future studies, such as discovery of menin's metabolic pathway, could lead to new opportunities in cell biology and in tumor therapy.

We analyzed constitutional and tumor DNA from 27 MEN1 kindreds not known to be related to each other. Disease allele haplotypes were constructed for each pedigree based on shared alleles from two or more affected members and from determination of allelic loss patterns in their tumors. Analysis of disease allele haplotypes showed unexpected linkage disequilibrium at marker PYGM. Further haplotype analysis indicated this could be explained by the presence of two founder chromosomes, one in four families, the other in three. A shared disease haplotype was not observed among two MEN1 kindreds with the prolactinoma phenotype of MEN1.

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.

Primary hyperparathyroidism is a common disorder with an annual incidence of approximately 0.5 in 1,000 (ref. 1). In more than 95% of cases, the disease is caused by sporadic parathyroid adenoma or sporadic hyperplasia. Some cases are caused by inherited syndromes, such as multiple endocrine neoplasia type 1 (MEN1; ref. 2). In most cases, the molecular basis of parathyroid neoplasia is unknown. Parathyroid adenomas are usually monoclonal, suggesting that one important step in tumour development is a mutation in a progenitor cell. Approximately 30% of sporadic parathyroid tumours show loss of heterozygosity (LOH) for polymorphic markers on 11q13, the site of the MEN1 tumour suppressor gene. This raises the question of whether such sporadic parathyroid tumours are caused by sequential inactivation of both alleles of the MEN1 gene. We recently cloned the MEN1 gene and identified MEN1 germline mutations in fourteen of fifteen kindreds with familial MEN1 (ref. 10). We have studied parathyroid tumours not associated with MEN1 to determine whether somatic mutations in the MEN1 gene are present. Among 33 tumours we found somatic MEN1 gene mutation in 7, while the corresponding MEN1 germline sequence was normal in each patient. All tumours with MEN1 gene mutation showed LOH on 11q13, making the tumour cells hemi- or homozygous for the mutant allele. Thus, somatic MEN1 gene mutation for the mutant allele. Thus, somatic MEN1 gene mutation contributes to tumorigenesis in a substantial number of parathyroid tumours not associated with the MEN1 syndrome.

Adriamycin (doxorubicin, AdM) is a potent antineoplastic agent which binds specifically and with high affinity to the acidic phospholipid cardiolipin (CL) [Goormaghtigh et al. (1980) Biochim. Biophys. Acta 597, 1]. Duramycin (DM), a polypeptide antibiotic, has been reported to interact selectively with phosphatidyl-ethanolamine (PE) and monogalactosyldiacylglycerol [Navarro et al. (1985) Biochemistry 24, 4645]. The selectivity of DM—PE interaction was confirmed. AdM and DM were then used to explore the roles of CL and PE in Ca 2+ translocation in a phosphatidylcholine (PC)/ PE/CL liposome system modeled on the inner mitochondrial membrane with the following results: (i) AdM (100–400 μ m ) altered Ca 2+ uptake by PC/PE/CL (4/4/1, mol/mol) liposomes in a concentration-dependent fashion which varied with temperature, external Ca 2+ concentration, and liposome PE content, (ii) Addition of AdM was qualitatively equivalent to increasing temperature, Ca 2+ concentration, or liposome PE content, and cooperative interactions among these parameters were observed. An increase in any one factor generally enhanced Ca 2+ uptake; simultaneous increases in several factors inhibited uptake, (iii) Inhibition of Ca 2+ uptake was correlated with efflux of Arsenazo III. (iv) Ca 2+ uptake by PC/PE/CL liposomes is biphasic [Kester and Sokolove (1989) Biochim. Biophys. Acta 980, 127]. DM suppressed the PE-dependent slow phase and stimulated the PE-independent initial phase. Ca 2+ uptake by PC/PE/CL liposomes in the presence of DM resembled uptake by PC/CL liposomes. These data confirm the ability of PE to enhance the slow, highly temperature-dependent component of CL-mediated Ca 2+ translocation and suggest that this process is sensitive to lipid phase behavior.

An improved, rapid and simplified HPLC method is presented for the quantitation of theophylline in serum. The internal standard, beta-hydroxypropyl theophylline (20 ul of a 100 ug/ml solution) is added to 50 ul of serum. Serum proteins are precipitated by the addition of 30 ul of 20% trichloroacetic acid solution. The mobile phase consists of a sodium acetate buffer with 8% acetonitrile. Chromatogram run time is 8 minutes. The sensitivity limit is 0.10 ug/ml. This method is interference free from the major metabolites of theophylline and other drugs commonly coadministered with theophylline.

A high-performance liquid chromatographic technique is presented for the simultaneous determination of theophylline, 3-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid, and caffeine in urine using beta-hydroxyethyl theophylline (BHET) as an internal standard. Following centrifugation of the sample, 2 mL of urine was placed onto a Sep-Pak C18 cartridge (Waters Associates, Milford, MA, USA) that was prepared with 5 mL of methanol followed by 10 mL of distilled water. The compounds were eluted with 2.5 mL of a methanol:water (50:50) mixture. An aliquot of the filtered urine sample (100 microL) was added to 40 microL of an internal standard solution (BHET; 100 micrograms/mL). Distilled water (60 microL) was added to yield a volume of 200 microL. The mixture was vortexed and centrifuged, and 100 microL was injected onto a C8 column maintained at 55 degrees C. Inter- and intraday variability of the assay for all compounds was less than 5.7%. Sensitivity ranged from 0.10 microgram/mL for caffeine to 0.68 microgram/mL for 1-methyluric acid. Drugs commonly coadministered with theophylline did not interfere with the assay.

An improved and simplified high-performance liquid chromatographic method is presented for the simultaneous determination of theophylline and dyphylline in serum. The internal standard, beta-hydroxyethyltheophylline (20 microliters of a 100 micrograms/ml solution) is added to 50 microliters of serum. Serum proteins are precipitated by the addition of 30 microliters of 40% trichloroacetic acid solution. The mobile phase consists of a sodium acetate buffer with 6% acetonitrile. Chromatogram run time is 6 min. The sensitivity limit for both compounds is 0.25 micrograms/ml. This method is interference-free from the major metabolites of theophylline and other drugs commonly coadministered with theophylline.

The polypeptide antibiotic duramycin has been reported to interact specifically with two lipids: phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol (Navarro et al. (1985) Biochemistry 24, 4645-4650). PE is a major component of mitochondrial membranes. Duramycin was used to examine the role of PE in maintenance of mitochondrial structure and membrane permeability properties with the following results: (1) Duramycin addition to isolated rat heart mitochondria produced abrupt organelle contraction which was followed, depending on composition of the suspending medium, by pronounced swelling. The most notable morphological effect of the antibiotic was ruffling or crenelation of the outer membrane, which resulted ultimately in its separation from the inner membrane. (2) Low concentrations (less than 5 microM) of the antibiotic selectively increased the permeability of the mitochondrial inner membrane to cations and small solutes. This effect was blocked by atractyloside, a highly specific inhibitor of the adenine nucleotide translocator, by palmitoyl coenzyme A, by N-ethylmaleimide, and by AMP, ADP and ATP but not GDP or GTP, implicating the adenine nucleotide translocator in the selective permeability increase. (3) Higher concentrations of duramycin induced a more generalized permeability increase which was not subject to inhibition by compounds capable of interacting with the adenine nucleotide translocator.

Ca2+ uptake by liposomes consisting of phosphatidylcholine (PC) and cardiolipin (CL) has recently been reported (Smaal, E.B. et al. (1987) Biochim. Biophys. Acta 897, 191-196). In eukaryotic cells, CL is localized exclusively in the inner mitochondrial membrane, where it occurs in the presence of equimolar amounts of PC and phosphatidyl-ethanolamine (PE). We have therefore re-examined CL-mediated Ca2+ translocation in liposomes of more nearly physiological composition, i.e., PC/PE/CL (2:2:1 and 4:4:1, mol/mol). In addition, the effect on Ca2+ uptake of plasmalogens of PE, which may account for up to 50% of mitochondrial PE, was determined. Our findings can be summarized as follows. (1) Ca2+ uptake into CL-containing liposomes was increased dramatically by PE. (2) Ca2+ entry into PC/CL liposomes was biphasic; in the presence of PE, uptake was dominated by a slow process. (3) Ca2+ uptake by PC/CL liposomes saturated at less than or equal to 2 mM external Ca2+, whereas uptake into PC/PE/CL liposomes increased with increasing Ca2+ concentration up to 10 mM or until Ca2+ release ensued. (4) Ca2+ translocation by PE-containing liposomes and the slow phase of Ca2+ uptake into PC/CL liposomes were similarly and highly dependent on temperature. It can therefore be proposed that PE amplifies the slow component of CL-mediated Ca2+ translocation. This process is characterized by a requirement for high external Ca2+ concentrations and a large apparent activation energy. Ca2+ uptake was not significantly modified by plasmalogens of PE.

A high-performance liquid chromatographic technique is presented for simultaneous determination of theophylline, 3-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid and caffeine in serum using beta-hydroxyethyltheophylline (BHET) as an internal standard. An aliquot of a serum sample (100 microliters) was added to 40 microliters of an internal standard solution (BHET; 100 micrograms/ml) and vortexed. A 20% trichloroacetic acid solution (60 microliters) was then added; the mixture was vortexed, centrifuged and 100 microliters were injected onto a C8 column maintained at 45 degrees C. Inter- and intra-day variability of the assay for all compounds was less than 4.3%. Sensitivity ranged from 10 ng/ml for 1-methyluric acid to 25 ng/ml for theophylline. Drugs commonly coadministered with theophylline did not interfere with the assay.

Arsenazo III-loaded liposomes are in wide use as model systems in the study of Ca2+ transport. The most sophisticated method for quantitation of Ca2+ uptake [E. B. Smaal et al. (1985) Biochim. Biophys. Acta 816, 418] utilizes the absorbance changes (650-700 nm) elicited by sequential additions of EDTA and A23187 to distinguish Ca2+-Arsenazo complexes which are outside and inside the liposomes. In this paper, the analytical approach of Smaal and co-workers is reevaluated and a straightforward treatment that allows calculation both of the concentration of Ca2+ inside liposomes and of total Ca2+ uptake (in moles/mole phospholipid) is developed.

Duramycin is a polypeptide antibiotic (MR = 2012) isolated from culture broth of Streptoverticillium cinnamomeus (formerly Streptomyces cinnamomeus forma azacoluta, NRRL B-1699) (Shotwell et al., 1958). The antibiotic interferes with the function of several membrane transport systems including the chloride transporter of clathrin-coated vesicles (Stone et al., 1984), the Ca2+-ATPase of rabbit sarcoplasmic reticulum (Navarro et al., 1985), and the proton secretion system of B. subtilis(Navarro et al., 1985). Recently duramycin has been reported to interact specifically with the non-bilayer lipids phosphatidylethanolamine (PE) and monogalactosyldiglyceride (MGDG) (Navarro et al., 1985).