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Illudins are novel low molecular weight natural products cytotoxic to human tumor cells in vitro. Illudin-derived analogs are effective against experimental human cancers nonresponsive to conventional anticancer agents. It is not known why some illudin analogs are more efficacious in vitro and in vivo than other analogs. Therefore, the in vitro cytotoxicity of the parent compound illudin S towards tumor cells was characterized using radiolabeled drug. Two cell lines sensitive at nanomolar concentrations using only a 15-min exposure period displayed a saturable, energy-dependent accumulation of illudins with relatively low K(m) and high Vmax values. A nonsensitive cell line, requiring millimolar concentrations to achieve in vitro toxicity, showed minimal illudin uptake with higher K(m) and lower Vmax values. No release of radioactivity could be demonstrated from tumor cells, indicating that there was no efflux of illudin S (or metabolites) from these cells. The number of intracellular illudin S molecules required to kill 50% of cells of different tumor cell lines varied from 78000 to 1114000 molecules per cell and was correlated with the 2-h IC50 value determined using a colony-forming assay. Illudin S was cytotoxic to a variety of multidrug-resistant tumor cell lines regardless of whether resistance was mediated by gp170/mdrl, gp180/MRP, GSHTR-pi, topoisomerase I, topoisomerase II, increased DNA repair capacity, or alterations in intracellular thiol content. Information obtained in this study could be used to design clinical phase I trials and to develop analogs with improved therapeutic indexes.

The successful results of a feasibility experiment for tracking underwater vehicles equipped with laser beacons by aircraft equipped with detectors are presented. The system design focuses on tracking limited payload vehicles such as torpedoes in shallow-water (0-200 m) environments during Navy test and evaluation exercises. A compact, battery operated, Q-switched, frequency-doubled, dual-pulse Nd:YAG laser operating at 532 nm was used. The upward-pointing laser with a diffuse 14° output beam was mounted to a stationary buoy at a depth of five attenuation lengths. Aboard an SH3 helicopter at 5000 ft (1524 m), a 14° field-of-view avalanche photodiode detector system detected the first pulse that triggered an image-intensified CCD camera to image the second pulse at the ocean surface. When the results were scaled, we concluded that a coverage diameter of 14,375 m could be achieved at an aircraft elevation of 1 mile (1.6 km) with a signal-to-noise ratio of 10:1 for depths of ten attenuation lengths.

Objective: The purpose of this paper was to review the research literature related to eating behaviors and disturbances among American minority groups., Method: A computer-based literature search was conducted to locate articles pertaining to this topic., Results: This review indicates that, compared to Caucasian females, eating disturbances are equally common among Hispanic females, more frequent among Native Americans, and less frequent among Black and Asian American females. Risk factors for eating disorders (EDs) are greater among minority females who are younger, heavier, better educated, and more identified with White, middle-class values., Discussion: Further studies of EDs among American minority groups are needed, especially studies that are longitudinal and developmental in nature and that focus more specifically on the effects of racism in the development of EDs.

Illudin analogs are cytotoxic to a variety of multidrug resistant cell lines, and display an unusual toxicity towards DNA helicase-deficient cell lines. Earlier illudin analogs demonstrated efficacy in several xenograft models, including a metastatic MV522 lung cancer model, resistant to conventional anticancer agents. These illudin analogs prolonged life span as compared to conventional agents, but did not induce complete remission of primary tumors. In vitro screening studies identified a semisynthetic derivative, hydroxymethylacylfulvene (HMAF, MGI-114), with increased selective cytotoxicity towards carcinoma cells. The HMAF analog was markedly effective in the experimental MV 522 metastasizing lung carcinoma xenograft system, a model refractory to treatment with existing anticancer agents. Treatment with paclitaxel, doxorubicin, or cisplatin failed to significantly inhibit primary tumor growth or prolong life span of MV522 tumor-bearing animals. Treatment with mitomycin C at the LD20 increased life span in surviving animals up to 61% (p = 0.04). Treatment with HMAF induced primary tumor regression in all animals and increased life span greater than 150% (p < 0.001). Thus, administration of HMAF inhibited development of lung metastasis in a model refractory to treatment with conventional anticancer agents. These results support further evaluation of HMAF as a therapeutic agent for treatment of solid tumors such as adenocarcinoma of the lung.

Objective: The purpose of this paper was to review and summarize the research literature on the spectrum of eating disturbances., Method: Literature was searched using a computer data base to identify recent articles related to the prevalence and occurrence of disturbed eating patterns as well as full and partial syndrome eating disorders (EDs)., Results: This review indicates that the prevalence of partial syndrome EDs in nonclinical populations is at least twice that of full syndrome EDs, and that there is a progression in some individuals from less to more severe disturbances in eating behavior., Discussion: These findings suggest the need for careful, scientific evaluation of risk factors for EDs in both children and adults. A longitudinal research program in progress is described which aims to identify the risk factors for EDs.

Four second-generation Illudin analogues were synthesized and tested for antitumor activity using a metastatic lung carcinoma xenograft model resistant to conventional antitumor agents. One analogue, the parent illudofulvene-derivative called Acylfulvene, inhibited xenograft primary tumor growth and prolonged life span of tumor-bearing animals when administered i.p. or i.v. The efficacy of Acylfulvene exceeded that of mitomycin C, cisplatin, paclitaxol, the parent compound Illudin S, and an earlier analogue, dehydroilludin M. Promising features of this new analogue are: (a) the retention of in vitro activity against a variety of mdr tumor phenotypes including gp170+, gp150+, GSHTR-Pi, topoisomerase I, and topoisomerase II mutants; and (b) an apparent selective cytotoxicity toward cells deficient in either ERCC2 or ERCC3 DNA helicase activity.

Psoriasis is a hyperproliferative and inflammatory skin disorder of unknown aetiology. A fusion protein composed of human interleukin-2 and fragments of diphtheria toxin (DAB389IL-2), which selectively blocks the growth of activated lymphocytes but not keratinocytes, was administered systemically to ten patients to gauge the contribution of activated T cells to the disease. Four patients showed striking clinical improvement and four moderate improvement, after two cycle of low dose IL-2-toxin. The reversal of several molecular markers of epidermal dysfunction was associated with a marked reduction in intraepidermal CD3+ and CD8+ T cells, suggesting a primary immunological basis for this widespread disorder.

The human lung carcinoma cell line MV522 was previously noted to produce extensive metastasis to the lungs, spleen and lymph nodes after subcutaneous transplantation into athymic nude mice. Animals eventually succumb to these metastases, and not primary tumor growth. The ability to produce extensive metastasis after a simple subcutaneous injection in 100% of animals (> 100 tested to date) would be an advantage when screening compounds for anticancer activity. To validate the utility of this xenograft model for testing anticancer agents, we tested the ability of 10 anticancer drugs to either inhibit primary tumor growth and/or prolong life span of MV522-bearing animals. Among the 10 antitumor conventional agents, only mitomycin C and taxol demonstrated primary tumor growth inhibition. Mitomycin C produced a mild increase in median life span of 41% to 63%, while taxol had inconsistent effects. The metastatic MV522 carcinoma model appears to reflect clinical resistance of primary non-small cell lung cancer to conventional chemotherapeutic agents and should be useful for testing new anti-cancer drugs.

Objective: To report a case of a perforated gastric ulcer associated with the use of injectable ketorolac tromethamine., Data Synthesis: A 77-year-old woman with no previous history of peptic ulcer disease developed a perforated gastric ulcer after four days of treatment with ketorolac. To date, six other cases of gastrointestinal (GI) perforation associated with the use of ketorolac have been reported to the manufacturer., Conclusions: Although ketorolac is an effective analgesic, it is a nonsteroidal antiinflammatory agent and thus has the propensity for causing GI ulceration. Caution should be used when administering this drug and patients should be monitored for GI adverse effects.

High performance liquid chromatography (HPLC) combined with effective preseparation steps (liquid extraction, solid-phase extraction, preconcentration procedures) was used in pharmacokinetic studies of a novel anticancer agent (acylfulvene) in serum. HPLC conditions were optimized for the analysis of model clinical samples and real serum samples with different contents of acylfulvene. Extraction recoveries for both extraction procedures were evaluated and linearity was achieved for a wide concentration range. Detection limit for acylfulvene in serum was 0.075 microgram/ml, preconcentration minimum 5 times was recommended, especially after the solid-phase extraction step. The HPLC assay was applied for pharmacokinetic studies of acylfulvene in dogs and rats and pharmacokinetic parameters were determined.

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.

Clonal subpopulations of neoplastic cells were derived, in soft agar, from the spontaneously metastazing variant (MV522) of a human lung carcinoma cell line. The ability of these clones to spontaneously metastasize from subcutaneous sites in athymic mice was then tested. A variation in metastatic ability was expected with the derivation of some low metastatic clones and some high metastatic ones. However, all of the derived clones, although equally tumorigenic, were less metastatic than the parental variant. These clonal cell lines can now be used in a systematic analysis of events associated with the reversion to a less malignant state.

Spontaneously occurring microscopic lung and lymph node metastases (in athymic mice) of a low metastatic human lung carcinoma cell line, UCP3, and its high metastatic variant, MV522, were isolated. Characterization of the variants included karyotypic and isoenzyme analyses; assessment of spontaneous metastatic capabilities in athymic mice, and monoclonal antibody analyses. The high metastatic variant LNT had barely detectable amounts of a glycoprotein molecule with apparent Mr 73 kd and 90 kd, which was present in the other cell lines. This molecule was detected in 20/24 primary human neoplasms but only in 3/18 metastatic neoplasms, suggesting a loss during the metastatic disease process.

For the characterization of the metabolic and biologic properties of oval cells (i.e., cells emerging in the livers of rats treated with chemical carcinogens due to proliferation of bile ductular and/or duct cells) and transitional cells (i.e., cells having properties intermediate between those of oval cells and hepatocytes), these cells were isolated from the livers of Sprague-Dawley rats fed DL-ethionine for 4-5 weeks. The livers were dissociated into single cells by perfusion in situ with collagenase, and total cell suspensions were allowed to stand at unit gravity for 10 minutes to separate parenchymal (hepatocytes) from nonparenchymal cells. Nonparenchymal cells were centrifuged in linear gradients of Metrizamide (8-24% wt/vol), and 2-ml fractions were collected from the gradients. The cells in the fractions were defined by light microscopy, electron microscopy, and histochemical and immunofluorescence methods. A cell isolate was thus obtained consisting of Kupffer's cells (approximately 20%), bile ductular and/or duct cells and oval cells (approximately 30%), and transitional cells (approximately 50%). A twofold enrichment of bile ductular and/or duct cells and their derivatives was achieved over that found in the nonparenchymal cell fraction before isopyknic gradient centrifugation.

A method is presented for the isolation of pulmonary surfactant from rat lung homogenates and lavages using differential discontinuous density gradient centrifugation. The isolated surfactant was characterized by electron microscopy, assay of enzyme markers for different subcellular organelles, and its chemical composition. Surfactant that was isolated from lung homogenates appeared to be free of other contaminating cellular organelles. Electron microscopic examination of surfactant isolated from whole homogenates showed the presence of lamellar bodies and tubular myelin figures and, hence, represented total lung surfactant. On the other hand, surfactant prepared from lung lavages had mostly tubular myelin figures and, thus, represented extracellular surfactant. Both preparations were rich in phospholipids, especially lecithins and phosphatidylglycerol, and showed high phospholipid to protein ratios. The method was used to study the quantity and composition of surfactant during lung development in rats. Surfactant was isolated from lung homogenates of fetuses at days 19 to 21 of gestation and from newborn and adult rats. During the period of 19 to 21 days of gestation, there is a 10- and 20-fold increase in the amount of protein- and phospholipids (milligrams per gram of wet lung), respectively. A further 2-fold increase occurs after birth. Phosphatidylcholines account for 65% of the total phospholipids at day 20, 71% on day 21, and 81% at term. A progressive increase in the phosphatidylcholine to sphingomyelin ratios and in the amount of phosphatidylglycerol occurs during this period. The amount of disaturated lecithins (expressed as percentage of total phosphatidylcholines) in the lung surfactant increases from 49% at day 20 to 53% at birth. There is no change in the amount of disaturated lecithins after the newborn stage. The present method for the isolation of lung surfactant is reproducible, is less time consuming, and can be used to isolate quantitatively surfactant from small lung aliquots.

The development of arteriosclerosis is the most serious and common complication in long-term survivors of cardiac transplantation. We have used a variety of inbred rat strains with selected histocompatibility differences to examine the influence of prolonged, mild rejection reactions on the development of pathological changes in long-term cardiac allografts. Heterotopic cardiac allografts were exchanged between rat strains that differed for MHC class I (RT1.A and/or RT1.E) antigens or groups of minor, non-MHC antigens in MHC-compatible congenic combinations. Our results demonstrate that in strain combinations in which the allograft reaction is mild and prolonged, the donor hearts exhibit pathological changes that include a diffuse, interstitial myocardial fibrosis, perivascular fibrosis, and intimal proliferation in arteries of the graft myocardium. The lesions were less prominent in animals with more active rejection and infrequent in strains that differ for class I histocompatibility antigens or syngeneic controls. These results suggest that the comparable pathological changes seen in long-term human cardiac survivors may reflect low-level, persistent allograft reactions rather than factors associated with graft anoxia or effects of immunotherapy to prevent graft rejection.

Purified splenic and thymic lymphocytes from the ACI and F344 strains of inbred rats were disrupted by controlled hypotonic treatment, and their plasma membranes were prepared by sucrose density gradient centrifugation. The plasma membrane preparations were highly purified as judged by the structural appearance of the smooth membrane vesicles, by the 10- to 15-fold enrichment of 5'-nucleotidase, which cytochemically localized exclusively in the plasma membranes of intact lymphocytes, by the high cholesterol to phospholipid molar ratio (0.7-1.0), and by the very low specific activities of the enzymes associated predominantly with mitochondria, lysosomes, and endoplasmic reticulum. The protein and the lipid contents of the membranes were 48-55 and 37-48%, respectively. The total lipid content of plasma membranes was characteristically higher in thymic than splenic lymphocytes from both ACI and F344 strains. The specific activity of 5'-nucleotidase was similar in splenic lymphocyte membranes of the ACI strain, and in both the thymic and splenic lymphocyte membranes of the F344 strain. In contrast, the thymic lymphocyte membranes in the ACI strain showed half as much 5'-nucleotidase specific activity. Cytochemical results indicated that the 5'-nucleotidase is located on the outside surface of the lymphocyte plasma membranes.

Female, albino mice were fed a choline-deficient diet containing 0.5% DL-ethionine. All animals died within 5 days due to the development of an acute hemorrhagic pancreatis with fat necrosis throughout the peritoneal cavity. The apancreatitis was characterized by a massive necrosis of the exocrine parenchyma with intense hemorrhage and inflammatory reaction of the stroma. The sequence of histologic and ultrastructural alterations occurring in the acinar cells of the pancreas were studied in mice fed the diet for 1, 2, and 3 days. Major findings consited of accumulation of zymogen granules, vacuolation due to foci of cytoplasmic degradation, and alterations in the morphology of the zymogen granules. The pancreatitis appears to be due to intraparenchymal activation of zymogens, resulting from a synergistic action of choline deficiency with the basic toxicity of ethionine toward the acinar cells of the pancreas. The experimental model simulates closely the acute hemorrhagic pancreatitis with fat necrosis occurring in humans and may prove useful for exploring the pathogenesis of this condition.

1. The response of the liver to phenobarbital administration was compared in rats fed either laboratory chow, a semipurified choline-supplemented diet or a semipurified choline-deficient diet. 2. The liver contents of proteins, lipids and cytochrome P-450, as well as the activity of aminopyrine and ethylmorphine demethylases, were measured after 5 days of feeding and five daily injections of phenobarbital. Liver sections were examined electron microscopically. 3. In rats fed the choline-supplemented diet, phenobarbital administration caused increases in cellular constituents, enzyme activities and smooth endoplasmic reticulum membranes as great as those seen in rats fed laboratory chow. However, in rats fed the choline-deficient diet, the response of the liver to phenobarbital administration was severely reduced in comparison to that in rats fed the other diets. 4. It is concluded that dietary choline and an adequate synthesis of lecithins are necessary for the induction of microsomal mixed-function oxidases and the concomitant accumulation of smooth endoplasmic reticulum in hepatocytes.

Fetal hepatic ultrastructure was examined from 18 days gestation to term. Such structure was compared to that of tissue obtained at 18 days gestation which had been maintained in organ culture for 24, 48, and 72 h. In addition, the effects of maternal glucocorticoid administration in vivo and in vitro organ culture glucocorticoid exposure upon fetal hepatic morphogenesis were examined. Enhanced fetal hepatocyte ultrastructural maturation, characterized by a reduction in hematopoietic percursors, an increased frequency of hepatocyte-hepatocyte contact, a complete compliment of intracellular organelles, and the development of bile canaliculi by either maternal in vivo or in vitro fetal organ culture glucocorticoid exposure, was demonstrated. These studies extend previous observations concerning bile salt synthesis and secretion by fetal hepatic tissue both in vivo and in vitro by providing an ultrastructural basis for the functional changes observed and reported to occur in response to steroid therapy

Livers of 6- to 7-week-old male C3H/He, CBA, A, and BALB/c mice were examined by electron microscopy for the presence of intracisternal A particles (ICAP) after administration of diethylnitrosamine (DEN) in drinking water. In control mice, ICAP were extremely rare; they were found in the livers of only 2 mice (strains C3H/He and A; none in the other strains). By contrast, the treatment of mice with DEN greatly enhanced the appearance of ICAP in the liver cells of all strains. Within 2 weeks of the treatment, ICAP were found in 8-26% of liver cells examined in all mice and the number of ICAP/cell ranged from 3 to 12. Aside from mild disorganization of the rough endoplasmic reticulum, such as segmentation and vesiculation, liver cells of carcinogen-treated mice showed none of the consistent abnormalities that characterize the appearance of ICAP. The reactivation of ICAP (which are usually suppressed in adult mice) by DEN may become a useful marker for analysis of the sequential alterations of the liver that lead to the development of hepatoma during carcinogenesis.

The nephrotoxic effect of ethanol feeding on renal structure and function was evaluated in rats and compared to that in dextrose-fed isocaloric control animals. Alcohol-fed animals had larger kidneys than their controls. Despite this increase in renal mass, the alcohol-fed animals had a 50% reduction in creatinine clearance and a 67% reduction in osmolar clearance compared to their controls. When specific renal constituents were compared, the alcohol-fed animals were found to have twice the renal protein and a 50% increase in renal lipid. Despite these marked structural and functional differences, the light microscopic appearance of the kidneys of the two groups did not appear significantly different. In contrast, the electron microscopic differences were substantial. The renal epithelial cells, particularly of the distal tubules and Henle's loops, were found to show varying degrees of cellular injury and were observed to be sloughing into the lumens. These electron microscopic observations are similar to those obtained in tubular necrosis due to a variety of nephrotoxic agents. We propose, therefore, that chronic alcohol feeding of rats produces significant renal dysfunction and abnormalities of structure such that ethanol should be considered a true nephrotoxin.

The effects of methionine and ethionine on the fine structure of hepatic cell nucleoli of guinea pigs and rats were investigated. A single intraperitoneal injection of methionine into guinea pigs results in the disruption of nucleolonema as early as 2 hours after the injection. By 4 hours, nucleoli show complete fragmentation consisting of many small fragments and small remnants of nucleoli. Large aggregates of interchromatinic granules and condensation of chromatin appear in the nucleoplasm. These changes are remarkably similar to the lesions induced by ethionine in the liver of the rat or the guinea pig. The methionine-induced nuclear and nucleolar lesions persist up to 10 hours after the injection. The administration of adenine 4 hours after the methionine injection reverses the nucleolar lesions by 8 hours. The appearance of incompletely reconstructed nucleoli with twisted ropelike structures suggests a pattern of recovery very similar to the adenine-induced nucleolar reformation in ethionine-treated rats. Injecting methionine into rats induced no nucleolar abnormalities. It is suggested that the mechanism of nucleolar fragmentation induced by methionine or ethionine is related to the accumulation of S-adenosyl compounds with concomitant ATP deficiency in the liver.

A cell line (MDCK) of dog kidney origin grows on a glass surface as a mosaic of epithelium with many multicellular hemispherical vesicles. The cells lining the blisters actively secrete into the cyst cavities. Suspensions of these cells injected intravenously in the chick embryo produce brain metastases resembling adenocarcinoma.