Intrinsic activity of precursor forms of HIV-1 proteinase. The wild-type -Phe*Pro- bond located at the N-terminus of the mature aspartic proteinase of HIV-1 was replaced by -Ile-Pro- or -Val-Pro-. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV-proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31-8959, was assessed. A hitherto unobserved cleavage junction (at approximately Ala-Phe*Leu-Gln approximately) in the frame-shift region of the gag-pol viral genome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self-processing at neural pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31-8959.


Studies of oxidative stress in cellular systems. The interaction of monocytes and erythrocytes. 1H spin echo NMR spectroscopy is used to follow the interaction of intact and viable erythrocytes and monocytes obtained from different sources in mixed cultures. After a lag time (270 min) erythrocyte glutathione is observed to become more oxidised. This result is believed to occur as a consequence of monocyte activation generating hydrogen peroxide or hypochlorous acid, which is targeted at the erythrocyte. The red cell in turn employs its sulphydryl system as an anti-oxidant defence.


The neuropeptide, neuromedin C, activates a potassium current in mouse macrophages. Neuromedin C (NmC) induced an outward current (Io(NmC)) in macrophages. Reversal potentials were dependent on external K+ concentrations ([K+]o) and independent of [Cl-]o. Tetraethylammonium (TEA) and quinidine effectively suppressed Io(NmC). Charybdotoxin (ChTX) and apamin had little effect. Io(NmC) was abolished in Ca(2+)-free EGTA-containing solution. These results suggest that MnC activates a Ca(2+)-dependent K+ current (IK,Ca) and can modulate activities in macrophages.


Identification of a MAP kinase kinase kinase in phaeochromocytoma (PC12) cells. A MAP kinase kinase kinase (MAPKKK) was identified in phaeochromocytoma (PC12) cells which reactivated homogeneous MAP kinase kinase (MAPKK) from rabbit skeletal muscle that had been inactivated by incubation with protein phosphatase 2A. Reactivation was accompanied by stoichiometric phosphorylation of MAPKK on a serine residue(s). Following stimulation of PC12 cells with nerve growth factor and chromatography of the extracts on Mono Q, MAP kinase and MAPKK were detected as active phosphorylated enzymes, whereas MAPKKK was inactive and only activated after prolonged storage at 4 degrees C. The results suggest that the activation of MAPKKK by growth factors is likely to occur by a non-covalent mechanism.


Role of liver-type glucose transporter (GLUT2) in transport across the basolateral membrane in rat jejunum. To obtain information on the regulation of glucose transport across the basolateral membrane (BLM) of intestinal epithelial cells, we measured the number of [3H]cytochalasin B binding sites and the level of liver-type glucose transporter (GLUT2) protein in the BLM in the jejunum of rats (i) with diabetes (ii) given a high-carbohydrate diet or (iii) with experimental hyperglycemia (12 h infusion of a high-glucose solution). A glucose uptake and the number of D-glucose inhibitable [3H]cytochalasin B binding sites in BLM vesicles were significantly increased in all three conditions. Western blot analysis showed that the amount of GLUT2 protein in BLM vesicles was increased in rats with diabetes and those given a high-carbohydrate diet, but not in those with experimental hyperglycemia. These results suggest that there is a mechanism for rapid regulation of glucose transport in the BLM that does not depend on change in the amount of GLUT2.


Uptake and metabolism of fluorescent ceramide analogs by rat oligodendrocytes in culture. We studied the metabolism of sphingolipids by oligodendrocytes derived from rat spinal cord by providing lipid vesicles with either N-lissamine-rhodaminyl-ceramide (LRh-Cer) or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ceramide (NBD-Cer) to the cells cultured in a chemically-defined medium. With both probes the major fluorescent product turned out to be sphingomyelin (SM). Most of LRh-SM was not cell-associated but recovered from the culture medium, probably due to back-exchange to the lipid vesicles. The accumulation of LRh-SM, both in the cells and in the medium, was inhibited in the presence of monensin or brefeldin A, whereas the production of NBD-SM was much less affected by these Golgi perturbing drugs. With LRh-Cer as substrate, LRh-labelled fatty acid (FA), galactosyl- and sulfogalactosyl-ceramides (GalCer and SGalCer) were also formed. NBD-Cer, however, was metabolized to glucosylceramide (GlcCer) and GalCer but not to SGalCer or NBD-FA. These data demonstrate that chemical modifications of ceramide alter its metabolism in oligodendrocytes and that the metabolites of LRh-Cer reflect the glycolipid composition of myelin more closely than those of NBD-Cer.


The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials. In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme-carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to HbI is cooperative and entails major quaternary rearrangements. The reaction of Cys92 with p-chloromercuri-benzoate (PMB) and p-nitro-o-chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen-linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN-reacted HbI cooperativity is maintained, albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.


The binding of the fluorescent ATP analogue 2'(3')-trinitrophenyladenosine-5'-triphosphate to rat liver fatty acid-binding protein. The less polar fluorescent analogue of ATP, 2'(3')-trinitrophenyl-5'-triphosphate bound to rat liver fatty acid-binding protein with high affinity (Kd 6.3 x 10(-6) M) and 1:1 molar stoichiometry. This probe bound to the fatty acid binding site of the protein and was displaced by oleic acid and oleoyl CoA. High concentrations of ATP did not cause significant displacement of the fluorescent ATP analogue. Since the anionic part of this molecule is the triphosphate group it is difficult to envisage this group being accommodated at an anion binding site within the non-polar core of this protein as is the case with other fatty acid binding proteins. Therefore it is anticipated that the ligand must bind to liver fatty acid-binding protein with this triphosphate group surface exposed. Caution must be exercised when using the more hydrophobic fluorescent analogue of ATP to investigate the ATP binding properties of proteins.


Evidence for receptor-linked activation of phospholipase D in rat parotid glands. Stimulation by carbamylcholine, PMA and calcium. In order to test if phospholipase D (PLD) activity exists in the rat parotid gland, we took advantage of the fact that, in the presence of ethanol, PLD generates phosphatidylethanol (PEth) via a transphosphatidylation reaction. Lipid extracts of parotid acini prelabelled with [3H]myristic acid were analyzed by thin layer chromatography to determine [3H]phosphatidylethanol ([3H]PEth) formation. Carbamylcholine (1 mM) stimulated [3H]PEth formation in the presence of 2% ethanol, this effect was completely inhibited by atropine (10 microM). PMA (0.1-1 microM) and ionomycine (10 microM) also caused [3H]PEth generation. We conclude that a phospholipase D activity is present in the rat parotid gland and is regulated by muscarinic cholinergic receptors. Protein kinase C and calcium could also modulate this activity. This report provides the first evidence for the existence and receptor-linked regulation of phospholipase D in an exocrine gland, the rat parotid gland.


[Endogenous levels and dynamics of estrogen sulfates--physiological and pathological roles of estrone sulfate and estradiol 17-sulfate]. Plasma ethinylestradiol increases 47.6% when taken with ascorbic acid because of competition in producing sulfate conjugation. Thus the role of sulfates may be important. Serum and urinary estrone sulfate (E1-S) in pregnancy and non-pregnancy were analyzed. Its serum peak during the menstrual cycle was 2.67 +/- 0.37 ng/ml (mean +/- SE) and about ten times that of estradiol-17 beta. E1-S showed lower levels in malignant tissues of breast cancer and endometrial cancer. Increased sulfatase activity in the malignant tissue hydrolyzes E1-S to E1, which may develop the tumors. Serum estradiol 17-sulfate (E2-17-S) in pregnancy was first measured. As E2-17-S decreased, lipid peroxides increased. E2-17-S is converted to 2-OH or 4-OH E2-17-S, which act as lipid peroxide scavengers. Pregnancy-induced hypertension showed lower levels of E2-17-S. In vitro study using the human endothelial cell of the aorta, E2-17-S and 2-OH E2-17-S strongly suppressed lipid peroxidation, which precedes atherosclerotic change.


[Diagnosis and treatment of primary hyperparathyroidism]. Primary hyperparathyroidism is nowadays not uncommon in Japan. The incidence is considered to be one in every 2,500 to 5,000 persons. Many advances have occurred in diagnosis, localization study, and treatment of the disease. Surgery remains the treatment of choice for patients with primary hyperparathyroidism. Notwithstanding, there are still controversies concerning the management of primary hyperparathyroidism. One of the current disputable points is whether the use of localization study in patients who are undergoing and initial operation is justified or not. Another is whether unilateral exploration is justified or not. The basic reasons for these persistent controversies is the lack of consensus about incidence of multiglandular hyperplasia or double adenomas. Our observation implies that the incidence of hyperplasia or double adenomas among all the patients with primary hyperparathyroidism in Japan is lower than that in western countries. Since diagnostic accuracy of noninvasive localization studies has recently improved, it seems reasonable to adopt unilateral neck exploration in our country. It is also characteristic in Japan that the incidence of parathyroid carcinoma is relatively high (6%) compared with that reported in many other countries.


[Prediction of ovulation by urinary LH surge]. To investigate the usefulness of a semi-quantitative assay of urinary luteinizing hormone (LH) for predicting ovulation, the relationship among urinary LH, serum LH, serum estradiol (E2) and ultrasonographic observations of follicles were examined in 32 infertile women (35 cycles) at spontaneous (7), clomiphene (13) or cyclofenil (3) induced or hMG (12) induced cycles. Their urine samples were collected 3 times a day for LH assay, starting from about the 10th cycle day or when follicles having a diameter of 10mm or more were detected to the day of ultrasonographical confirmation of follicle ruptures. In the morning on these days, blood was taken twice daily for LH and E2 determination at intervals of 30 min. In spontaneous cycles, urinary LH was (P < 0.001) correlated to a significant extent with the serum LH and E2 levels, and follicle rupture was observed 1.9 days after the urine LH surge. In contrast, there was no significant correlation between the urine LH and serum LH or E2 levels in the ovulation induced cycles. In addition, the period from urine LH positive to follicle rupture was significantly (P < 0.05) prolonged in those cycles compared with that in spontaneous cycles. These results suggested that the urine LH determination was good for predicting ovulation in spontaneous cycles, but it was also necessary to monitor the follicle sizes by ultrasonography in ovulation induced cycles.


[Athyreotic congenital hypothyroidism in two sisters]. Two sisters with athyreotic congenital hypothyroidism are described. This is the fifth report on athyreotic congenital hypothyroidism in siblings. The elder sister is 14 years old and the younger one is 12. The parents have no consanguinity or family history of thyroid disease. Both of the patients were born before the start of neonatal screening tests for congenital hypothyroidism. After birth, they had jaundice, abdominal distention and constipation, which are typical symptoms of congenital hypothyroidism. Serum T4 levels were decreased, and the serum TSH levels were markedly increased. Therefore we diagnosed them as having congenital hypothyroidism. They have received replacement therapy with thyroxine since diagnosis and have shown normal development physically and psychologically. They were tested for thyroid scintigram when the elder sister was 9 years old and the younger one was 7. 123I thyroid uptakes were 0.69% and 0.64%, respectively. The thyroid scans demonstrated no focus of accumulation of 123I. They do not have trapping defect of iodine, because 123I ratio of saliva and serum were 41.3 and 46.3, respectively. From these results, we diagnosed them as having athyreotic congenital hypothyroidism. They and their mother do not have any antithyroid antibodies. We suppose that some genetic factor is responsible for the athyreotic congenital hypothyroidism.


[Studies on thyroid hormone autoantibody in two euthyroid cases with spuriously high value of serum free triiodothyronine]. Spuriously high value of serum free triiodothyronine (FT3: Amerlex free T3 kit, Amersham, UK.) was noted accidentally on routine laboratory examination of two clinically euthyroid patients (case 1: FT3; 18.5 pg/ml, FT4; 1.1 ng/dl, T3; 103 ng/dl, T4; 8.2 micrograms/dl, TSH; 1.74 microU/ml, case 2: FT3; 8.5 pg/ml, FT4; 1.1 ng/dl, T3; 137 ng/dl, T4; 8.9 micrograms/dl, TSH; 1.45 microU/ml), the former with poorly controlled diabetes (FBG 253 mg/dl, HbA1c 12.1%) and the latter with essential hypertension (184/108 mmHg). Although the hypertensive patient showed mild diffuse goiter, there was no evidence that the patients had autoimmune thyroid diseases because anti-thyroglobulin antibody tests measured by radioimmunoassay and MCHA, TGHA or TBII were all negative. Their serum levels of TBG were within the normal range. Further studies revealed that both patients' sera had unusual binding activity to labelled polyaminocarboxy T3 (125I-aT3) but not labelled T3 (125I-T3). Furthermore, this binding protein was precipitated by goat anti-human immunoglobulin G (IgG). The IgG purified from both patients' sera also showed strong binding activity to 125I-aT3, which was inhibited by unlabelled T3 in a dose dependent manner. In conclusion, we found anti-T3 antibody in two clinically euthyroid patients with no apparent evidence of complicating autoimmune thyroid diseases. The stronger binding activity to polyaminocarboxy T3 rather than T3 may lead to the spuriously high value of serum FT3. The mechanisms of the production of such autoantibodies in our cases should be further investigated.


[A case of Hashimoto's thyroiditis associated with renal tubular acidosis, Sjögren syndrome and empty sella syndrome]. This report describes a 48-year old female patient with Hashimoto's thyroiditis, distal-type renal tubular acidosis (d-RTA), Sjögren syndrome (SjS), and empty sella syndrome (ESS). She has been receiving replacement of thyroxine for Hashimoto's thyroiditis since 1967. She felt muscle weakness and numbness in the extremities and was found to have low serum potassium (2.9 mEq/l) in 1987. Since then she has been administrated potassium chloride orally. She was admitted to our hospital because of recurrence of muscle weakness and numbness of the extremities in November 1990. Laboratory examination revealed that her serum levels of antimicrosomal antibody and anti-thyroglobulin antibody were highly positive (MCHA: x 2(10) x 100, and TGHA: x 100). Furthermore, she was revealed to have 1) d-RTA by oral tolerance tests with the administration of NH4Cl and NaHCO3, 2) SjS by Schirmer test and sialography, and 3) ESS by computed tomography and magnetic resonance imaging examinations of the pituitary. Association of Hashimoto's thyroiditis, d-RTA, SjS and ESS in this case may possibly be caused by common autoimmune mechanism.


Adrenal renin: regulation and function.

Adrenal renin: regulation and function. Extrarenal renin has been identified in a variety of tissues. All of the components of the renin-angiotensin system have been detected in some organs, including the adrenal gland. A number of hypotheses have been expressed regarding the physiological function of these extrarenal renin systems, but no specific function has been clearly identified. This review focuses on the renin system in the adrenal cortex. Renin has been found in a variety of species, including the human adrenal cortex and adrenal tumors. Our investigations have focused on the renin-angiotensin system in the adrenal cortex of the rat, and have shown that renin is mainly present and synthesized in the zona glomerulosa cells. Renin production is under physiological control and can be altered by changes in electrolyte intake, adrenocorticotrophic hormone (ACTH), and the genetic background of the rat. In these experiments, a positive correlation between adrenal renin and aldosterone concentration is found. Furthermore, in primary culture of zona glomerulosa cells, a converting enzyme inhibitor can reduce the aldosterone stimulation by ACTH and potassium. However, these studies by themselves are not convincing evidence of the physiological role of the adrenal renin system. Further investigations are necessary to determine its role, if any, in adrenal function.


Immune-neuroendocrine circuits: integrative role of cytokines. Several efficient autoregulatory mechanisms confer a certain degree of autonomy to the immune system. However, increasing evidence shows that immune processes operate in a coordinated fashion with other body systems. In this article, we discuss concepts and facts concerning interactions between immune and neuroendocrine mechanisms. There are clear examples that immune cells can be influenced by hormones, neurotransmitters, and neuropeptides and also by alterations in brain functions. Conversely, immune-derived products such as lymphokines and monokines can affect endocrine, autonomic, and central mechanisms. Neuroendocrine responses occur during the activation of the immune system. These responses can be elicited by innocuous antigens; they can also be detected during pathological conditions involving immune activation, and in many cases are dissociable from the effects of the disease itself and from the stress of being sick. On this basis, we emphasize the multidirectional nature of the communication processes between the immune, endocrine, and nervous systems. The role of lymphokines and monokines as messengers able to convey information to neuro and endocrine structures about the present state of activity of the immune system is stressed. The relevance of immune-neuroendocrine interactions for immunoregulation and host defenses is discussed as well as the active role of the immune system in mediating metabolic and homeostatic adjustments or derangements during the course of certain infectious, inflammatory, and neoplastic processes. The evidence available suggests that complex immune-neuroendocrine networks operate under both physiological and pathological conditions.


Neurotrophic factors and female sexual development. The concept is proposed that polypeptide neurotrophic factors contribute to the developmental regulation of ovarian and hypothalamic function in mammals. Nerve growth factor (NGF) and neurotrophin-3, two members of the neurotrophin family, have been identified in the rat ovary and one of its receptors has been localized to the innervation and thecal cells of developing follicles. Although NGF supports the sympathetic innervation of the gland, the extent to which follicles are innervated appears to be defined by the differential expression of NGF receptors in the theca of developing follicles. The presence of NGF receptors in steroid-producing cells suggests a direct involvement of neurotrophins in the regulation of gonadal endocrine function. Evidence is beginning to emerge suggesting that development of the reproductive hypothalamus is affected by insulin-like growth factor 1 secreted by peripheral tissues, and transforming growth factor alpha (TGF alpha) produced locally. In the rat hypothalamus, TGF alpha appears to be synthesized in both neurons and glial cells. In glial cells it may interact with epidermal growth factor (EGF) receptors to further enhance TGF alpha synthesis and to, perhaps, stimulate eicosanoid formation. In turn, one of these eicosanoids, prostaglandin E2, may act on luteinizing hormone-releasing hormone (LHRH) neurons to stimulate the release of LHRH in a genomic-independent manner. This provides the basis for the notion that during development LHRH secretion is regulated by a dual mechanism, one that involves transsynaptic effects exerted by neurotransmitters, the other that requires a glial-neuronal interaction and that may predominantly regulate release of the neuropeptide. An increased expression of the TGF alpha and EGF receptor genes in reactive astrocytes is postulated to contribute to the process by which hypothalamic injury causes sexual precocity. Morphological maturation of the reproductive hypothalamus is thought to occur during sexual development. The process is accelerated by estradiol, which exerts its neurotrophic effects by enhancing the expression of genes encoding cytoskeletal proteins involved in neuronal development and regeneration. It is suggested that acquisition of functional competence by both the ovaries and the reproductive hypothalamus requires the participation of specific, but not similar, neurotrophic factors. The relevance of these concepts to the process of sexual development in other species, particularly primates, remains to be defined.


The 5 alpha-reductase in the brain: molecular aspects and relation to brain function. All the classes of hormonal steroids physiologically produced in the body (androgens, estrogens, progestagens, and corticosteroids) are able to exert important effects on the brain, but the mechanisms of their actions are not always well understood. Steroids may interact with intracellular receptors to activate the genome, but some of their effects are probably extragenomic and involve interactions with cellular membranes. Moreover, not all the steroids act always in their native molecular form; a large group of them must actually be transformed into "active" metabolites. This may occur at the level of their respective target structures. For example, androgens are metabolized in the brain into estrogens and into 5 alpha-reduced androgens, like 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone; DHT) and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol). Progesterone, and possibly corticosteroids, may also be transformed into their corresponding 5 alpha-reduced metabolites. Also the cellular target (neurons and/or glial cells) of the hormonal steroids in the brain is not always clear. This review analyzes in detail one of the two major enzymatic systems that transform steroids in the brain, namely the 5 alpha-reductase-3 alpha-(3 beta)-hydroxysteroid dehydrogenase pathway. An active 5 alpha-reductase-3 alpha-hydroxysteroid dehydrogenase system is widely distributed in practically all CNS structures in all phases of development. In the brain, this enzymatic system is not regulated by castration or sex steroid administration; furthermore, neural inputs seem to be ineffective at the hypothalamic level. A recent interesting finding is the presence of high concentrations of the 5 alpha-reductase in the white matter. This probably is due to the fact that the white matter is particularly rich in myelin membranes, with which the enzymatic activity appears to be associated. An active 5 alpha-reductase activity has also been shown to be present in peripheral myelinated nerves. The localization in myelin membranes may suggest a possible involvement of 5 alpha-reduced metabolites of the different steroids in the process of myelination. The presence of the 5 alpha-reductase was analyzed in neurons, astrocytes, and oligodendrocytes isolated from the brains of male rats, as well as in neurons and glial cells grown in culture. Neurons appear to be more active than glial cells in converting testosterone into DHT. Only neurons possess aromatase activity.(ABSTRACT TRUNCATED AT 400 WORDS)


Gonadotropin-releasing hormone neuronal cell lines. Gonadotropin-releasing hormone (GnRH) cell lines were developed by genetically targeted tumorigenesis in transgenic mice. The cell lines designated GT1 cells have a neuronal phenotype, express neuronal but not glial markers and express the GnRH gene at high levels. The GnRH prohormone is processed in the cells to multiple molecular forms including biologically active GnRH and GnRH-associated peptide. Basal secretion of GnRH from the cells is regulated in part by fast Na+ channels necessary for propagated action potentials. In many instances, basal GnRH release is pulsatile with an interpulse frequency similar to that seen in castrated rodents, suggesting that GnRH neurons are the pulse generator and are capable of synchronizing their secretion in vitro. The secretion of GnRH is stimulated by depolarization and by the neurotransmitter norepinephrine. In related studies we have demonstrated that expression of Simian virus 40 T antigen in GnRH neurons of transgenic mice leads to hypothalamic hypogonadism due to the inability of GnRH nerve terminals to organize in the median eminence. These findings support the use of genetically-directed tumorigenesis to establish highly differentiated GnRH neuronal cell lines that are a valuable model to study the cell biology and regulation of the neurons.


Long-term prognosis of patients with achalasia treated by cardial dilatation therapy. The long-term prognosis of expanding bag dilatation therapy using a Matsuo pneumatic bag dilator was evaluated in 163 cases of esophageal achalasia treated by this method over the 26-year period from 1964 to 1989. In all these cases, one year or more had passed since therapy. Practically no correlation was found between the efficacy of the therapy and the grade of esophageal dilation prior to therapy, the previous history of symptomatic distress or the number of dilatations performed. The efficacy of expanding bag cardial dilatation was most obvious in the increase of body weight, 59 cases (36.2%) showing an increase of 1-5 kg and 48 cases (29.4%) showing an increase of 6-10 kg. The therapy was rated "highly effective" in 61 cases (37.4%) and "effective" in 60 cases (36.8%), i.e. it was effective in a total of 121 cases (74.2%). It was rated as being "ineffective" in 16 cases (9.8%) including 4.3% of cases in which an operation had been performed. This indicated that surgical operation of esophageal achalasia should be performed in those cases in which good long-term results were not obtained even after expanding bag dilatation therapy had been carried out several times.


Bowel preparation for the total colonoscopy by 2,000 ml of balanced lavage solution (Golytely) and sennoside. One of disadvantages of the Golytely preparation is that examinees have to drink as much as 4,000 ml of Golytely. To overcome this disadvantage, we designed a modified preparation regimen in which examinees have to drink only 2,000 ml of Golytely by taking sennoside orally. Bowel preparation was carried out in 297 examinees by this modified method. Examinees ate their usual diet and took 36 mg of sennoside orally on the night before the examination. On the day of the examination, the examinees drank a total of 2,000 ml of Golytely. No severe complications were noted and 97% of the examinees were able to drink the dose of 2,000 ml. Subjects who had also experienced bowel preparation by the modified method of Brown were asked to compare the two regimens, and only 1% preferred Brown's method while 73% preferred bowel preparation by our Golytely method. The result of bowel preparation by this method was excellent or good in 90 to 97% of the subjects at all sites in the colon and rectum. We conclude that bowel preparation for total colonoscopy using 2,000 ml of Golytely and sennoside is superior because it is highly acceptable to the examinees and provides excellent gut irrigation.


Electron microscopic study of hepatitis B virus-associated antigens on the infected liver cell membrane in relation to analysis of immune target antigens in chronic hepatitis B. To clarify the characteristics of cell surface Pre-S2 Ag, HBcAg and HBeAg immunohistochemically and to explore their relationship with a cellular immune target antigen, 31 liver biopsy specimens from chronic HBV carriers were examined by immunoperoxidase staining. By immune light microscopy, Pre-S2 Ag was detected on the liver cell membrane in 18 (58%) of the 31 cases, HBcAg in 4 cases (13%) and HBeAg in 4 cases (13%). Pre-S2 Ag frequently showed a honeycomb-like membrane expression pattern which was present regardless of liver inflammation, whereas HBcAg and HBeAg exhibited a scattered membrane expression pattern detected in areas of marked inflammation. Of the 18 cases showing a honeycomb-like Pre-S2 Ag expression, 3 concomitantly showed a scattered membrane expression pattern. Immunoelectron microscopy revealed these two distinct membrane expression patterns. In areas showing a honeycomb-like membrane expression pattern, Pre-S2 Ag was demonstrated in the intercellular space and on the basolateral membranes of hepatocytes, but was not detected on the cell membranes in areas of the intercellular space lacking an immunoreaction. Cytoplasmic expression of Pre-S2 Ag was less extensive in these hepatocytes. These findings suggest that the honeycomb-like membrane expression of Pre-S2 Ag results from attachment of extracellular antigen to the liver cell membrane. In contrast, in areas showing a scattered membrane expression pattern, Pre-S2 Ag, HBcAg and HBeAg were each detected as single-layered linear deposits along the cell membrane, but were absent in the intercellular space. Each antigen was also expressed abundantly in the cytoplasm, and the immunoproducts appeared to fuse with the cell membrane. These findings suggest that the scattered membrane expression of these antigens results from intrahepatic transfer of antigen synthesized in the liver cell to the cell membrane, possibly serving as a target for the host immune-mediated response in connection with inflammation.


Obliteration of portal systemic shunts as therapy for hepatic encephalopathy in patients with non-cirrhotic portal hypertension. The effects of obliteration of portal systemic shunts (PSS) in 5 patients with non-cirrhotic hepatic encephalopathy is reported. All patients had a history of disturbance of consciousness for several years, and examinations revealed large PSS, most of which connecting the left gastric vein to the left renal vein. After the obliteration of PSS, portal vein pressure elevated, the shunt ratio of the portal blood flow decreased, the indocyanine green disappearance rate increased, and serum albumin increased. Blood ammonia (NH3) decreased significantly accompanied by disappearance of hepatic encephalopathy. This treatment may open a way to improve the quality of life in patients with large PSS without severe hepatic injury.


Carcinoma in situ of the gallbladder with superficial extension into the Rokitansky-Aschoff sinuses and mucous glands. Twenty-four cases of carcinoma in situ of the gallbladder encountered over the past 29 years were evaluated histopathologically. Macroscopic classification disclosed 12 protruding types and 12 superficial types (7 elevated types and 5 flat types). Histologically, 18 cases had papillary growth and 6 had tubular patterns. Epithelial dysplasia of varying degrees was observed around the carcinoma in situ in all except one case and was seen more extensively in superficial type cases, although it was often hard to distinguish dysplasia from carcinoma in situ in some cases. Metaplasia of the mucous glands was observed in 20/24 cases (83.3%). Superficial extension of carcinoma in situ into the Rokitansky-Aschoff sinuses (located below the muscle layer) was observed in 11 cases (45.8%), and extension into the mucous glands was seen in 8 cases (33.3%). These results indicate that it is very important to be able to differentiate invasive carcinoma from cases with superficial extension of dysplasia and/or carcinoma in situ deep into the mucous glands or Rokitansky-Aschoff sinuses. Histological assessment should also be conducted carefully while considering these factors.


Leiomyosarcoma of the esophagus: report of a case and preoperative evaluation by CT scan, endoscopic ultrasonography and angiography. A case of esophageal leiomyosarcoma, for which CT scan, endoscopic ultrasonography (EUS) and angiography were employed for preoperative diagnosis is reported. CT scan identified an exophytic mass which had no rim-enhancement in the lower end of the esophagus. EUS revealed a homogeneous mass originating from the muscularis propria. Angiography showed a mildly hypervascular tumor. Histologically, the resected specimen was a leiomyosarcoma. These results suggest that the evaluation by EUS combined with CT scan and angiography is useful in differentiating smooth muscle tumors from other submucosal tumors.


Caroli's disease in three siblings.

Caroli's disease in three siblings. Three sisters with cystic dilatation of the intrahepatic bile ducts (Caroli's disease) are reported. The index case, a 41-year-old woman with remittent high fever and right upper quadrant abdominal pain, was diagnosed as Caroli's disease with hepatic lithiasis and cholangitis based on findings of ultrasonography, computed tomography and endoscopic retrograde cholangiography. Her two older sisters were also examined and found to have the same disease without clinical symptoms. Their symptoms, locations of the dilated ducts and complications all varied. The hereditary mode of Caroli's disease in 13 families (32 cases) reported in the world literature including our study was examined. While Caroli's disease is thought to be an autosomal recessive disease, a conclusion on the hereditary mode of transmission could not be made in this study because of an insufficient investigation of family members, especially the parents.


Conjugal transfer of plasmid-borne bacteriocin production in Enterococcus faecalis 226 NWC. Enterococcus faecalis 226 NWC, isolated from natural whey cultures utilized as starter in water-buffalo Mozzarella cheese manufacture, produces a bacteriocin, designated Enterocin 226 NWC, which is inhibitory to Listeria monocytogenes. Plasmid analysis of E. faecalis 226 NWC showed a single 5.2-kb plasmid, pEF226. In conjugation experiments, pEF226 was transferred into a plasmid-free strain of E. faecalis JH2-2. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The identity of conjugation was confirmed by digestion with SmaI restriction endonuclease and subsequent pulsed-field gel electrophoresis (PFGE) of the genomic DNA of E. faecalis 226, E. faecalis JH2-2 and of the isolates after the mating. The data indicate that the ability of E. faecalis 226 NWC to produce the bacteriocin is linked to the 5.2-kb conjugative plasmid pEF226.


Two different beta-lactamase genes are present in Streptomyces cacaoi. Two beta-lactamase genes called blaL and blaU have been cloned independently in Liège and in Umeå, from Streptomyces cacaoi. Genes blaL and blaU were found to differ largely in their nucleotide sequences, although the encoded proteins both belonged to the class A of beta-lactamases (active-site serine penicillinases). DNA-hybridization and polymerase chain reaction assays have now demonstrated that both blaL and blaU genes were present in the S. cacaoi strains used in Liège and in Umeå.


Kinetic properties of Helicobacter pylori urease compared with jack bean urease. The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.


Effect of flash photoreactivation on Escherichia coli recA induction by ultraviolet light. Excision-deficient Escherichia coli, carrying the gene for the photolyase on a multicopy plasmid, were irradiated with ultraviolet (UV) light then photoreactivated by illumination delivered from a camera flash unit. Such instantaneous illumination monomerizes only cyclobutane pyrimidine dimers already bound by the photolyase. Whereas the lethal effect of UV light and the number of C-to-T transition-type mutations induced by UV irradiation were both significantly reduced by subsequent irradiation with a single flash of light, single-flash photoreactivation did not reverse the induction of the recA gene by UV light. The results indicate, therefore, that non-photoreactivable DNA lesions play a role in recA induction.


Correlation between cell-surface hydrophobicity of Candida albicans and adhesion to buccal epithelial cells. Adhesion of four isolates of Candida albicans to buccal epithelial cells was determined after growth of the yeasts in defined medium containing 50 mM glucose or 500 mM galactose as the carbon source. With each isolate, adhesion of galactose-grown yeasts was significantly higher than that of glucose-grown organisms. Yeast cell-surface hydrophobicity was assessed by two methods, a modified hydrocarbon adhesion assay and a more sensitive polystyrene microsphere assay. All four isolates were significantly more hydrophobic after growth on 500 mM galactose than after growth on 50 mM glucose. Overall, a strong positive correlation between adhesion and surface hydrophobicity was observed (r = 0.965). These results are discussed in relation to the role of yeast-surface hydrophobicity in pathogenesis.


Cadmium-resistance plasmid in Staphylococcus lugdunensis. Plasmids that confer resistance to cadmium (MIC > or = 125 microM), were found in 18 out of 30 independent Staphylococcus lugdunensis strains from clinical specimens. Variants that were cured of their plasmid were cadmium sensitive. Restriction endonuclease sites of a 3.2-kb cadmium-resistance plasmid of S. lugdunensis, designated pLUG10, were similar to those of pOX6, a S. aureus cadmium-resistance plasmid containing the cadB gene. Southern-blot hybridisation was performed with a probe intragenic to cadB. Hybridisation was found with the cadB probe in the cadmium-resistant S. lugdunensis isolates to the 2.9-, 3.2- and 3.7-kb plasmids. These findings suggest that cadmium-resistance in some S. lugdunensis strains is due to a gene sharing homology with the cadB gene of S. aureus.


The cloning and nucleotide sequence of the serine protease gene (aspA) of Aeromonas salmonicida ssp. salmonicida. The gene for Aeromonas salmonicida serine protease has been cloned into phagemid pTZ18R in two restriction fragments, 2.0-kb PstI and 2.3-kb KpnI, of genomic DNA. The nucleotide sequences of the two fragments have been determined, in both directions, after subcloning, by double-stranded sequencing of nested deletions. An open reading frame of 1863 bp translated into a sequence of 621 amino acids, a 24-amino acid signal peptide and a 597-amino acid mature enzyme of molecular mass 64,173 Da. The consensus sequence, NGTS, of a serine protease substrate primary binding site was identified and a putative ribosome-binding site GGAG occurred 6 bp upstream of the ATG initiation codon.


Immunological characterization of a 17-kDa antigen from Cryptosporidium parvum recognized early by mucosal IgA antibodies. Cryptosporidium parvum antigens were characterized by immunoblot analysis of sera and intestinal secretions of BALB/c mice orally infected with 10(5) oocysts. A major band at 17 kDa under non-reduced conditions and at 18 kDa under reduced conditions was recognized by anti-C. parvum IgA and IgG in serum and intestinal secretions from day 15 post-infection. This recognition persisted throughout the experiment (day 30). Mouse-serum antibodies raised against the 17-kDa purified antigen (P17) showed no cross-reactivity with other C. parvum antigens. Immunofluorescence study revealed that this antigen is located on the sporozoite. It is suggested that this antigen could be a good candidate for studies of mucosal immune response to C. parvum and for vaccination.


Inactivation of the fis gene leads to reduced growth rate. The FIS protein (factor for inversion stimulation) is known to activate the transcription of rRNA and tRNA operons in Escherichia coli. In this report the growth behaviour of a fis bacterium was studied under steady-state conditions and in competition experiments with a wild-type bacterium in a chemostat. The growth rate of the fis bacterium was reduced as compared to that of an isogenic wild-type and this reduction increased linearly with increasing growth rate. A similar result was obtained in competition experiments in a chemostat, with the selection factor increasing linearly with the growth rate.


Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction. A fast and simple method to detect bacterial and especially mycoplasma contamination in tissue culture by means of polymerase chain reaction (PCR) amplification is described. In a first step the universal primer pairs P1/P2 (190-bp fragment) and P3/P4 (120-bp fragment) directed to different conserved parts of the prokaryotic 16S rRNA gene are used. A positive signal after amplification on cell culture DNA with these primers provides an indication of bacterial infection. Using the internal primers IP1, IP3 and IP'3 complementary to a part of the V4 and V8 variable regions of the 16S rRNA gene, in combination with a universal primer, cultures contaminated with mycoplasma could be identified. Six mycoplasma species, typical contaminants in tissue cultures, were investigated: Mycoplasma orale, M. fermentans, M. arginini, M. hyorhinis, M. hominis and Aeromonas laidlawii. This mycoplasma test is an easy, specific and sensitive assay which should be extremely useful in any tissue culture setting.


N-terminal mutations modulate yeast SNF1 protein kinase function. The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-delta N kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-delta N protein showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell.


XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination. The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that XRS2 also encodes an essential meiotic function. Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids. The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway. In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains. Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion. Moreover, xrs2 strains display a mitotic hyper-rec phenotype. Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions. Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.


Construction of a GAL1-regulated yeast cDNA expression library and its application to the identification of genes whose overexpression causes lethality in yeast. We have constructed a galactose-inducible expression library by cloning yeast cDNAs unidirectionally under control of the GAL1 promoter in a centromeric shuttle vector. Eleven independent libraries were made each with an average size of about 1 x 10(6) clones, about 50 times larger than the reported mRNA population in a yeast cell. From this library, LEU2 and HIS3 cDNAs were recovered at a frequency of about 1 in 10(4) and in 12 out of 13 cases these were expressed in a galactose-dependent manner. Sequence analysis of leu2 and his3 complementing cDNAs indicates that they contain all the coding sequence and much of the 5' untranslated region. To test the utility of the library for the identification of genes whose overexpression confers a specific phenotype, we screened 25,000 yeast transformants for lethality on galactose. Among 15 clones that showed galactose inducible lethality were cDNAs encoding structural proteins, including ACT1 (actin), TUB2 (beta-tubulin) and ABP1 (actin-binding protein 1), and genes in signal transduction pathways, including TPK1 (a cAMP-dependent protein kinase) and GLC7 (type 1 protein phosphatase). cDNAs overexpressing NHPB (nonhistone protein B) and NSR1 (nuclear sequence recognition protein) were also found to be lethal. Among these, ACT1 was isolated four times, and NSR1 three times. The useful features of this library for cDNA cloning in yeast by complementation, and for the identification of genes whose over-expression confers specific phenotypes, are discussed.


Analysis of the Caenorhabditis elegans axonal guidance and outgrowth gene unc-33. Mutations in the unc-33 gene of the nematode Caenorhabditis elegans lead to severely uncoordinated movement, abnormalities in the guidance and outgrowth of the axons of many neurons, and a superabundance of microtubules in neuronal processes. We have cloned unc-33 by tagging the gene with the transposable element Tc4. Three unc-33 messages, which are transcribed from a genomic region of at least 10 kb, were identified and characterized. The three messages have common 3' ends and identical reading frames. The largest (3.8-kb) message consists of the 22-nucleotide trans-spliced leader SL1 and 10 exons (I-X); the intermediate-size (3.3-kb) message begins with SL1 spliced to the 5' end of exon V and includes exons V-X; and the smallest (2.8-kb) message begins within exon VII and also includes exons VIII-X. A gamma-ray-induced deletion mutation situated within exon VIII reduces the sizes of all three messages by 0.5 kb. The three putative polypeptides encoded by the three messages overlap in C-terminal sequence but differ by the positions at which their N termini begin; none has significant similarity to any other known protein. A Tc4 insertion in exon VII leads to alterations in splicing that result in three approximately wild-type-size messages: the Tc4 sequence and 28 additional nucleotides are spliced out of the two larger messages; the Tc4 sequence is trans-spliced off the smallest message such that SL1 is added 13 nucleotides upstream of the normal 5' end of the smallest message.


Dynamics of cytoplasmic incompatibility and mtDNA variation in natural Drosophila simulans populations. In Drosophila simulans a cytoplasmically transmitted microorganism causes reduced egg hatch when infected males mate with uninfected females. The infection is rapidly spreading northward in California. Data on a specific mtDNA restriction site length polymorphism show that changes in the frequency of mtDNA variants are associated with this spread. All infected flies possess the same mtDNA allele, whereas the uninfected flies are polymorphic. Given that both paternal inheritance of the infection and imperfect maternal transmission have been demonstrated, one might expect instead that both infected and uninfected flies would possess both mtDNA variants. Our data suggest that imperfect female transmission of the infection (and/or the loss of the infection among progeny) is more common in nature than paternal transmission. A simple model of intrapopulation dynamics, with empirically supported parameter values, adequately describes the joint frequencies of the mtDNA variants and incompatibility types.


Characterization of Suppressor of fused, a complete suppressor of the fused segment polarity gene of Drosophila melanogaster. fused (fu) is a maternal effect segment polarity gene of Drosophila melanogaster. In addition, fu females have tumorous ovaries. Two ethyl methanesulfonate mutageneses were carried out in order to isolate suppressors of the fu phenotype. A new gene, Suppressor of fused (Su(fu)), was identified. It is located in the 87C8 region of the third chromosome. Su(fu) displays a maternal effect and is also expressed later in development. Although Su(fu)LP is a complete loss-of-function mutation, it is homozygous viable and has no phenotype by itself. Su(fu) fully suppresses the embryonic and adult phenotypes of fu mutants. Su(fu) mutations are semidominant and a Su(fu)+ duplication has an opposite effect, enhancing the fused phenotype. It is proposed therefore that the Su(fu)+ product is involved in the same developmental step as the Fu+ kinase. Thus, a new gene interacting with the segment polarity pathway was identified using an indirect approach.


The distribution of spontaneous mutations on quantitative traits and fitness in Drosophila melanogaster. Starting from a completely homozygous population of Drosophila melanogaster, two groups of 100 inbred lines each were established and maintained for 46 generations, by a single brother-sister mating and two double first cousin matings, respectively. Sternopleural bristle number, wing length and wing width were simultaneously scored in all lines every 4-5 generations. The means of four lines in each group departed significantly from the overall mean and, in each case, this was attributed to a single mutation of relatively large effect on at least one trait (0.3-1.4 environmental standard deviations in absolute value). Further analyses revealed widespread pleiotropy, similar gene action of a given mutation for all traits affected, and predominant additive action. No apparent association was found between the magnitudes of mutational effects on the traits and fitness. However, all recessive mutations were deleterious. The distribution of mutant effects was asymmetrical (positive for bristles and negative for wing measurements). Moreover, these distributions had a high variance and may be leptokurtic, due to the presence of major genes. Estimates of the ratio of new mutational variance to environmental variance ranged within (0.7-3.4) x 10(-3), those for wing measurements being generally larger. In agreement with theory, the rate of between-line differentiation was independent of population size.


Direct measurement of in vivo flux differences between electrophoretic variants of G6PD from Drosophila melanogaster. Demonstrating that naturally occurring enzyme polymorphisms significantly impact metabolic pathway flux is a fundamental step in examining the possible adaptive significance of such polymorphisms. In earlier studies of the glucose-6-phosphate dehydrogenase (G6PD) polymorphism in Drosophila melanogaster, we used two different methods, exploiting both genotype-dependent interactions with the 6Pgd locus, and conventional steady-state kinetics to examine activity differences between the two common allozymes. In this report we use 1-14C- and 6-14C-labeled glucose to estimate directly genotype-dependent flux differences through the pentose shunt. Our results show that G6pdA genotype possesses statistically lower pentose shunt flux than G6pdB at 25 degrees. We estimate this to be about a 32% reduction, which is consistent with the two former studies. These results reflect a significant responsiveness of pentose shunt flux to activity variation at the G6PD-catalyzed step, and predict that the G6PD allozymes generate a polymorphism for pentose shunt flux.


The evolution of duplicate glyceraldehyde-3-phosphate dehydrogenase genes in Drosophila. In Drosophila melanogaster there are two genes which encode the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Gapdh-43E and Gapdh-13F. We have shown that Gapdh-43E codes for the GAPDH subunit with an apparently larger molecular weight while Gapdh-13F encodes the GAPDH subunit having an apparently smaller molecular weight. Immunoblots of sodium dodecyl sulfate gels were used to survey species from throughout the genus and results indicated that two classes of GAPDH subunits are present only in Drosophila species of the melanogaster and takahashi subgroups of the melanogaster group. Only the smaller subunit is found in species of the obscura group while all other species have only a large subunit. Drosophila hydei was analyzed at the DNA level as a representative species of the subgenus Drosophila. The genome of this species has a single Gapdh gene which is localized at a cytogenetic position likely to be homologous to Gapdh-43 E of D. melanogaster. Comparison of its sequence with the sequence of the D. melanogaster Gapdh genes indicates that the two genes of D. melanogaster are more similar to one another than either is to the gene from D. hydei. The Gapdh gene from D. hydei contains an intron following codon 29. Neither Gapdh gene of D. melanogaster has an intron within the coding region. Southern blots of genomic DNA were used to determine which species have duplicate Gapdh genomic sequences. Gene amplification was used to determine which species have a Gapdh gene that is interrupted by an intron. Species of the subgenus Drosophila have a single Gapdh gene with an intron. Species of the willistoni and saltans groups have a single Gapdh gene that does not contain an intron.(ABSTRACT TRUNCATED AT 250 WORDS)


Germ line variable regions that match hypermutated sequences in genes encoding murine anti-hapten antibodies. We asked whether there are germ line immunoglobulin variable (V) segments that match sites of hypermutation in V regions encoding murine antibodies. Murine germ line DNA was probed with a panel of short deoxyoligonucleotides identical in sequence to segments of hypermutated V regions from hybridomas generated in the BALB/c response to the hapten 2-phenyloxazolone (Ox). Germ line sequences that match mutations in both heavy and kappa light chain V regions were identified, and clones of some of these germ line V segments were obtained. Comparison of these clones with hypermutated V regions revealed regions of identity ranging in size from 7 to over 50 nucleotides. In an effort to separate the effects of antigen selection from the mutagenic process, we also searched for matches to a panel of silent mutations in VH regions from germinal center B cells. Fourteen silent mutations occur among a collection of 36 hypermutated VH regions from two separate germinal centers of C57BL/6 mice stimulated with the hapten 4-hydroxy-3-nitrophenyl. Matches to nine of these silent mutations can be found among published sequences of C57BL/6 VH regions of the J558 family. Taken together, these data are consistent with the possibility that a template-dependent mutational process, like gene conversion, may contribute to somatic hypermutation.


Identification of genetic factors contributing to heterosis in a hybrid from two elite maize inbred lines using molecular markers. The use of molecular markers to identify quantitative trait loci (QTLs) affecting agriculturally important traits has become a key approach in plant genetics-both for understanding the genetic basis of these traits and to help design novel plant improvement programs. In the study reported here, we mapped QTLs (and evaluated their phenotypic effects) associated with seven major traits (including grain yield) in a cross between two widely used elite maize inbred lines, B73 and Mo17, in order to explore two important phenomena in maize genetics-heterosis (hybrid vigor) and genotype-by-environment (G x E) interaction. We also compared two analytical approaches for identifying QTLs, the traditional single-marker method and the more recently described interval-mapping method. Phenotypic evaluations were made on 3168 plots (nearly 100,000 plants) grown in three states. Using 76 markers that represented 90-95% of the maize genome, both analytical methods showed virtually the same results in detecting QTLs affecting grain yield throughout the genome, except on chromosome 6. Fewer QTLs were detected for other quantitative traits measured. Whenever a QTL for grain yield was detected, the heterozygote had a higher phenotype than the respective homozygote (with only one exception) suggesting not only overdominance (or pseudooverdominance) but also that these detected QTLs play a significant role in heterosis. This conclusion was reinforced by a high correlation between grain yield and proportion of heterozygous markers. Although plant materials were grown and measured in six diverse environments (North Carolina, Iowa and Illinois) there was little evidence for G x E interaction for most QTLs.


The evolution of tandemly repetitive DNA: recombination rules. Variable numbers of tandem repeats (VNTRs), which include hypervariable regions, minisatellites and microsatellites, can be assigned together with satellite DNAs to define a class of noncoding tandemly repetitive DNA (TR-DNA). The evolution of TR-DNA is assumed to be driven by an unbiased recombinational process. A simulation model of unequal exchange is presented and used to investigate the evolutionary persistence of single TR-DNA lineages. Three different recombination rules are specified to govern the expansion and contraction of a TR-DNA lineage from an initial array of two repeats to, finally, a single repeat allele, which cannot participate in a misalignment and exchange process. In the absence of amplification or selection acting to bias array evolution toward expansion, the probability of attaining a target array size is a function only of the initial number of repeats. We show that the proportions of lineages attaining a targeted array size are the same irrespective of recombination rule and rate, demonstrating that our simulation model is well behaved. The time taken to attain a target array size, the persistence of the target array, and the total persistence time of repetitive array structure, are functions of the initial number of repeats, the rate of recombination, and the rules of misalignment preceding recombinational exchange. These relationships are investigated using our simulation model. While misalignment constraint is probably greatest for satellite DNA it also seems important in accounting for the evolution of VNTR loci including minisatellites. This conclusion is consistent with the observed nonrandom distributions of VNTRs and other TR-DNAs in the human genome.


[Translocation 11q;22q: a clinico-cytogenetic study]. Seven families with translocations t(11; 22) identified at our Institute and analysis of the literature showed that the imbalance resulted from such translocations is always due to nondisjunction 3:1. Nondisjunction occurs more often in the 1st meiotic division, and is more rare in the second one. Expressed prezygotic selection against spermia with an additional chromosome greatly increases the risk of having an imbalanced child for the women-carriers as compared to men-carriers. The phenotype of the patients with +der(22)t(11; 22) is composed of the features characteristic for trisomy 22q (cleft lip and palate, preauricular papillomas and fistulas, rectal atresia or stenosis) and trisomy 11q (long philtrum with the upper lip hanging over, renal al; asia and hypoplasia). Diaphragmatic hernias are found to be common for the patients with +der(22)t(11; 22).


[Age and chromosome nondisjunction].

[Age and chromosome nondisjunction]. The parental age in 77 families of Down syndrome (DS) children with the known origin of extra chromosome 21 and in 12 families of DS children resulting from de novo translocation (more probable than not in 2 meiotic division) was studied. It was shown that when nondisjunction occurred in the 1st meiotic division, both in oogenesis (n = 30) and spermatogenesis (n = 12), mean parental ages and age distributions were different from that of control (400 couples with normal children). The mean age and age distribution were found to differ from control when nondisjunction occurred in the 2nd meiotic division of oogenesis (n = 19). On the basis of our information and the previously published data, lack of the effect of parental age on chromosome segregation in the Ist meiosis may be inferred. It is chromatid disjunction in the 2nd meiosis which is more probably age-dependent. The reasons preventing elucidation of real associations are under debate.


[Preparation and analysis of mutants of rhizospheric pseudomonads, resistant to toxic analogs of tryptophan and phenylalanine]. The absence of plasmids in strains of fluorescent pseudomonads characterized by high level of synthesis of phytohormone indole-3-acetic acid (IAA) as well as invariability of this feature in plasmid and non-plasmid variants of strain BSP8 suggests chromosomal control of IAA synthesis by the rhizosphere bacteria tested. Using toxic analogues of aromatic amino acids -5-fluorine-tryptophan and 5-methyl-tryptophan variants were obtained which synthesized and secreted only anthranilic acid. Mutants with resistance to p-fluorine-phenylalanine and capable of secreting tryptophan and/or phenylalanine were found. Testing of the secreting variants failed to reveal any differences between the levels of IAA biosynthesis in comparison with the wild-type strains.


[Characteristics of a positive regulatory element in the first intron of the mts1 gene]. The first intron of the mts1 gene, a gene which is selectively expressed in metastatic cells and in normal cells that are motile, was found to be highly homologous to the CD3 delta enhancer element. Because of the homology between the CD3 delta enhancer and the first intron of mts1, we analysed the first intron of the mts1 gene to determine whether it functions as a transcriptional regulatory element. Highly metastatic CSML-0 cells transfected with chloramphenicol acetyltransferase containing plasmids demonstrated the ability of the mts1 first intron to function as a positive regulatory element. In vitro footprinting analysis using extracts from CSML-0 cells (which express mts1 at low levels) of CSML-100 cells (which express mts1 at high levels) identified a protected 16 nucleotide element in the first intron of mts1, regardless of the extract used. However, in vivo footprinting analysis of the same region identified the protected 16 nucleotide fragment only in the mts1 intron from CSML-100 cells, not from CSML-0 cells. Differences in the methylation pattern of the mts1 gene in CSML-100 cells and CSML-0 cells are known to exist, and may in part be responsible for the mts1 footprinting differences observed in vivo from the different cells lines.


[Plasmid transduction using the D3112DeltaH miniphage in Pseudomonas aeruginosa cells]. Plasmid DNA transduction with mini-D3112 delta H, deletion derivative of phage D3112, which lost the genes essential for phage growth but retained the sites required for transposition and packaging was studied. Unlike D3112, mini-D3112 delta H element can transduce plasmids and plasmid markers at frequencies of 10(-5)-10(-8) in rec+ cells of Pseudomonas aeruginosa. Plasmids R1162 and R388 of the size smaller than phage genome were transduced intact. Large plasmids, like RP4 and R151, were deleted under transduction. By this way, we isolated deletion derivatives of RP4. The smallest derivative pN2 contained a 4.5 kb fragment of RP4. Unlike the latter, pN2 plasmid had narrow host range and did not maintain in Escherichia coli cells.


[Distributive pairing of chromosomes and aneuploidy in man]. The up-to-date state of human cytogenetics allows to turn back to hypothesis of distributive pairing as a strongly fruitful for resolving a number of problems concerning etiology of chromosome aneuploidy. Distributive pairing could account for such phenomena as: 1. Prevalence of nondisjunction (ND) in the first meiosis, compared with the second; 2. Excess of males among children with the Down syndrome; 3. Recurrent cases of aneuploidy, including aneusomies for different chromosomes; 4. Appearance of individuals with double aneusomies; 5. High recurrent risk for young parents; 6. Increased chance of ND for chromosomes not involved in rearrangement in carriers of balanced translocations; 7. Increased frequency of ND of autosomes in individuals with quantitative and structural sex chromosome anomalies; 8. The role of heterochromatic regions in ND; 9. Increased frequency of spontaneous abortions in couples having children with chromosome anomalies and in persons with unusual heterochromatic variants. The hypothesis could predict: 1. Essential contribution of errors in gonial cells to the origin of aneuploidy; 2. Important role of the factors influencing the prophase; 3. The possibility of offering forecast for sex chromosome anomalies rate on the basis of trisomy 21 rate, due to the fact that both autosomal and gonosomal aneuploidies have to be induced by the same factors.


[Change in the genetic structure of an experimental population of Drosophila by electrophoretic locus under the effect of ecological conditions]. Dynamics of the genetic structure of experimental Drosophila populations being kept over 50 generations at 25 and 17 degrees C6 and on the medium with ethanol (12%) was studied. In all experimental populations alteration in allele frequencies of Adh, Gpdh, Hex and Est-6 loci took place in the first 20-25 generations on monitoring under the influence of the temperature and ethanol. Later on, an equilibrium relation between allele frequencies will be established.


[New genes interacting with the zeste gene in Drosophila melanogaster]. We have found that mutations in the enhancer of yellow, 1,2 and 3 loci strongly enhance the effect of zv77h-mutation (full inactivation of the zeste locus) on the white locus expression. Their effect is realized through the distal white enhancer which is located 1,1 kb upstream to the promoter. It is suggested that the protein products of enhancers of yellow 1,2 and 3 represent a family of proteins which, like zeste protein, are responsible for formation of contacts between elements located at a large distance in the genome.


[[A dual system of superinstability at the yellow and scute loci in Drosophila melanogaster]. We have characterized the phenomenon of super-unstability in yellow and scute loci of Drosophila melanogaster. A few derivatives with different combinations of yellow and scute phenotypes appeared after dysgenic cross between potentially super-unstable stock y2nsscme and P[ry+, (delta 2-3)] (99B). Essentially, the double alterations of yellow and scute phenotypes constitute more than 40% of all derivatives. Most frequently the mutations in both yellow and scute loci change coordinately giving rise to the y+nssc+ allele. Lethal derivatives were not observed. The spectrum and the frequency of the y2nsscme mutagenesis in females differ considerably from the analogous in males. Possibly, the neutral homologous chromosome in females changes contacts between two insertions, and because of that mutagenesis changes too. Thus, all alterations in the double super-unstable system seem to be connected with the recombination between two unstable insertions A genetic exchange may by initiated by a double-strand breaks induced by the transposase of the ends of insertions.


Supernova (spno), a new maternal mutant producing variable-sized cleavage nuclei in Drosophila. This paper describes a new recessive maternal lethal which disrupts normal nuclear division and migration during cleavage in Drosophila. We have named this gene locus supernova. Deletion mapping and in situ hybridization have located this gene to 88 F9/89 A1 on the polytene chromosome map. The terminal mutant phenotype is characterized by the presence of many variable-sized nuclei scattered throughout the cytoplasm of the unhatched egg. Following fertilization, the initial cleavage divisions appear delayed and are often accompanied by the formation of ring-like association of chromosomes and/or chromosome bridges. Although the polymerization of tubulin into spindles occurs during the initial cleavage divisions, there appears to be both a spatial and temporal uncoupling of DNA replication from the formation and proper functioning of spindles. Eventually no functional spindles are formed, but nuclei continue to increase in size and number with increasing age of the embryo following fertilization.


Phenotypic plasticity for life-history traits in Drosophila melanogaster. III. Effect of the environment on genetic parameters. We estimated genetic and environmental variance components for developmental time and dry weight at eclosion in Drosophila melanogaster raised in ten different environments (all combinations of 22, 25 and 28 degrees C and 0.5, 1 and 4% yeast concentration, and 0.25% yeast at 25 degrees C). We used six homozygous lines derived from a natural population for complete diallel crosses in each environment. Additive genetic variances were consistently low for both traits (h2 around 10%). The additive genetic variance of developmental time was larger at lower yeast concentrations, but the heritability did not increase because other components were also larger. The additive genetic effects of the six parental lines changed ranks across environments, suggesting a mechanism for the maintenance of genetic variation in heterogenous environments. The variance due to non-directional dominance was small in most environments. However, there was directional dominance in the form of inbreeding depression for both traits. It was pronounced at high yeast levels and temperatures but disappeared when yeast or temperature were decreased. This meant that the heterozygous flies were more sensitive to environmental differences than homozygous flies. Because dominance effects are not heritable, this suggests that the evolution of plasticity can be constrained when dominance effects are important as a mechanism for plasticity.


Lambdoid phages as elements of bacterial genomes (integrase/phage21/Escherichia coli K-12/icd gene). The lambdoid phages are a group of related temperate bacteriophages that lysogenize by site-specific recombination with the bacterial chromosome. Various members of the group have different specific chromosomal insertion sites, despite the fact that the enzymes catalyzing the insertion (integrases) appear to be all descended from a common ancestor. Insertion sites are not located randomly on the E. coli chromosome but are restricted to one segment of the map; also, most prophages are oriented in the same direction along the chromosome. Lambdoid phage 21 inserts within the isocitrate dehydrogenase gene and introduces an alternative 165 bp 3' end for that gene. A defective element (e14) inserts at the same position. We suggest that this mode of insertion arose from insertion of an ancestral phage to the right of icd which then picked up part of the icd gene by abnormal excision and speculate that, at an earlier time, phages may have arrived at their present locations by a process of chromosomal walking.


The intracisternal A particle derived solo LTR promoter of the rat oncomodulin gene is not present in the mouse gene. The rat gene encoding oncomodulin, a small calcium-binding protein related to parvalbumin, is under the control of a solo long terminal repeat (LTR) derived from an endogenous intracisternal A-particle (IAP). This gene was the first example of a mammalian gene regulated in normal cells by a promoter of retroviral origin (see also article by D. Robins and L. Samuelson in this volume). We show here that the oncomodulin LTR is a member of a small subset of sequence related solo LTR elements present in the rat genome and that a full length IAP genome containing LTRs of this type is no longer present in the rat genome. We have assayed the transcriptional activity of the oncomodulin LTR coupled to the human growth hormone gene as a reporter. Transfections in both Hela cells and 293 cells indicate the oncomodulin LTR promoter is sufficient to efficiently initiate transcription. In 293 cells (human embryo kidney cells transformed with human adenovirus type 5 DNA), the oncomodulin LTR is a strong promoter, capable of bidirectional transcription. Finally, we have determined the structure and the sequence of the mouse oncomodulin gene. Our results suggest that the integration of the IAP particle genome within the rat oncomodulin gene occurred after the rat and the mouse became distinct species.


[Hygienic substantiation of maximum allowable concentration of isatrin in the soil]. Isatrin (bioresmethrin) maximum allowable concentration in soil at level 0.05 mg/kg based on translocation and sanitary and toxicologic standardization is offered. Mathematical model taking account of soil factors (humidity, temperature and type of soil) and initial concentration of isatrin has been derived.


[Hygienic standardization of methyl salicylate in the air of workplace]. A complex toxicological study of methyl salicylate was done. Threshold level at 8 mg/m3, and maximum allowable concentration of methyl salicylate at 1 mg/m3 (2-nd class of chemical hazard, with remark of'harmful in contact with skin') for the air of working zone were estimated. Maximum single and daily average allowable concentrations in ambient air at level 0.01-0.005 mg/m3 were calculated.


[Environmental pollution and congenital abnormalities according to the data of environmental health monitoring]. Study of the environmental pollution (ambient air, drinking water, food and fodder) in southern Ukraine industrial region and study of congenital developmental defects were carried out. 78,678 newborns were examined. The most significant environmental factors responsible for congenital developmental defects, as authors proposed, were the ambient air pollution and environmental radioactivity.


Vascular cells under peroxide induced oxidative stress: a balance study on in vitro peroxide handling by vascular endothelial and smooth muscle cells. Enzymes such as glutathione peroxidase and catalase play an important role in the cellular defence against (per)oxidative stress. Balance- and inhibitor-studies were undertaken with in vitro cultured human vascular endothelial cells (EC) and smooth muscle cells (SMC) to assay the relative importance of these enzymes in the handling of cumene hydroperoxide (Chp) and hydrogen peroxide (H2O2). Low concentrations of Chp (up to 80 microM) could be removed to near completion within the first hour of incubation by stimulation of the hexose monophosphate shunt (HMS) of both cell types. The HMS activity reached a plateau upon incubation with higher concentrations of Chp (> 80 microM). The non-converted Chp in the higher concentrations could be detected quantitatively in the incubation solution. After inhibition of the glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), incubation with Chp (40 microM) did not result in a stimulation of the HMS activity. Moreover the added Chp could be recovered from the medium. So Chp is exclusively handled by the GSH-redox cycle. When low concentrations of H2O2 (up to 80 microM) were added to EC or SMC approximately 50% of the peroxide loss could not be accounted for. Inhibitor studies with aminotriazole proved that catalase was responsible for the handling of this unaccounted H2O2. In both ECs and SMCs at lower concentrations of H2O2 the GSH-redox cycle was as effective as catalase and at higher H2O2 concentrations the catalase pathway plays the major role.


Application of methionine as a detector molecule for the assessment of oxygen radical generation by human neutrophils and endothelial cells. Diverse cell types can generate reactive oxygen species (ROS) which are implicated in many disease processes and are ascribed both beneficial and deleterious roles. In vitro studies of this phenomenon indicate that properties of the microenvironment in culture influence the cells' behaviour with regard to ROS generation in vivo. To date, however, the assessment of cellular ROS generation has been limited to techniques which are invasive of the culture environment, or require cells to be in suspension. This study describes the application of NMR spectroscopy to the detection of ROS generation, a technique which is non-invasive of the cell culturing environment.


Effects of high fat-, cholesterol-enriched diet on the antioxidant defence mechanisms in the rabbit heart. In 7 rabbits fed on hyperlipidic diet (0.5% cholesterol, 5% peanut oil and 5% lard) for 4 weeks, the ventricular myocardium was tested for antioxidant defences and thiobarbituric acid reactive substances. Seven age-matched rabbits served as controls. The hearts were previously subjected to 45 min Langendorff perfusion to study coronary flow, developed tension and resting tension; coronary effluent values of CPK activity, pH and UV absorbance at 250 nm (i.e., low molecular weight ATP catabolites) were also investigated. After 4 weeks of diet, a significant rise of plasma cholesterol (P < 0.0001) and triglycerides (P < 0.0001) was observed. Total superoxide dismutase, catalase and glutathione transferase activities underwent a significant increase (P < 0.05) in the hyperlipidemic animals. On the contrary, a depression of glutathione reductase (P < 0.01) and selenium-dependent glutathione peroxidase (P < 0.01) activities, associated with decreased levels of non proteic thiol compounds (P < 0.01), was assessed. The selenium-independent glutathione peroxidase activity was not detectable in both groups. Thiobarbituric acid reactive substances levels were significantly increased in the hyperlipidemic rabbit myocardium (P < 0.01). Even though heart hemodynamics, CPK release and perfusate pH did not differ in control and experimental animals, higher 250 nm absorbance values (P < 0.05) were detected in the myocardial effluent of hyperlipidemic rabbits. In conclusion, high fat-, cholesterol-enriched diet induces an imbalance in the rabbit heart antioxidant defences, some of which are increased, whereas others are depressed, eventually resulting in enhanced myocardial lipid peroxidation. These biochemical changes are associated with higher perfusate values of UV absorbance at 250 nm, but not with significant CPK leakage or myocardial hemodynamics derangement.


[Substantiation of maximum allowable concentration of p-chlorophenol in the ambient air of settlements]. A complex toxicological study of the p-chlorophenol was carried out. Single permissible p-chlorophenol MAC in the ambient air at level 0.015 mg/m3, and daily average one 0.003 mg/m3 were derived.


Molecular genetic changes in human epithelial ovarian malignancies. The frequent finding of loss of heterozygosity (LOH) for a specific chromosomal marker in tumor DNA compared to normal DNA suggests the presence of a closely linked tumor-suppressor gene. Using Southern blot analysis, 34 primary ovarian epithelial tumors were examined for the presence of tumor-specific allelic losses, using six probes for chromosomes 6q, 11p, 13q, 16q, and 17p. A high incidence of LOH was observed on 11p, 13q, and 17p. LOH for 17p was present in 3 of 4 (75%) informative benign ovarian tumors, 1 of 5 (20%) borderline tumors, and 16 of 24 (67%) invasive ovarian cancers. Allelic loss with the H-ras1 probe on 11p was present in 10 of 19 (53%) invasive tumors but was not identified in 6 benign or borderline tumors. LOH on 13q was present in 18 of 31 (58%) informative cases including 8 of 10 (80%) Stage 1 tumors. This preliminary study suggests that loss of tumor-suppressor genes on chromosomes 13q and 17p may be early events in ovarian tumorigenesis and that changes on chromosome 11p are later events.


Lymph node sampling in patients with epithelial ovarian carcinoma. Lymph node sampling is part of the FIGO staging of patients with ovarian carcinoma and is usually part of a meticulous second look operation. We analyzed the primary lymph node status of patients and compared this to the lymph node status at second look operation. From 3/86-3/91, 97 patients with epithelial ovarian tumors were treated at this institution. Seventy-one of the 97 patients (73.2%) had lymph node sampling at primary surgery. Thirty of the 71 patients had positive lymph nodes (42.2%) and 41 patients were lymph node negative (57.8%). Of the initial 97 patients, 58 were eligible for second look operation (59.8%), and 48 of these patients had lymph nodes sampled at second look operation. Nine of the 48 patients had positive lymph nodes (18.7%) and 39 had negative lymph nodes at second look operation (81.3%). Of the patients with negative lymph nodes at primary surgery, 25 patients had second look operation and 24 of these patients had lymph node sampling at second look operation. All patients with negative lymph nodes at primary surgery had negative lymph nodes at second look operation. Of the 30 patients with positive lymph nodes at primary surgery, 12 underwent second look operation. Four patients had persistent positive lymph nodes and 8 patients had negative lymph nodes. Our data suggest that patients with negative lymph nodes at primary surgery are unlikely to have positive lymph nodes at second look operation. Therefore, we believe that lymph node sampling under these circumstances is unnecessary.


Intraperitoneal P-32 is not an effective consolidation therapy after a negative second-look laparotomy for epithelial carcinoma of the ovary. Thirty-four patients with epithelial carcinoma of the ovary were entered into a trial of adjuvant intraperitoneal P-32 following induction chemotherapy and a negative second-look laparotomy. The breakdown by initial Stage was Stage IC, 5; Stage II, 3; Stage III optimal, 22; and Stage III suboptimal, 4. Previous treatment consisted of 4-12 cycles (median 6) of cisplatin or carboplatin-based combination chemotherapy. Fifteen millicuries of P-32 were instilled via a Tenckhoff catheter placed at the time of second-look laparotomy. Because of a 22% incidence of bowel injury in the first 23 patients, the P-32 dose was reduced to 12 mCi in the last 11 patients. To date, there have been no bowel injuries at the lower dose. Eighteen of the 34 (53%) patients have relapsed with a median time to relapse of 20 months and a median follow-up for all patients of 31 months. There has been no difference in the relapse rate between a dose of 12 and 15 mCi. Intraperitoneal P-32 does not appear to reduce the relapse rate following a negative second-look laparotomy. The incidence of bowel injury is dose dependent and is higher than that seen in patients treated as an adjuvant following initial surgery without subsequent chemotherapy or second-look laparotomy.


Long-term follow-up of serous ovarian tumors of low malignant potential. The biologic behavior of serous tumors of low malignant potential (LMP) is of significant interest, yet most large series lack extended follow-up. This study consists of 200 patients: 106 patients were diagnosed with serous tumors of LMP at our institution between 1979 and 1984 and an additional 94 patients were identified in the referred tumor registry. The patients ranged in age from 6 to 98 years (median, 34 years). The stage distribution was Stage I in 135 patients (67.5%), Stage II in 24 patients (12%), and Stage III in 41 patients (20.5%). Follow-up information from 4 to 27 years (median, 10 years; mean, 11.2 years) revealed 155 patients (77.5%) were alive without further evidence of disease and 11 patients (5.5%) died of unrelated conditions without recurrent tumor. Thirty-four patients (17%) developed recurrent neoplasms at 6 to 145 months (median, 26 months). Patients with Stage III disease developed recurrent neoplasms more commonly (54%) than did patients with Stage I or II disease (6 and 17%, respectively). Following treatment of recurrence, 15 patients are free of disease, 6 patients are alive with disease, and 13 (6.5% overall) patients have died of disease 1 to 15 years (median, 5 years) after their initial diagnosis. Mortality was also stage dependent: 0.7, 4.2, and 26.8% of patients with Stages I, II, and III disease, respectively, died secondary to tumors of LMP. Clinical life table analysis demonstrated 5-, 10-, and 15-, and 20-year survival rates for all stages of 97, 95, 92, and 89%, respectively. These findings confirm the excellent prognosis for patients with serous tumors of LMP, even when long-term follow-up is extended to 20 years. Additionally, these data suggest that those with more advanced or recurrent disease can enjoy extended survival.


The influence of cytoreductive surgery on recurrence-free interval and survival in small-volume stage III epithelial ovarian cancer: a Gynecologic Oncology Group study. Gynecologic Oncology Group Protocol 52, a randomized trial of cisplatin and cyclophosphamide with or without doxorubicin in "optimal" Stage III epithelial ovarian cancer, failed to demonstrate a significant difference in the outcome in 349 evaluable patients. Additional review of the records was carried out to determine the influence of cytoreductive surgery on survival. Since eligibility for the study was the presence of residual cancer of 1 cm or less, the influence of cytoreductive surgery could be evaluated by comparing outcome in patients presenting with large-volume extrapelvic disease, but who were cytoreduced to small-volume disease. Factors evaluated were age, cell type, grade, size, and location of disease at exploration, size, and location of residual disease after cytoreduction, number of residual nodules, ascites, type of surgery, blood loss, and hospital days. Univariate analysis revealed that age, size of residual disease, mucinous or clear cell histologic type, histologic grade, and number of residual lesions were significant prognostic factors. By univariate analysis patients found to have extrapelvic disease of 1 cm or less had a better recurrence-free interval and survival than those patients with large-volume disease who were cytoreduced to disease of 1 cm or less. Multivariate analysis revealed that older age, histologic grades 2 and 3, and 20 or more residual lesions were unfavorable. The volume of initial extrapelvic disease remained significant when gross disease was present in the omentum and in other extrapelvic sites. This study failed to prove the hypothesis that initial cytoreductive surgery would allow a patient presenting with large-volume ovarian cancer to have the same chance for survival as a patient found to have small-volume disease. Factors other than cytoreductive surgery are important in predicting survival.


Endometrial adenocarcinoma: genetic analysis suggesting heritable site-specific uterine cancer. Genetic factors are clearly integral to the etiology of neoplasia. A cancer family syndrome (Lynch syndrome II) consisting of uterine, colon, and ovarian cancer is recognized, but the heritability of isolated endometrial adenocarcinoma has not otherwise been thoroughly investigated. We have performed pedigree studies in index cases with endometrial adenocarcinoma, using spouses as controls. Preliminary results from 64 probands showed four families in which endometrial adenocarcinoma was diagnosed in at least one first-degree relative of the proband (mother, daughter, sister); none showed relatives with colon or ovarian cancer. In none of the 34 control pedigrees did either a mother or sister have endometrial adenocarcinoma. In four other families, multiple first- and second-degree relatives of probands had adenocarcinoma of the uterus, colon, or ovary, presumably representing a cancer family syndrome (Lynch syndrome II). Our preliminary data not only show familial and probably heritable tendencies for endometrial adenocarcinoma, but further suggest that there are at least two distinct forms: (1) the previously described Lynch syndrome II (cancer family syndrome), and (2) a heretofore unemphasized entity characterized by a tendency to endometrial adenocarcinoma alone.


Chemosensitivity to the new anthracycline pirarubicin and other chemotherapeutic agents in primary and recurrent ovarian tumors in vitro. We analysed the in vitro sensitivity of 50 fresh gynecologic cancer specimens to the new anthracycline-analogue pirarubicin (Pira). Nine endometrial and cervical tumors were excluded from the evaluation in this study. The in vitro sensitivities of 41 ovarian cancer specimens to Pira were evaluated with the adenosine triphosphate chemosensitivity assay (ATP-CSA). The results were compared to the sensitivity of drugs used in gynecologic oncology: Adriamycin, cisplatin, and the metabolite of cyclophosphamide, 4-hydroxycyclophosphamide (4-HC). Sensitivity (S) is defined as > or = 70%, partial sensitivity as 50-69% ATP decrease compared to controls at 20% of the peak plasma concentration. In vitro response is defined as S + PS. Twenty-two primary ovarian tumors were assayed, with 77% response to Pira, 12% to Adriamycin, 29% to cisplatin, and 38% to 4-HC. In 19 recurrent ovarian tumors, Pira showed 53% response; Adriamycin, 25%; cisplatin, 8%; and 4-HC, 67%. The in vitro data are partly consistent with reported data from the literature. Pira reveals a significantly higher degree of cytotoxicity compared to the three drugs listed above. The IC50 of Pira is less than 20% of the peak plasma concentration achievable in patients and is significantly lower compared with the IC50s of other drugs. We conclude from our in vitro data that Pira is more active than Adriamycin, cisplatin, and 4-HC.


Comparison of U-73,975 and cisplatin cytotoxicity in fresh cervical and ovarian carcinoma specimens with the ATP-chemosensitivity assay. U-73,975 (U-73), a closely related synthetic analogue of the antitumor agent CC-1065, acts by binding tightly in the minor groove of DNA. A comparison was made between the cytotoxicity of U-73 and cisplatin (DDP) on 11 fresh cervical and 7 fresh ovarian carcinoma specimens. The ATP-chemosensitivity assay as previously described (Sevin et al. Gynecol. Oncol. 31, 191-204, 1988) was used to determine the cytotoxic effect of U-73 and DDP. IC 50s were calculated using regression analysis. The mean IC 50s for U-73 and DDP were 519 pg/ml and 2918 ng/ml, respectively, for the cervical carcinoma specimens and 324 pg/ml and 2649 ng/ml, respectively, for the ovarian carcinoma specimens. Significance comparing U-73 and DDP for cervical and ovarian tissue was demonstrated with P < 0.001. U-73 was 4000 times as cytotoxic per unit of mass as DDP on cervical carcinoma compared to over 8000 times for ovarian carcinoma. Based on these in vitro data, U-73 appears to be a very promising antitumor agent for cervical and ovarian carcinoma.


Effects of radiation on TNF alpha-mediated cytolysis of cell lines derived from cervical carcinomas. The effect of radiation, a primary mode of treatment for cervical malignancies, on the tumor necrosis alpha (TNF alpha)-mediated cytolysis of five cell lines derived from human cervical carcinoma cell lines (C-33 A, ME-180, HT-3, MS751, and SiHa) was analyzed. Results of this analysis showed that all of the cell lines were resistant to the cytolytic effects of TNF alpha. Although resistant when protein synthesis proceeds normally, ME-180, HT-3, MS751, and SiHa cells were sensitive to TNF alpha-mediated cytolysis in the presence of protein synthesis inhibitors. The cytolytic response of these cells to radiation was heterogeneous, with C-33 A cells being the most radiosensitive and SiHa cells being the least radiosensitive. The cell lines ME-180, MS751, and HT-3 were intermediate in their sensitivities to radiation. Because radiation is known to inhibit protein synthesis, the ability of radiation to enhance TNF alpha cytolytic activity was examined. The cell lines with intermediate sensitivities to radiation (ME-180, HT-3, and MS751) demonstrated statistically significant synergistic increases in cytolysis when exposed to TNF alpha in combination with radiation. Neither the radioresistant SiHa cell line nor the radiosensitive C-33 A cell line displayed increased cytolysis with increasing concentrations of TNF alpha at any dose of radiation. Possible mechanisms which may explain the synergy in ME-180, HT-3, and MS751 cells and lack of synergy in C-33 A and SiHa cells by TNF alpha and radiation are discussed.


The impact of subspecialty training on the management of advanced ovarian cancer. A retrospective study was conducted to determine the influence of subspecialty training in gynecologic oncology as well as several other covariates on the feasibility, operative mortality, and survival benefits of cytoreductive surgery for 263 patients with stages IIIC and IVA epithelial ovarian cancer. Covariates most predictive of an optimal (< or = 1 cm) cytoreductive outcome were the diameter of the largest metastases before cytoreduction (< or = 10 cm vs > 10 cm, P < 0.001) and the specialty training of the physicians present at surgery (gynecologic oncologists vs other, P < 0.001). Age influenced operative mortality most (< 60 vs > or = 60, P < 0.001). Covariates found to most significantly influence survival time include the specialty training of the physicians present at surgery (gynecologic oncologists vs other, P < 0.0001), cytoreductive outcome (complete vs optimal, P = 0.001, optimal vs suboptimal, P < 0.0001), grade of tumor (grade 1 vs grades 2 and 3, P = 0.01), and pelvic disease status (frozen pelvis vs mobile primary tumor, P = 0.03). We conclude that patients with advanced epithelial ovarian cancer should undergo aggressive cytoreductive surgery by gynecologic oncologists, with the objective to remove all macroscopic disease. Subsequent treatment with platinum-based chemotherapy offers the best chance for long-term survival or cure.


Proposal of a modified (FIGO 1985) classification of stages I and II cancer of the uterine cervix. A modified FIGO (1985) classification of cancer of the uterine cervix has been suggested. The changes refer to stages I and II of the disease. The already accepted system of cervical cancer staging has been used. Stages Ib, IIa, and IIb have been subdivided, similar to stage Ia. The criterion used in staging is the size of the tumor up to, or more than 2 cm (Ib 1 and Ib 2) and, for stage II, the extent of penetration into adjacent tissue. The condition of lymph nodes should be noted by LN(+) or LN(-) along with the stage of the disease. Such staging should demonstrate an objective evaluation of treatment results obtained by different methods and their combinations.