Hematopoietic growth factors.

Recent advances in our understanding of the hemolymphopoietic growth factors has revolutionized knowledge of blood cell development, the immune system, and of tumor cell biology. However, the rapid translation of these insights from basic research to the clinic has been perhaps the most dramatic part of the story. Commercially available erythropoietin has become established for the treatment of the anemia of end-stage renal disease, and promises to be of value in the supportive care of patients with cancer and perhaps other chronic diseases. It likely will be increasingly utilized for enhancing autologous blood donation and for perioperative management. Both GM-CSF and G-CSF only recently released by the FDA for specific clinical indications, though there are a variety of potential applications (Table 12). It is clear that G-CSF is the therapy of choice for most neutropenias and that both agents have effects in diminishing the myelotoxicity and mucositis seen after aggressive chemoradiotherapy. However, it is important to note that as yet there is no evidence that the use of either G-CSF or GM-CSF has resulted in increased cure rates or, in fact, increased survival in patients with various malignancies. It would appear that both G-CSF and GM-CSF will, in fact, allow dose escalation and/or diminished toxicity of various chemotherapeutic regimens. However, there are important considerations in the overall place of these cytokines with regard to treatment of human disease. A major goal in the therapy of patients with malignancy is obviously prolongation of life and cure. If, in fact, escalation of doses of chemotherapeutic agents does not result in increased tumor responses or cures then the use of these growth factors will have a relatively trivial impact on the care of cancer patients. In addition, the disturbing observations of receptors for these growth factors on various tumor cell lines and of varying degrees of in vitro tumor cell proliferative responses raises the possibility that in some situations they may actually stimulate tumor growth. This is an unknown which has not been adequately evaluated in any clinical study to date and which may vary from tumor to tumor. For example, if these cytokines increase tumor growth rate by 20-30% (an effect which would probably not be detected in the clinical studies to date) while allowing an escalation of chemotherapy doses it is possible that there would be no significant beneficial effect.(ABSTRACT TRUNCATED AT 400 WORDS)


T-cell activation-associated antigen expression by neoplastic T-cells.

In this study, a clinicopathologically and immunophenotypically diverse group of T-cell neoplasms were evaluated by one- and two-color flow cytometry and/or immunohistochemistry for the presence of eight antigens (T10, T9, IL2-R, EMA, HLA-DR, LeuM1, Ki-1, and LeuM5) which are expressed in a hierarchical manner by phytohemagglutinin (PHA)-activated benign T cells. We found that 70 of the 72 T-cell neoplasms (97%) expressed at least one of these eight T-cell activation-associated antigens (T-AAgs) and that the number and type of T-AAgs expressed by the neoplastic T cells varied according to the clinicopathologic category of T-cell neoplasia. All 5 T-cell lymphoblastic malignancies expressed T10 and T9; 2 also expressed LeuM1. Twelve of 14 (86%) T cell chronic lymphocytic leukemias (T-CLL) expressed two to four T-AAgs, most frequently T10 (86%) and HLA-DR (79%). The 26 cutaneous T-cell lymphomas (CTCL) expressed between 2 and 5 T-AAgs, most commonly T9 (92%) and HLA-DR (92%), and least often T10 (12%) and EMA (15%). Twenty-six of 27 (96%) peripheral T-cell lymphomas (PTCL) expressed more than 4 T-AAgs. Each of the T-AAgs were expressed by between 22% (LeuM5) and 85% (T9) of the PTCLs. Some T-AAgs were preferentially expressed by the PTCLs in association with other T-AAgs, such as EMA in association with IL2-R and Ki-1. In addition, LeuM5 was preferentially expressed by CD4- CD8+ T-cell neoplasms. However, only 19 of the 72 (26%) T-cell neoplasms (3/5 lymphoblastic malignancies, 3/14 CLLs, 0/26 CTCLs, 13/27 PTCLs) expressed T-AAg immunophenotypic profiles paralleling those expressed by normal peripheral blood T cells activated in vitro with PHA. These results suggest that T-AAg expression by neoplastic T cells does not often mirror the hierarchical order of expression by activated benign T cells, implying that neoplastic T cells do not usually represent the precise malignant counterpart of activated benign, normal T cells.


In vitro study of erythropoiesis in patients with aplastic anemia and myelodysplastic syndromes: a possible tool for prospective determination of the clinical effectiveness of growth factors.

Effects of recombinant human erythropoietin (rhEpo) and the combination of recombinant human interleukin-3 (rhIL-3) or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) with rhEpo on erythroid colony formation were examined in vitro in 13 patients with aplastic anemia and 16 with myelodysplastic syndromes (MDS). The methylcellulose cultures of marrow cells from normals and the patients yielded no erythroid colonies in the absence of rhEpo. In normals, CFU-E and BFU-E colony formation was significantly increased by adding either rhIL-3 or rhGM-CSF with rhEpo, compared with rhEpo alone, and rhIL-3 was more potent than rhGM-CSF to form colony-forming units and burst-forming units of erythroid (CFU-E) (BFU-E) colonies. By adding rhIL-3 with rhEpo, CFU-E colony formation was increased in half of patients with RA, compared with rhEpo alone, and by rhGM-CSF, in one third. Approximately one third or one fourth of the patients with MDS showed increased BFU-E colonies when rhIL-3 or rhGM-CSF were added to rhEpo. Cultures containing rhIL-3 or rhGM-CSF with rhEpo yielded larger numbers of BFU-E colonies in half of the patients with nonsevere aplastic anemia than those containing rhEpo alone. These observations suggest that the combination of these growth factors, especially rhIL-3 with rhEpo, is applicable to the treatment of anemia in some patients with aplastic anemia and MDS.


Primary splenic presentation of plasma cell dyscrasia: report of two cases.

Primary splenic presentation of plasma cell tumors is extremely rare. Recently we observed two female patients with primary (initially solitary) plasmacytoma of the spleen. While the pathoanatomical diagnosis of plasmacytoma could be established easily, the clinical picture in both cases was puzzling and allowed no definitive diagnosis to be made. One of the patients exhibited a long-standing monoclonal gammopathy. Repeated bone marrow examinations in both patients revealed slight increase in plasma cells (between 5 and 10% of all nucleated cells), but no infiltrates of multiple myeloma. The leading clinical feature in both cases was pronounced splenomegaly (780 g and 1600 g). Histologically both spleens exhibited marked infiltration by pleomorphic plasma cells, with monotypic expression of IgG kappa in one case and of the light chain lambda in the other. A broad panel of monoclonal antibodies detecting various hemopoietic and nonhemopoietic antigens was used to determine the immunophenotype of the neoplastic plasma cells, but in both cases they reacted only with a minority of the antibodies applied. The bone marrow in both cases remained free of tumorous infiltrates, but the disease progressed a few months after splenectomy with infiltration of the liver in one case and of lymph nodes in the other. To summarize, these two cases are definitely not multiple myelomas but could represent a distinct entity among the plasma cell dyscrasias for which the preliminary term "disseminated plasmacytoma with primary splenic presentation" is proposed.


Trisomy 4: clinical picture, hematology, and survival. Presentation of two cases and review of the literature.

Two patients with acute nonlymphocytic leukemia (ANLL) and trisomy 4 as the only cytogenetic aberration are presented. Including the two, a total of 31 cases with this karyotype have been described till now. A review of the 31 cases shows that they fall into two distinct age groups, a younger group of 10 patients (5-34 years) and an older group of 21 patients (50-75 years). With four exceptions the diagnosis was ANLL in all cases, mostly FAB classes M2 and M4. At the time of diagnosis 18 had clinical signs of active infection. In 20 cases the peripheral leukocyte count was above 10 x 10(9)/L but not associated with presence/absence of infection. The median survival was 17 months from diagnosis. Independent prognostic factors were diagnosis (p = 0.01), peripheral leukocyte count (p = 0.03), and percentage of metaphases with trisomy 4 (p = 0.04). The two age groups as well as presence/absence of infection at the time of diagnosis were without significant prognostic consequences.


Predicting individual differences in complex skill acquisition: dynamics of ability determinants.

Substantial controversy exists about ability determinants of individual differences in performance during and subsequent to skill acquisition. This investigation addresses the controversy. An information-processing examination of ability-performance relations during complex task acquisition is described. Included are ability testing (including general, reasoning, spatial, perceptual speed, and perceptual/psychomotor abilities) and skill acquisition over practice on the terminal radar approach controller simulation. Results validate and extend Ackerman's (1988) theory of cognitive ability determinants of individual differences in skill acquisition. Benefits of ability component and task component analyses over global analyses of ability-skill relations are demonstrated. Implications are discussed for selection instruments to predict air traffic controller success and for other tasks with inconsistent information-processing demands.


The meaning of occupational stress items to survey respondents.

This study tested the effect of using the word stress in the measurement of self-reported occupational stressors and strains. Employees from two organizations responded to a questionnaire that included specific occupational stressors, strains, and 16 items in which the word stress was used. Survey respondents tended to interpret the word stress to refer both to employees' strains or reactions to the work environment and to job stressors or elements of the environment itself. Implications of these findings for occupational stress research are discussed.


The effects of goal difficulty on physiological arousal, cognition, and task performance.

Two laboratory experiments with 117 undergraduate students were conducted to examine (a) the effect of assigned goal difficulty on arousal (self-report and heart rate), cognition (perceived norm, self-efficacy strength, and personal goal), and behavioral (task performance) measures and (b) the role of heart rate as a mediator of the goal-difficulty-performance relation. All Ss performed a task requiring cognitive and physical responses. Results of both experiments demonstrated that assigned goal difficulty affected heart rate, cognition, and task performance and that heart-rate change was positively related to the cognitive and behavioral measures. Regression analyses suggested that a cognitive-affective mechanism may mediate the goal-difficulty-performance relation. Discussion is focused on the theoretical and practical implications of integrating an arousal concept within goal-setting theory.


Detection measures in real-life criminal guilty knowledge tests.

The present study provides a first attempt to compare the validity of the respiration line length (RLL) and skin resistance response (SRR) amplitude in real-life criminal guilty knowledge tests (GKTs). GKT records of 40 innocent and 40 guilty Ss, for whom actual truth was established by confession, were assessed for their accuracy. When a predefined decision rule was used and inconclusive decisions were excluded, 97.4% of the innocent Ss and 53.3% of the guilty Ss were correctly classified with the SRR measure. For the RLL measure, the respective results were 97.2% and 53.1%. The combination of both measures improved detection of guilty Ss to 75.8% and decreased detection of innocent Ss to 94.1%. The combined measure seems to be a more useful means of identifying guilty suspects than each physiological measure alone. The results elaborate and extend those obtained in a previous field study conducted by Elaad (1990).


Subliminal self-help audiotapes: a search for placebo effects.

Subliminal self-help audiotapes to aid weight loss were evaluated to determine if their apparent effectiveness is due to a placebo effect. All subjects were female students or staff who were both overweight and believed in the possible effectiveness of subliminal audiotapes. Three different groups of subjects were tested: One group listened to subliminal audiotapes purchased directly from a manufacturer, the 2nd group listened to comparable placebo tapes, and the 3rd group did not listen to any tapes at all. Each subject in each group was weighed once each week for 5 weeks. All 3 groups of subjects lost weight, and the average amount of weight lost by each group was approximately equivalent. These results provide no evidence that regular use of subliminal audiotapes leads to a placebo effect. Rather, the results suggest that regular use of subliminal audiotapes may simply make subjects more conscious of their weight.


Isolation and long-term maintenance of differentiated adult chicken hepatocytes in primary culture.

Adult chicken hepatocytes were obtained by an adaptation of the two step in situ collagenase perfusion. Usually 0.5 to 1 x 10(9) cells were obtained, with 75 to 95% viability. Hepatocytes attached within 2 h when plated on plastic cell culture dishes and spread in 4 h, surviving for several months in a specific serum-free medium. These cells retained a typical parenchymal cell morphology and the ability to produce a specific protein (albumin) throughout the culture period. We hereby provide a suitable model for studying hepatic metabolism in birds.


Nerve growth factor binding sites on hepatic parenchymal cells.

Nerve growth factor (NGF) binding sites on rat hepatocytes (HCs) in culture for 24 to 48 h were characterized using 125I-NGF. Specific binding of 125I-NGF to HCs was saturable. Scatchard analysis indicated a single population of binding sites with a Kd of 5.5 nM and a Bmax of 540 fmol/mg protein. In isolated hepatocyte membranes, specific binding of 125I-NGF was also apparent with Kd and Bmax values of 10.8 nM and 3740 fmol/mg protein, respectively. Specific binding of 125I-NGF to HCs was displaced by excess, unlabeled NGF but not by up to 1000-fold excess of either insulin or epidermal growth factor. Internalization/sequestration of 125I-NGF into HCs was measured as radioactivity present in solubilized cells after exposure to high salt and acid. These studies indicated 83 +/- 11% of 125I-NGF was accumulated by internalization/sequestration at a concentration of 1 nM 125I-NGF. Internalization was reduced to 43 +/- 4% when incubations were carried out at 4 degrees C. These results indicate the presence of a specific, low-affinity binding site for NGF on hepatocytes in culture.


Optimization of estrogen growth response in MCF-7 cells.

The factors involved in estradiol-17 beta induced growth stimulation of MCF-7 human breast cancer cells have been examined. Wild type MCF-7 cells (and clone E3) were shown to undergo slow growth in phenol-red-free medium containing specific calf sera. The E3 clone was used to document a mean 6-day growth stimulation of 3.35-fold (doubling time = 33 +/- 3 h) in cultures supplemented with 10(-11) M estradiol-17 beta. The serum batch utilized in the culture medium is most important in acquiring significant growth stimulation of MCF-7 cells by estradiol-17 beta. Regardless of the absence of phenol-red, only selected sera (2 out of 14 tested) supported minimal growth of MCF-7 cells in the absence of added estradiol 17 beta (doubling time = 55 +/- 11 h). When a calf-serum-supplemented culture failed to display a complete growth response to estradiol-17 beta, it was due to the rapid growth of the cells in the control (minus estradiol-17 beta) flasks. Sera that promoted shorter doubling times for MCF-7 cells cultured in the absence of estradiol-17 beta were rendered less supportive of growth if treated with dextran-coated charcoal or when cultures were supplemented with the estrogen antagonist ICI 164,384 (10(-7) M). Pooled extracts of these sera were shown to contain stimulatory levels of estradiol-17 beta. Dextran-coated charcoal treatment of sera removed or deactivated factors (other than estradiol-17 beta) which were not only required for the growth of MCF-7 cells, but were necessary for estrogen-stimulated growth. Varying the serum-containing medium, buffer, and nutrient mix or the addition of insulin has no effect on the growth response of these cells to estradiol-17 beta. These investigations document the culture conditions required to produce a maximal and consistent proliferative effect of E2 on MCF-7 cells without exposing the serum constituent to damaging chemical or absorbent agents.


Hormonal induction of functional differentiation and mammary-derived growth inhibitor expression in cultured mouse mammary gland explants.

A method for the cultivation of organ explants from abdominal mammary glands of virgin mice has been established. In a serum-free medium containing aldosterone, prolactin, insulin, and cortisol (APIH medium) mammary gland development was documented by lobuloalveolar morphogenesis. The hormonal requirements for in vitro expression of beta-casein and of the mammary-derived growth inhibitor (MDGI) were tested. To this end, a full length cDNA coding for mouse MDGI was prepared displaying strong homologies to a mouse heart fatty acid binding protein, which is also expressed in the mammary gland. MDGI and beta-casein transcripts were found to be absent in the mammary tissue from primed virgin mice, and were induced upon culture of mammary explants in the APIH medium. An immunohistochemical analysis with specific antibodies against MDGI and casein revealed a different pattern of expression for the two proteins. In the APIH medium, MDGI was expressed mainly in differentiating alveolar cells of the lobuloalveolar structures, whereas beta-casein was present in both ductules and alveoli. The relationship between functional differentiation and MDGI expression was further studied in explants from glands of late-pregnant mice. At this stage of development, MDGI is found both in ducts and in alveoli. If explants were cultured with epidermal growth factor (EGF) and insulin, the lobuloalveolar structure was still present, whereas MDGI disappeared. Reinduction of MDGI expression was achieved by subsequent PIH treatment. Independent on developmental stage, EGF strongly inhibits MDGI mRNA expression. It is concluded that MDGI-expression is associated with functional differentiation in the normal gland.


Two unique human leukemic T-cell lines endowed with a stable cytotoxic function and a different spectrum of target reactivity analysis and modulation of their lytic mechanisms.

We have reported the establishment of two interleukin (IL)-2-dependent human leukemic cell lines (TALL-103/2 [CD3+TCR gamma delta +] and TALL-104 [CD3+ TCR alpha beta +]) which display major histocompatibility complex nonrestricted tumoricidal activity. Whereas TALL-103/2 cells lyse only natural killer cell-susceptible targets, TALL-104 cells display a broad range of tumor target reactivity. In reverse antibody-dependent cell-mediated cytotoxicity (ADCC), lysis by both cell lines is triggered by monoclonal antibodies (mAb) recognizing CD3 and, to a lesser extent, CD2, but not CD8 or CD56 antigens. In conventional cytotoxic assays, the lytic activity of both cell lines is strictly Ca(2+)-dependent. In reverse ADCC, lysis by TALL-103/2 cells is highly dependent on the presence of Ca2+, whereas TALL-104 cells seem to only partially require extracellular Ca2+. The cytoplasm of both cell lines contains azurophilic granules typical of cytotoxic cells. Northern blot analysis demonstrates mRNA expression of pore-forming protein (PFP; perforin) and serine esterases (SE). The magnitude of expression of these transcripts and of lytic activity depends on the doses of IL-2. Upon deprivation of IL-2, TALL-103/2 cells completely lose cytotoxic granules and function within 16 h, whereas TALL-104 cells progressively lose expression of PFP and SE mRNA, as well as killer activity, within 4 wk. Both anti-CD3 mAb and lysable target cells induce efficient BLT-esterase secretion from TALL-103/2 and TALL-104 cells analogous to findings with conventional cytotoxic T lymphocytes. The stable expression of tumoricidal activity over 2 yr in culture renders these cell lines unique and very useful for studies on the regulation of cell-mediated lysis in vitro and in animal models.


RB tumor suppressor gene expression responds to DNA synthesis inhibitors.

Expression of the RB tumor suppressor gene, whose function is putatively in controlling cell growth, may be regulated by S-phase specific inhibitors of DNA synthesis that are commonly used in cell synchronization and cancer chemotherapy. Relatively low concentrations of the agents, cytosine arabinoside, bromodeoxyuridine, 5-fluorouracil, hydroxyurea, methotrexate and retinoic acid, were tested. At low concentrations still permitting submaximal cell growth, these drugs all changed RB gene expression, causing either up or down regulation of RB expression to varying degrees. Despite their potential similarity as a class, the nucleotide analogues elicited differential effects. The drug-induced up or down regulation of RB expression did not correlate with changes in c-myc expression indicating that the changes are not a manifestation of general metabolic changes potentially associated with altered proliferation. Amongst the agents considered, retinoic acid was the only one that caused a significant parallel reduction in RB and c-myc expression in the HL-60 human promyelocytic leukemia cells tested. The results thus show that even low concentrations of DNA synthesis inhibitors can have unpredictable affects on expression of growth regulatory genes.


Antioxidant enzymes in the differentiated Caco-2 cell line.

Injury to the gastrointestinal tract by oxygen dependent processes is important in ischemia, inflammatory bowel disease, and necrotizing enterocolitis. The Caco-2 cell line is an important tool in assessing various gastrointestinal functions and offers a unique opportunity to assess gastrointestinal oxidant metabolism on a cellular level. However, some Caco-2 cell functions change with time after confluence. To determine if antioxidant enzyme activity changes during differentiation, Caco-2 cells were grown to confluence, and superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase activities and specific mRNA content were quantitated. With time after confluence the enzymes demonstrated a small, but statistically significant increase in activity. Neither superoxide dismutase nor glutathione peroxidase mRNA levels correlated with enzyme activity changes. Catalase mRNA levels increased as catalase activity increased. Thus, differentiated Caco-2 cells express superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase activities and the superoxide dismutase, glutathione peroxidase, and catalase genes. Superoxide dismutase activity and glutathione peroxidase activity do not correlate with mRNA levels, and suggest that regulation may be at a level other than transcription. The correlation between catalase activity and catalase mRNA suggests differentiation may occur at transcription. If Caco-2 cells are used to elucidate oxidative metabolism, changes in activities of antioxidant enzymes as a function of cell differentiation should be considered.


A simplified method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids.

A simple and highly reproducible method was established for the culturing of adult rat and human hepatocytes as multicellular aggregates (spheroids). Purified rat and human liver parenchymal cells were cultured on nontissue culture (bacteriological) polystyrene petri dishes on a rotating platform. After an overnight incubation, the cells were found to form multicellular aggregates. The aggregates became spheroidal in shape after several days in culture. Histological sections of the spheroids showed an organized structure consisting of squamated cells on the outermost layer and cuboidal cells in the interior. Cellular structures characteristic of hepatocytes in the liver in vivo including bile canaliculi, peroxisomes, Golgi bodies, abundant mitochondria, and rough and smooth endoplasmic reticulum were observed with electron microscopy. The spheroids were found to be viable up to the longest time studied of approx. 1 month in culture as demonstrated by their adherence and growth on collagen-coated substratum. The morphological resemblance between hepatocytes cultured as spheroids and the liver in vivo suggests that the spheroids may be a useful in vitro experimental model of the liver. Our simple method should allow hepatocytes to be cultured as spheroids easily in any laboratory equipped for cell culture. Our study here also is the first to report the culturing of human hepatocytes as spheroids.


Inducible expression of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and interferon-gamma in two human cytotoxic leukemic T-cell lines.

We investigated the ability of the TALL-103/2 and TALL-104 leukemic cell lines to produce lymphokines in response to activation signals, such as tumor cells and anti-CD3 (OKT3) or -CD2 (B67.1) monoclonal antibodies (mAb) or both. Both cell lines were found to produce high levels of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF). The latter lymphokine is induced by lysable tumor cells and by immobilized OKT3 and B67.1 mAb only in the presence of interleukin (IL-2). IFN-gamma and TNF-alpha are induced upon CD3 but not CD2 stimulation, both in the presence and absence of IL-2. Interestingly, the B67.1 mAb amplifies the OKT3-induced responses by 2- to 10-fold, bringing the IFN-gamma and TNF-alpha levels of production up to 200 U/ml. Thus, simultaneous triggering of the CD2 and CD3 signaling pathways results in a very efficient lymphokine release. Of all the tumor cell lines tested as inducers, only K562 cells are able to stimulate the production of IFN-gamma and TNF-alpha in TALL-103/2 and TALL-104 cells, especially upon culture in IL-2. Lymphokine mRNA expression after stimulation with mAb or K562 cells peaks at 2 h in both cell lines. No messages are detectable in TALL-103/2 cells at 8 h, whereas in TALL-104 cells, IFN-gamma and GM-CSF transcripts are still present at 8 and 20 h, respectively. The inducible and highly regulatable expression of lymphokine release by these cell lines provides a unique model for studying mechanisms of lymphokine induction by different biological agents.


Phospholipids regulate growth and function of MDCK cells in hormonally defined serum free medium.

The effects of the simple phospholipids phosphatidic acid (PA) and lysophosphatidic acid (LPA) on the growth and function of Madin Darby Canine Kidney (MDCK) cells has been studied. We observed that PA and LPA not only stimulated the growth of MDCK cells (at 20 microM), but also stimulated the growth of normal rabbit kidney cells in serum free medium (albeit at a lower dosage of 5 microM). Evidence was obtained that PA interacts synergistically with insulin so as to elicit a growth stimulatory effect. Recently, extracellular PA and LPA were proposed to stimulate mitogenesis in several types of animal cells by binding to particular sites on the plasma membrane which are coupled to signaling mechanisms such as adenylate cyclase via a pertussis toxin sensitive, inhibitory guanosine triphosphate binding protein (Gi protein) (15). However, even when the pertussis toxin dosage was increased to 50 ng/ml, LPA still had a dramatic growth stimulatory effect on MDCK cells. In the absence of LPA pertussis toxin was slightly growth stimulatory to MDCK cells. Phospholipids such as PA and LPA have been observed to prevent prostaglandin-induced increases in adenylate cyclase activity in other cell types via their effects on such a pertussis toxin sensitive Gi protein. If PA and LPA act on MDCK cells in this manner, then these phospholipids may possibly prevent the effect of PGE1 on the growth of normal MDCK cells. However PGE1 was still growth stimulatory to normal MDCK cells. The effects of PA on PGE1 independent variants of MDCK cells, which have elevated intracellular cyclic AMP levels (22), were also examined. In the presence of PA, PGE1 remained growth inhibitory, rather than growth stimulatory to the PGE1 independent cells. However, the PA dosage required to elicit an optimal growth response (5 microM) was dramatically reduced, as compared with normal MDCK cells (20 microM). This altered dosage requirement could be explained by the elevated intracellular cyclic AMP levels in the PGE1 independent variants. Like PGE1 and 8-bromocyclic AMP, PA and LPA also significantly increased the initial rate of Rb+ uptake by confluent monolayers of MDCK cells. The increase in the initial rate of Rb+ uptake could be explained by an increase in the ouabain-sensitive component of Rb+ uptake. An increase in the initial rate of ouabain-insensitive Rb+ uptake was also observed in LPA treated MDCK cell cultures.


The untoward effects of steroid treatment on the musculoskeletal system and what to do about them.

The focus of this review is the musculoskeletal side effects of corticosteroids. These untoward effects can be divided into high-dose phenomena (myopathy and aseptic necrosis) and low-dose problems (growth suppression and osteoporosis). For the clinician, the former group may be an uncommon experience whereas the latter group is highly predictable. The low-dose problems are subtle and asymptomatic, and they can occur in spite of alternate day dosing with steroids. Treatments available to prevent or treat these side effects in patients who are unable to discontinue steroids are reviewed.


Airway response to inhaled fenoterol in hyperthyroid patients before and after treatment.

Bronchodilatory response to inhaled fenoterol was studied in 15 hyperthyroid patients before and after successful treatment with antithyroid drugs. Baseline forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) were lower than the predicted values in 12 and 11 patients, respectively. Improved values were seen after treatment for hyperthyroidism although statistical significance was not reached. Even if some improvement occurred in PEFR (a rise by 0.24-0.48 L/s) and FVC (increase of 73-78 ml) in the hyperthyroid state in response to fenoterol inhalation after various time intervals, the increase in different parameters of lung function was significantly more after the patients achieved euthyroid state (increases in FVC by 290-165 ml; in FEV1 by 333-193 ml; in peak expiratory flow (PEFR) by 0.75-0.52 L/s and in forced expiratory flow (FEF50%) by 0.55-0.31 L/s). In the euthyroid state the mean absolute improvements from the baseline values were significantly higher (< 0.05-0.001). These observations indicate that bronchodilatory response is impaired in the presence of excess thyroid hormones and improves after euthyroid state is achieved.


Airway responses to repeated exercises detected by krypton-81m in asthmatic children.

A repeated exercise program was used to test 7 asthmatic children for changes in ventilation. These changes were examined by continuously inhaled Krypton-81m and compared in subjects with positive and negative refractoriness, as defined by forced expiratory volume in 1 second (FEV1). Three of the seven patients showed significant refractoriness (% reduction in FEV1 > 50%). After the first exercise, they showed one or two ventilation defects which improved after the second exercise. The patient with incomplete refractoriness showed similar results. On the contrary, subjects without refractoriness showed several ventilation defects which fluctuated after the second or third exercise program. One defected area improved after the second exercise session, but deteriorated after the third; and another area deteriorated after the second exercise and improved after the third. It was concluded that approximately half of the patients were refractory in view of FEV1, but that they were all refractory in view of regional ventilation 81mKr images.


House-dust mite allergy and eosinophilia in patients with asthma in Rangoon (Yangon).

Skin tests were done by prick and intradermal techniques, using house dust mite (Dermatophagoides pteronyssinus) antigen on 35 patients with early onset asthma, 33 with late onset asthma, 43 with asthma and frequent cough with sputum production (chronic bronchitis) and 30 control subjects. Absolute blood eosinophil and sputum eosinophil (as percentage of leukocytes) counts were performed on each patient. Positive skin tests to house dust mite antigens were significantly more frequent in each of the patient groups (35-75%) than in control subjects (0%), but were not significantly different among the three groups of asthmatics. All three groups of asthmatics had significantly higher mean blood absolute eosinophil counts and sputum eosinophil counts than control subjects, eosinophilia being most frequent in patients with early onset asthma. These findings suggest the importance of exposure to house dust mite antigens in the development of asthma in patients in Rangoon (Yangon), Burma (Myanmar).


A life activities questionnaire for adult asthma.

The development, testing, and applicability of an instrument, the Life Activities Questionnaire for Adult Asthma is described that can be used to assess activity restriction in adults with asthma. It explains how items for the instrument were selected to insure the validity of the developed instrument. The reliability of the questionnaire is also discussed. The result is a valid and reliable paper-and-pencil instrument that should have wide applicability as a component of quality of life measurement. This instrument has potential for functional use in clinical settings, research environments, and policy-making procedures.


A protocol for aerosol therapy in acute hospitalized asthmatics.

The most important element in the treatment of acute hospitalized asthmatics is salbutamol administered via wet nebulization prescribed usually on a subjective evaluation of the patient's status. This study outlines a novel protocol that incorporates the FEV1 variable as the major objective parameter in a feedback loop. Data from patients admitted in the year prior to the initiation of the protocol (N = 348) were compared with similar data from patients hospitalized the year following institution of the protocol (N = 238). There were no withdrawals from the protocol due to adverse effects nor were there any deteriorations requiring ICU admission. A significant decrease in the length of hospitalization and the number of salbutamol prescriptions per hospitalization was observed after, relative to before, institution of the protocol (2.2 vs. 2.6 days; 2.3 vs. 3.8; p < .05, respectively). It is concluded that this protocol is safe and efficient, and may also be more efficacious than a nonstandardized approach for treating the hospitalized asthmatic child.


Debunking the myths of adolescence: findings from recent research.

This review summarizes some of the important research findings on adolescence that have accumulated during the past two decades. Current understanding of the adolescent age-period is first discussed with particular attention to the previously held myths about adolescence. Second, a review of existing studies that have examined the problems and help-seeking behaviors of adolescents is presented. Overall, the majority of recent research findings suggest adolescence should not be characterized as a time of severe emotional upheaval and turmoil because the majority (80%) of adolescents manage this transition quite well. Nevertheless, a sizable proportion of youth (20%) do not fare so well, with many not receiving the help they may need.


Hospitalized children of substance-abusing parents and physically and sexually abused children: a comparison.

The symptoms and diagnoses of 58 psychiatrically hospitalized children aged 12 or under who were children of substance abusing parents (COSAPs) were compared with a matched group of 51 children who were not COSAPs. These two groups were further subdivided according to history or lack of history of physical and/or sexual abuse. Symptoms and diagnoses of COSAPs and physically and/or sexually abused (P/SAs) children differed significantly from the hospitalized group who were neither COSAPs nor P/SAs. Being a COSAP and/or P/SA are strong correlates of psychiatric hospitalization for the children studied. Both high-risk groups should be awarded special diagnostic and treatment consideration that would include their systematic identification and focused treatment.


Childhood psychopathology in families with multigenerational alcoholism.

The present study examined psychopathology in high-risk children (ages 8 to 18) from families with a multigenerational history of alcoholism and contrasted them with low-risk children from community control families. Similar rates of childhood disorders were found for the high- and low-risk groups whether or not the children lived with an alcoholic parent. These findings suggest that the increased psychopathology commonly reported for children of alcoholics arises from comorbidity within the extended family as a result of assortative mating. When comorbidity is reduced through the selection of families with only alcoholism, a different symptom picture emerges.


Characteristics of adolescent psychiatric patients who engage in problematic behavior while intoxicated.

Although many adolescent psychiatric patients drink and use drugs, not all engage in problematic behaviors or experience social difficulties while intoxicated. The goal of this study was to identify characteristics of adolescent psychiatric patients for whom intoxication leads to behavioral, social, and medical problems. Alcohol-involved adolescent psychiatric inpatients were divided into high and low problem severity groups and compared on a number of measures. The groups did not differ in alcohol or drug consumption or psychiatric diagnoses. High problem severity was primarily associated with drinking in response to unpleasant affect and the belief that alcohol enhances social behavior. Explanations for the results are discussed, along with clinical implications.


Psychiatric disorders in substance-abusing adolescent inpatients: a pilot study.

In a pilot study of 226 adolescents entering inpatient treatment because of a primary substance use disorder, 82% met DSM-III-R criteria for an Axis I psychiatric disorder. Of this population, 74% had two or more psychiatric disorders. Mood disorders were found in 61%, conduct disorders in 54%, and anxiety disorders in 43%. Substance induced organic mental disorders were found in 16%. The data argue strongly for the simultaneous evaluation of both substance use and psychiatric disorders in this type of adolescent population. The pilot study also demonstrates the relative frequency of dually diagnosed subgroups. The authors propose that the dual diagnosis subgrouping may have specific treatment implications involving a combined psychiatric and substance abuse treatment approach.


Patterns of affective comorbidity in a clinical population of dually diagnosed adolescent substance abusers.

Patterns of affective comorbidity with substance abuse are examined in a sample of 156 adolescent psychiatric inpatients, ages 13 to 18 years old. Affective disorders, including adjustment disorder with depressed mood, were observed in 51.3% of patients. A total of 30.7% of patients had comorbid major depression. In both males and females, secondary major depressive disorder was more common than its primary form. In this population, the primary-secondary paradigm did not predict either acute remission for depressive symptoms or distinct family history of comorbid disorders. Consistent with previous studies of adults, significantly more females had comorbid affective disorder and significantly more males had conduct disorder.


Comparison between treatment completers and noncompleters among dually diagnosed substance-abusing adolescents.

The psychiatric and demographic characteristics that may distinguish treatment completers from noncompleters among hospitalized adolescents with substance abuse and comorbid psychiatric disorders were examined. Affective and adjustment disorders were more prevalent among treatment completers whereas non-completers were more likely to be assigned a conduct disorder diagnosis. There were no differences between the groups with respect to demographic and legal status, education level and lifetime psychiatric diagnosis in the parents or caretakers, living arrangements, treatment history, and perception of treatment benefits. A higher percentage of treatment completers than noncompleters received psychotropic medications. The factors contributing to treatment termination as well as the clinical and research implications of the findings are discussed.


Obsessions and compulsions across time in 79 children and adolescents with obsessive-compulsive disorder.

Individual symptoms of 79 children and adolescents with severe obsessive-compulsive disorder were obtained from chart review of at least two in-persons evaluations and recorded across an average of 7.9 years (range, 2 to 16). Symptoms were grouped according to the categories of the Yale-Brown Symptom Checklist. No significant age related trends were found with any one type of symptom, although patients with a very early onset of illness (less than 6 years old) were more likely to have compulsions than obsessions. Across the study period, patients reported symptoms from many different symptom categories, with 47% of the patients displaying both washing and checking compulsions at some time during their illness. No patient maintained the same constellation of symptoms from presentation to follow-up. These data support the concept of obsessive-compulsive disorder as an illness with varied clinical manifestations that individually change over time.


An epidemiological study of obsessive-compulsive disorder and related disorders in Israeli adolescents.

Five hundred and sixty-two, 16- to 17-year-old consecutive inductees into the Israeli Army, constituting a random sample of their cohort, were screened for obsessive-compulsive disorder (OCD), Tourette's syndrome, transient tics (TT), chronic multiple tics (CMT), and attention-deficit hyperactive disorder (ADHD). Two child psychiatrists interviewed the subjects, using screening items from structured interviews that implement DSM-III-R diagnostic criteria. For OCD, a point prevalence of 3.6% was found, 3.9% for ADHD, 1.8% for CMT, and 1.6% for TT. For ADHD, TT, and CMT, but not for OCD, there was a significantly higher prevalence for males than for females. Among the OCD individuals, there was an elevation of TT, CMT, and Tourette's syndrome relative to the population rates.


Double-blind, crossover trial of fluoxetine and placebo in children and adolescents with obsessive-compulsive disorder.

Rigorously designed clinical trials have demonstrated the efficacy and safety of fluoxetine in adults with major depressive disorder and obsessive-compulsive disorder (OCD) but not in patients below 18 years old. This report describes a randomized, double-blind, placebo-controlled, fixed-dose (20 mg qd) trial of fluoxetine in 14 children and adolescents with OCD, ages 8 to 15 years old; the study was 20 weeks long with crossover at 8 weeks. Obsessive-compulsive symptom severity was measured on the Children's Yale-Brown Obsessive Compulsive Scale (CY-BOCS) and the Clinician's Global Impression-Obsessive Compulsive Disorder scale (CGI-OCD). The CY-BOCS total score decreased 44% (N = 7, p = .003) after the initial 8 weeks of fluoxetine treatment, compared with a 27% decrease (N = 6, p = .13) after placebo. During the initial 8 weeks, the magnitude of improvement for the fluoxetine group significantly exceeded that for the placebo group as measured by the CGI-OCD (p = .01) but not by the CY-BOCS (p = .17). The most common drug side effects were generally well tolerated. The results suggest that fluoxetine is a generally safe and effective short-term treatment for children with OCD.


DSM-III-R anxiety disorders in children: sociodemographic and clinical characteristics.

This study investigated the characteristics of each of the specific DSM-III-R (American Psychiatric Association, 1987) anxiety disorders in a clinic sample of 188 anxiety disordered children. Characteristics examined included sociodemographic variables (age-at-intake, gender, and race of the child, and family marital and socioeconomic status) and clinical variables (disorder age-at-onset and severity, and history of additional disorders). Findings are discussed in light of the contemporary literature on childhood anxiety disorders.


Clinical phenomenology of child and adolescent dissociative disorders.

A comparison of two separately diagnosed samples of children and adolescents with dissociative disorders demonstrates good construct validity for these diagnoses in childhood. Descriptive analyses of the total sample reveal a clinical profile characterized by a plethora of affective, anxiety, conduct, posttraumatic, and dissociative symptoms. Children with multiple personality disorder (MPD) differ from those with dissociative disorder not otherwise specified (DDNOS) in having more amnesias, identity disturbances, and hallucinations. Adolescents were more symptomatic than children age 11 or younger and more likely to receive a diagnosis of multiple personality disorder.


Avoidant disorder and social phobia in children and adolescents.

The diagnoses of avoidant disorder and social phobia in children have received little research attention. Although DSM-III-R describes avoidant disorder and social phobia as data are available to support this notion. The current study examined characteristics of avoidant disorder and social phobia by comparing outpatient youngsters with avoidant disorder, social phobia, and avoidant disorder plus social phobia on demographic variables and patterns of comorbidity. The psychiatric groups were compared with matched normal controls on symptom measures of depression and fear. Findings indicated that the three psychiatric groups were strikingly similar on all but one variable, age at intake. These findings question the notion of avoidant disorder and social phobia as distinct disorders in children and adolescents.


An integrated medical and psychiatric approach to conversion symptoms in a four-year-old.

Conversion disorder is a challenging diagnosis in children and adolescents. Medical and psychiatric diagnoses need to be evaluated both separately and in relation to each other. This case highlights both the diagnostic criteria for a conversion disorder in a young child and the need for an integrated medical and psychiatric approach by physicians.


Buspirone-associated mental status changes.

The pharmacological management of anxiety in children primarily has used antidepressants, such as imipramine. Buspirone, an atypical anxiolytic, has been shown to be of benefit in both adults and children. It has relatively few side effects and is generally well tolerated. Two cases are reported here involving children treated for anxiety with buspirone who subsequently suffered a possible psychotic deterioration.


Recruitment and training of child and adolescent psychiatry residents from pediatrics.

This article describes a 21-year experience of recruiting and training child and adolescent psychiatry residents with pediatric backgrounds. Three grounds of physicians are compared: those with previous training in general psychiatry alone, those with previous training in both general psychiatry and pediatrics, and those with previous training in pediatrics alone. Residents with pediatric backgrounds compared favorably with those with general psychiatry training. Those with only pediatric training were not only able to successfully complete the residency, but also the majority entered the field of child psychiatry. The findings support accepting residents directly from pediatrics as a viable pathway into child psychiatry.


Screening for psychosocial dysfunction in inner-city children: further validation of the Pediatric Symptom checklist.

A sample of 123 6- to 12-year-old outpatients at an inner-city pediatric clinic was screened for psychosocial dysfunction using the Pediatric Symptom Checklist (PSC), a brief parent-completed questionnaire. The prevalence of positive screening scores on the PSC was 22%, significantly higher than the rate found in lower middle to upper middle-class samples. Comparing PSC case classifications with comprehensive assessments made by clinicians, overall agreement was 92% (kappa = 0.82; sensitivity = 88%; specificity = 100%); a comparison with several other measures provided additional support for the validity of the PSC. The PSC's reliability over time was also acceptable. These findings provide preliminary evidence that the PSC is as valid and reliable for screening children from economically disadvantaged and minority backgrounds as it is for middle and upper middle-class populations.


Prevalence and predictors of pervasive noncompliance with medical treatment among youths with insulin-dependent diabetes mellitus.

School-age children were assessed longitudinally for up to 9 years, after the onset of their insulin-dependent diabetes mellitus (IDDM), to determine the time-dependent risk of the psychiatric diagnosis of noncompliance with medical treatment and to examine protective and risk factors. The cumulative risk for this diagnosis over the 9 years was .45. Noncompliance tended to emerge in middle adolescence and was found to be protracted. Social competence, self-esteem, and aspects of family functioning at IDDM onset and initial psychiatric status did not predict noncompliance. However, noncompliance was associated with having major psychiatric disorder later in the course of IDDM.


Depressive symptoms and adaptive style in children with cancer.

This article explores the relationship between a repressive style of adaptation and depressive symptomatology. Thirty-one adolescent cancer patients were compared with 83 healthy high school students. Subjects with repressive adaptation were identified as those reporting low anxiety and high defensiveness. Depressive symptomatology was measured by self-report. Patients with cancer reported significantly lower levels of depression, and a significantly higher proportion were identified as repressors. Repressor status accounted for significant variance in depression over that explained by illness. Adaptive style may be common in medically ill children and may directly and indirectly influence psychological and medical outcome.


Profiles, correlates, and trajectories of intelligence in Prader-Willi syndrome.

Three aspects of intellectual functioning in persons with Prader-Willi syndrome were examined in two, related studies. In study 1, 21 subjects were evaluated with a psychometric instrument that assesses neuropsychological styles of cognitive processing, the Kaufman Assessment Battery for Children. Prader-Willi subjects showed deficits in sequential processing, and strengths in academic achievement tasks such as reading and vocabulary. In contrast to previous reports on the syndrome, no relationship was found between weight and degree of intellectual impairment. Study 2 included a cross-sectional examination of the trajectory of IQ in 21 subjects aged 13 to 46 years, as well as a longitudinal analysis of 31 subjects aged 5 to 30 years who were tested twice with the same IQ test. No evidence of the previously described decline in IQ over time was noted in either the cross-sectional or longitudinal analyses. The implications of these findings for interventions are discussed.


Adaptive and maladaptive behavior in Prader-Willi syndrome.

The development and profiles of adaptive and maladaptive behavior of 21 adolescents and adults with Prader-Willi syndrome were cross-sectionally examined with the Vineland Adaptive Behavior Scales and Achenbach's Child Behavior Checklist (CBCL). Adaptive strengths emerged for the group as a whole in daily living skills, and this strength became more pronounced with increasing age. A relative weakness was found in socialization, most notably in coping skills. CBCL findings indicated that externalizing behaviors were particularly heightened in adolescence and that many behaviors previously described as either emerging or worsening in adolescence also persist into the adult years (e.g., temper tantrums, arguing, irritability, stubbornness, lying, skin picking, obsessions, defiance). Certain elevated CBCL behaviors were unique to young versus old age groups, and aging in this syndrome may be associated with heightened confusion, withdrawal, and fatigue. The need to study adaptive and maladaptive features in a wider age range of subjects with Prader-Willi syndrome was emphasized.


Psychopharmacogenetic aspects of Prader-Willi syndrome.

The study of genes, drugs, and behavior in three male adolescents with Prader-Willi syndrome (PWS) revealed a clinical profile that raises questions about the indications for neuroleptic and appetite-suppressing medications in this condition. Evidence of the inadvisability of neuroleptic medication and of the pathophysiology of PWS has led to a remarkable control of violent outbursts and hyperphagia by carbamazepine in one patient afflicted with both PWS and Klinefelter's syndrome. Testosterone and behavioral therapy proved to be useful in the management of two patients. The present observations, which are supported by recent advances in the pathophysiology of satiety, suggest that PWS should be understood as a metabolic disorder and subjected to psychopharmacogenetic study.


Specific frontal lobe deficits among women with the fragile X gene.

The neurocognitive phenotype of fragile X and its relation to cytogenetic expression were examined among 10 fragile X women with > or = 2% expression, 10 0% obligate carriers, and 10 controls. Measures were obtained for intellectual ability, achievement, and verbal, nonverbal, memory, and frontal lobe functions. Results show that no group demonstrated deficits on verbal, nonverbal, or memory measures. In contrast, when controlling for effects of IQ, the expressing fragile X women exhibited (1) deficits on measures of frontal lobe functioning, and (2) enhanced performance on verbal, but not figural, memory. Frontal lobe deficits may account for behavioral and cognitive manifestations of fragile X.


Clomipramine treatment of stereotypic behaviors and self-injury in patients with developmental disabilities.

The authors report an open clinical trial of clomipramine for chronic stereotypic and self-injurious behaviors in 11 consecutive patients with concomitant developmental disorders. Ten patients (91%) had marked decreases in rates of target behaviors. No seizures occurred despite the inclusion of six patients with previous histories of epileptic events, and improvement was evident regardless of level of mental retardation. These findings support both the clinical use of serotonergic medications in this population and the need for further research.


Neuroleptic-induced catatonia as a stage in the progression toward neuroleptic malignant syndrome.

Neuroleptic-induced catatonia is reported in an adolescent patient who responded successfully to lorazepam. The authors propose five discrete stages toward the progression of neuroleptic malignant syndrome, each with a separate treatment.


Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.

Clinical isolates of the fungal respiratory and systemic pathogen Histoplasma capsulatum have been placed in several different classes by using genomic restriction fragment length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RFLP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA fragments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not distinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsulatum are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.


Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing, and expression in Escherichia coli.

Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system.


Mutants carrying conditionally lethal mutations in outer membrane genes omsA and firA (ssc) are phenotypically similar, and omsA is allelic to firA.

We have previously identified the gene (the ssc gene) defective in the thermosensitive and antibiotic-supersusceptible outer membrane permeability mutant SS-C of Salmonella typhimurium and shown that this gene is analogous to the Escherichia coli gene firA (L. Hirvas, P. Koski, and M. Vaara, EMBO J. 10:1017-1023, 1991). Others have tentatively implicated firA in a different function, mRNA synthesis. Here we report that the defect in the thermosensitive outer membrane omsA mutant of E. coli (T. Tsuruoka, M. Ito, S. Tomioka, A. Hirata, and M. Matsuhashi, J. Bacteriol. 170:5229-5235, 1988) is due to a mutation in firA; this mutation changed codon 271 from serine to asparagine. The omsA-induced phenotype was completely reverted by plasmids containing wild-type firA or ssc. Plasmids carrying the omsA allele, or an identical mutant allele prepared by localized mutagenesis, under the control of lac elicited partial complementation. Transcomplementation studies with plasmids carrying various mutant alleles of the S. typhimurium gene indicated that the ability of these plasmids to complement the omsA mutation was similar to their ability to complement the ssc mutation. The antibiotic-supersusceptible phenotype of the omsA mutant closely resembled that of the ssc mutant, i.e., the omsA mutant was supersusceptible to hydrophobic antibiotics and large-peptide antibiotics against which the intact outer membrane is an effective permeability barrier. As previously demonstrated with the omsA mutant, the outer membrane of the ssc mutant became selectively ruptured after incubation for 1 h at the growth-nonpermitting temperature; 82% of the periplasmic beta-lactamase and less than 3% of the cytoplasmic marker enzyme were released into the medium. All of these findings are consistent with our concept that firA is an essential gene involved in generation of the outer membrane.


Regulation of narK gene expression in Escherichia coli in response to anaerobiosis, nitrate, iron, and molybdenum.

The regulation of the narK gene in Escherichia coli was studied by constructing narK-lacZ gene and operon fusions and analyzing their expression in various mutant strains in response to changes in cell growth conditions. Expression of narK-lacZ was induced 110-fold by a shift to anaerobic growth and a further 8-fold by the presence of nitrate. The fnr gene product mediates this anaerobic response, while nitrate control is mediated by the narL, narX, and narQ gene products. The narX and narQ gene products were shown to sense nitrate independently of one another and could each activate narK expression in a NarL-dependent manner. We provide the first evidence that NarL and FNR interact to ensure optimal expression of narK. IHF and Fis proteins are also required for full activation of narK expression, and their roles in DNA bending are discussed. Finally, the availability of molybdate and iron ions is necessary for optimal narK expression, whereas the availability of nitrite is not. Although the role of the narK gene product in cell metabolism remains uncertain, the pattern of narK gene expression is consistent with a proposed role of NarK in nitrate uptake by the cell for nitrate-linked electron transport.


Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli.

A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.


Repression of Escherichia coli purB is by a transcriptional roadblock mechanism.

Escherichia coli purB is regulated by a repressor-operator interaction. The purB operator is 242 bp downstream from the transcription start site and overlaps condons 62 to 67 in the protein-coding sequence (B. He, J. M. Smith, and H. Zalkin, J. Bacteriol. 174:130-136, 1992). The mechanism by which the repressor-operator interaction functions to repress transcription was investigated by a combination of promoter replacement experiments and RNA analyses. By using a trp promoter replacement that deleted 5' flanking DNA to position -986, purB expression was increased sevenfold, yet normal two- to threefold regulation was maintained. This indicates that repressor-operator control is independent of the purB promoter and other 5' flanking sequences. Transcriptional regulation was likewise independent of coupled translation. An approximately 260-nucleotide truncated in vivo purB mRNA was identified which was dependent upon repressor-operator interaction. Thus, binding of purine repressor to the purB operator inhibits transcription elongation by a roadblock mechanism. The roadblock was not influenced by a sevenfold increase in promoter strength or by an operator mutation resulting in a 2.5-fold increase in repressor-operator affinity.


Formation of the LuxR protein in the Vibrio fischeri lux system is controlled by HtpR through the GroESL proteins.

The transcription of the luminescence (lux) system of Vibrio fischeri is regulated by the LuxR protein and an autoinducer. We previously showed that apart from these regulatory elements, the transcription of the lux system is negatively controlled by the LexA protein and positively controlled by the HtpR protein (sigma 32). This study was conducted in order to elucidate the mode of action of the HtpR protein. Using luxR-lacZ fused genes, we showed that the HtpR protein is essential for the maximum expression of beta-galactosidase activity in Escherichia coli lac mutant cells. Using this construct, we also demonstrated that luxR is preferentially expressed toward the end of the logarithmic phase of growth. Starvation and addition of ethanol significantly advanced the appearance of beta-galactosidase activity in htpR+ cells. The luminescence system of E. coli htpR+ cells harboring the pChv1 plasmid with a deletion in the luxI gene is induced in the presence of low and constant concentrations (150 pg/ml) of the inducer only at a late stage of the logarithmic phase of growth. When the cellular LuxR content is reduced, following 23 generations of exponential growth in Luria broth, a mid-log-phase culture does not respond to the inducer (150 pg/ml). On the basis of the above observations we suggest that the HtpR protein controls the formation of V. fischeri LuxR protein. Preliminary findings indicate that the HtpR protein acts through the chaperonins GroESL. E. coli htpR/pChv1 cells retained their full level of in vivo and in vitro luciferase activities in the presence of multiple copies of groESL genes. The possibility that GroESL proteins stabilize the native form of LuxR protein is discussed.


Activity of mutant sigma F proteins truncated near the C terminus.

sigma F, the product of the spoIIAC gene of Bacillus subtilis, is homologous in amino acid sequence throughout most of its length with several other sigma factors of B. subtilis and Escherichia coli. However, 8 residues from the C terminus the homology abruptly breaks down, suggesting that the C-terminal tail of the protein may be dispensable. It is known that an amber mutation at the 11th codon (wild-type glutamine 245) from the C terminus abolishes the function of the sigma factor. We have now placed chain-terminating codons at the ninth codon (wild-type lysine 247), the eighth codon (wild-type valine 248), or the seventh codon (wild-type glutamine 249) from the C terminus. We have tested the resulting mutants for their capacity to sporulate and for their ability to transcribe from a promoter (spoIIIG) that is normally read by RNA polymerase bound to sigma F (E sigma F). The results indicate that a mutant sigma F lacking the terminal 7 residues functions almost normally, which suggests that glutamine 249 is dispensable. By contrast, lysine 247 is crucial for the activity of sigma F: deletion of the 9 C-terminal residues totally inactivates the protein. When the terminal 8 residues were deleted, placing lysine 247 at the C terminus, the transcriptional activity of the factor is reduced by about 80%: we attribute this effect to neutralization of the positive charge of lysine 247 by formation of a salt bridge with the -COO- terminus.


Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.


Fatty acids are precursors of alkylamines in Deinococcus radiodurans.

Deinococcus radiodurans contains novel phospholipids of which the structures of three have been previously described. These three lipids contain both fatty acids and alkylamines. Both the fatty acid and alkylamine constituents were found to be composed of a mixture of species, of which C15, C16, and C17 saturated and monounsaturated alkyl chains predominated. Alkylamines contained a relatively higher proportion of saturated species. Progression of bacterial growth through the mid-log to stationary phases was accompanied by an increase in the proportions of C15 and C17 alkyl chains in both fatty acid and alkylamine constituents. Radiolabeled palmitic acid was found to be rapidly incorporated into both fatty acid and alkylamine components of phosphatidylglyceroylalkylamine, which is the precursor of the more-complex phosphoglycolipids found in major amounts in D. radiodurans. After culturing D. radiodurans in the presence of a mixture of palmitic acids labeled with 14C and 3H in the 1 and 9,10 positions, respectively, the same 14C/3H ratio was recovered in both fatty acid and alkylamine constituents, strongly suggesting that alkylamines are derived from intact fatty acids rather than by a de novo pathway. The results identify a novel product of fatty acid metabolism which has not to date been observed in any other organism.


An internal region of rpoB is required for autogenous translational regulation of the beta subunit of Escherichia coli RNA polymerase.

In order to delineate the region involved in feedback regulation of the RNA polymerase beta subunit (encoded by rpoB), a collection of rpoB-lacZ translational fusions with different endpoints both upstream and downstream of the rpoB start site was assembled on lambda phage vectors. The extent of translational repression of beta was monitored by measuring beta-galactosidase levels in monolysogens of the fusions under conditions of increased intracellular concentrations of beta and beta' achieved via the induction of rpoBC expression from a multicopy plasmid. A construct containing as little as 29 bp upstream of the start of rpoB exhibited repression of beta-galactosidase activity to the same extent as a construct encoding the full upstream region. A construct which carried only 70 bp of the rpoB structural gene exhibited very little repression, while constructs which carried 126 or 221 bp of rpoB exhibited approximately the same degree of repression as a construct which carried 403 bp. These data suggest that the sequences important for feedback regulation of beta translation extend more than 70 bp into rpoB but are completely contained within a region which spans the sequences from 29 bp upstream to 126 bp downstream of the translational start site.


Phenotypes of sphingolipid-dependent strains of Saccharomyces cerevisiae.

To study sphingolipid function(s) in Saccharomyces cerevisiae, we have investigated the effects of environmental stress on mutant (SLC) strains (R. C. Dickson, G. B. Wells, A. Schmidt, and R. L. Lester, Mol. Cell. Biol. 10:2176-2181, 1990) that either contain or lack sphingolipids, depending on whether they are cultured with a sphingolipid long-chain base. Strains lacking sphingolipid were unable to grow at low pH, at 37 degrees C, or with high salt concentrations in the medium; these environmental stresses are known to inhibit the growth of some S. cerevisiae strains with a defective plasma membrane H(+)-ATPase. We found that sphingolipids were essential for proton extrusion at low pH and furthermore found that cells lacking sphingolipid no longer exhibited net proton extrusion at normal pH after a 1-min exposure to pH 3. Cells lacking sphingolipid appeared to rapidly become almost completely permeable to protons at low pH. The deleterious effects of low pH could be partially prevented by 1 M sorbitol in the suspension of cells lacking sphingolipid. Proton extrusion at normal pH (pH 6) was significantly inhibited at 39 degrees C only in cells lacking sphingolipid. Thus, the product of an SLC suppressor gene permits life without sphingolipids only in a limited range of environments. Outside this range, sphingolipids appear to be essential for maintaining proton permeability barriers and/or for proton extrusion.


A locus that contributes to colonization of the intestinal tract by Bacteroides thetaiotaomicron contains a single regulatory gene (chuR) that links two polysaccharide utilization pathways.

Previously, we isolated two Tn4351-generated mutants of Bacteroides thetaiotaomicron (46-1 and CS3) that were unable to grow either on heparin or on chondroitin sulfate. This phenotype was unexpected, since the heparin and chondroitin sulfate utilization pathways had appeared from earlier studies to be independent of each other. Mutants 46-1 and CS3 were also of interest because both were unable to compete successfully with wild-type B. thetaiotaomicron in the intestinal tracts of germfree mice. Thus, both appeared to have a colonization defect. We have now cloned the chromosomal locus in which the transposon insertions in 46-1 and CS3 occurred. Southern blot analysis showed that the Tn4351 insertions in 46-1 and CS3 were about 100 bp apart. Using complementation and insertional mutagenesis, we localized the region affected by the 46-1 and CS3 insertions to within 2.5 kbp. This DNA segment was sequenced and found to contain a 401-codon open reading frame (ORF1) and the N-terminal segment of a second open reading frame (ORF2), which was downstream of ORF1 and transcribed in the same direction. The deduced amino acid sequence of ORF1 showed significant homology to that of a putative positive regulator of an arylsulfatase gene in Klebsiella aerogenes. ORF2 was at least 381 amino acids long and did not exhibit homology to any proteins in the data bases searched. Transposon insertions in both mutants 46-1 and CS3 disrupted ORF1. The results of insertional mutagenesis and complementation experiments indicated that ORF2 was not essential for growth on chondroitin sulfate or heparin. Thus, the chondroitin sulfate-negative and heparin-negative phenotypes of 46-1 and CS3 appear to be due to the interruption of a regulatory gene encoded by ORF1 and not to a polar effect of the insertions on a downstream gene(s). The gene encoding ORF1 has been designated chuR, for regulation of chondroitin sulfate and heparin utilization. Transcriptional fusion studies showed that the expression of chuR occurred at the same level under inducing and noninducing conditions, in contrast to the regulated expression of structural genes of the chondroitin sulfate utilization system. chuR was not autoregulated, nor was its expression affected by a mutation (46-4) that eliminated the expression of all chondroitin sulfate utilization genes but did not affect the utilization of heparin.


Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis.

BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC. The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. The small open reading frame has been designated bamHIC (for BamHI controlling element). It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli. Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated. The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans. The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome. In B. subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.


Characterization of the Escherichia coli membrane domain responsible for binding oriC DNA.

It has previously been shown that hemimethylated DNA from the Escherichia coli replication origin (oriC) binds with high specificity to membrane fractions isolated from disrupted cells. In this article, the membrane localization of oriC-binding activity was studied by subjecting crude membrane preparations to successive cycles of sedimentation and flotation gradient analysis. This revealed that approximately two-thirds of the membrane-associated oriC-binding activity of the cell was not associated with the outer membrane fraction as previously suggested but was recovered instead in a unique membrane fraction (OCB1) whose buoyant density and protein profile differed from those of both inner and outer membranes. The specific activity of oriC binding in OCB1 was approximately fivefold higher than the activity of the isolated outer membrane peak. It is likely that membrane fraction OCB1 includes the membrane domain responsible for the binding of hemimethylated oriC to the cell envelope in intact cells.


Chromosomal structure of the halophilic archaebacterium Halobacterium salinarium.

The chromosomal structure of the extremely halophilic archaebacterium Halobacterium salinarium was examined. Sheared chromosomes prepared from the bacteria in the late exponential phase were separated into two peaks (peaks I and II) by sucrose gradient centrifugation, suggesting that the chromosomes consist of two parts differing in quality. The UV spectra of peaks I and II resembled those of DNA and eukaryotic chromatin, respectively. Electron microscopic observations revealed that the major component of peak I was protein-free DNA, while the major components of peak II were rugged thick fibers with a diameter of 17 to 20 nm. The rugged fibers basically consisted of bacterial nucleosome-like structures composed of DNA and protein, as demonstrated in experiments with proteinase and nuclease digestion. Whole-mount electron microscopic observations of the chromosomes directly spread onto a water surface revealed a configuration in which the above-described regions were localized on a continuous DNA fiber. From these results it is concluded that the H. salinarium chromosome is composed of regions of protein-free DNA and DNA associated with nucleosome-like structures. Peaks I and II were predominant in the early exponential phase and stationary phase, respectively; therefore, the transition of the chromosome structure between non-protein-associated and protein-associated forms seems to be related to the bacterial growth phase.


Multiple forms of bile salt hydrolase from Lactobacillus sp. strain 100-100.

Four isozymes of bile salt hydrolase (BSH) have been purified from the cytosol of cells of Lactobacillus sp. strain 100-100. The four proteins were designated BSH A, B, C, and D. They eluted from anion-exchange high-pressure liquid chromatography columns at 0.15, 0.18, 0.21, and 0.25 M NaCl, respectively. They are catalytically similar, except that the Vmax of BSH D is about 10-fold lower than those of the other three isozymes. All four proteins consist of one or two polypeptides. The peptides have molecular weights of 42,000 and 38,000 and are designated alpha and beta, respectively. The approximate native molecular weights of BSH A, B, C, and D are 115,000, 105,000, 95,000, and 80,000, respectively. The native proteins are probably trimers; the four isozymes are the array of possible subunit combinations alpha 3, alpha 2 beta 1, alpha 1 beta 2, and beta 3 for A, B, C, and D, respectively. The two subunits are antigenically distinct. Polyclonal antibodies raised against BSH A (all alpha peptide) react in Western blots (immunoblots) only with proteins containing the alpha peptide; such antibodies raised against BSH D (all beta peptide) react only with proteins containing the beta peptide. The amino acid compositions of the two peptides differ. This is the first report of a bacterium that makes four BSH isozymes.


The helix-turn-helix motif of sigma 54 is involved in recognition of the -13 promoter region.

Residue Arg-383 in the proposed helix-turn-helix motif of the novel RNA polymerase sigma factor sigma 54 has been changed by site-directed mutagenesis to all possible alternative amino acids. Only two mutants, RK383 and RH383, are active in promoting transcription from either the glnAp2 promoter or the nifL promoter. We constructed a set of mutant derivatives of glnAp2 such that each base in the conserved GG and GC doublets at -24 and -12 was changed to all possible alternatives. All 12 mutant glnAp2 promoters showed a marked promoter-down phenotype with wild-type sigma 54, but RK383 suppressed changes of both G to C and G to T at -13. This result suggests that the sigma 54 helix-turn-helix is involved in recognition of the -13 region of sigma 54-dependent promoters.


Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.


Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum.

Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.


Use of chromosomal gene fusions to investigate the role of repetitive DNA in regulation of genes involved in lipopolysaccharide biosynthesis in Haemophilus influenzae.

The lic3 locus of Haemophilus influenzae consists of four open reading frames. The derived amino acid sequences of orf2 and orf4 exhibit homology to Escherichia coli GalE and AdK, respectively. The functions of orf1 and orf3 remain unknown. orf1 contains multiple tandem repeats of the tetrameric DNA sequence CAAT near the 5' end. Two possible translational starts (ATG1 and ATG2) lie upstream. We have used lacZ fusions to investigate whether changes in the number of CAAT repeats in conjunction with differential usage of the upstream frames control the expression of lic3-orf1. Phase-variable expression of lacZ was observed for individual colonies and could be related to variable numbers of CAAT repeats. Of the three possible upstream frames, only one, containing the more downstream of the two possible ATG start codons (ATG2), is used for strong expression of lacZ. Utilization of the more upstream ATG (ATG1) or ATG2 was observed with medium-level expression, while utilization of any of the three possible frames was observed when lacZ was expressed at low to undetectable levels, indicating that other mechanisms may affect expression. To investigate this, lacZ was fused in frame with ATG2 of lic3-orf1, with concomitant deletion of the repeats. Phase-variable expression was still observed, supporting the view that an alternative level of control operates in conjunction with the repeat mechanism.


Identification of a new gene, tmoF, in the Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase.

Five genes, tmoABCDE, encoding toluene-4-monooxygenase (T4MO) were previously mapped to a 3.6-kb region of a 10.2-kb SacI DNA fragment isolated from Pseudomonas mendocina KR1 (K.-M. Yen, M. R. Karl, L. M. Blatt, M. J. Simon, R. B. Winter, P. R. Fausset, H. S. Lu, A. A. Harcourt, and K. K. Chen, J. Bacteriol. 173:5315-5327, 1991). In this report, we describe the identification and characterization of a DNA region in the SacI fragment whose expression enhances the T4MO activity determined by the tmoABCDE gene cluster. This region was mapped immediately downstream of the putative transcription termination sequence previously located at the end of the tmoABCDE gene cluster (Yen et al., J. Bacteriol., 1991) and was found to stimulate T4MO activity two- to threefold when expressed in Escherichia coli or Pseudomonas putida. Determination of the nucleotide sequence of this region revealed an open reading frame (ORF) of 978 bp. Expression of the ORF resulted in the synthesis of an approximately 37-kDa polypeptide whose N-terminal amino acid sequence completely matched that of the product predicted from the ORF. The ORF thus defines a gene, which has now been designated tmoF. The TmoF protein shares amino acid sequence homology with the reductases of several mono- and dioxygenase systems. In addition, the reductase component of the naphthalene dioxygenase system, encoded by the nahAa gene of plasmid NAH7 from P. putida G7, could largely replace the TmoF protein in stimulating T4MO activity, and TmoF could partially replace the NahAa protein in forming active naphthalene dioxygenase. The overall properties of tmoF suggest that it is a member of the T4mo gene cluster and encodes the NADH:ferredoxin oxidoreductase of the T4MO system.


Regulation of partitioned sterol biosynthesis in Saccharomyces cerevisiae.

Using yeast strains with null mutations in structural genes which encode delta-aminolevulinic acid synthetase (HEM1), isozymes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG1 and HMG2), squalene epoxidase (ERG1), and fatty acid delta 9-desaturase (OLE1), we were able to determine the effect of hemes, sterols, and unsaturated fatty acids on both sterol production and the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in Saccharomyces cerevisiae. We found that the HMGR isozymes direct essentially equal amounts of carbon to the biosynthesis of sterols under heme-competent conditions, despite a huge disparity (57-fold) in the specific activities of the reductases. Our results demonstrate that palmitoleic acid (16:1) acts as a rate-limiting positive regulator and that ergosterol acts as a potent inhibitor of sterol production in strains which possess only the HMGR1 isozyme (HMG1 hmg2). In strains which contain only the HMGR2 isozyme (hmg1 HMG2), sterol production was inhibited by oleic acid (18:1) and to a lesser degree by ergosterol. The specific activities of the two reductases (HMGR1 and HMGR2) were found to be differentially regulated by hemes but not by ergosterol, palmitoleic acid, or oleic acid. The disparate effects of unsaturated fatty acids and sterols on these strains lead us to consider the possibility of separate, compartmentalized isoprenoid pathways in S. cerevisiae.


Construction of an EcoRI restriction map of Mycoplasma pneumoniae and localization of selected genes.

A restriction map of the genome of Mycoplasma pneumoniae, a small human pathogenic bacterium, was constructed by means of an ordered cosmid library which spans the complete bacterial chromosome. The positions of 143 endonuclease EcoRI restriction fragments were determined and aligned with the physical map. In addition, restriction sites for the rare-cutting enzymes XhoI (25 sites), ApaI (13 sites), NotI (2 sites), and SfiI (2 sites) were included. The resulting map consists of 185 restriction sites, has a mean resolution of 4.4 kbp, and predicts a genome size of 809 kbp. In addition, several genes were identified and mapped to their respective genomic EcoRI restriction fragments.


The terminal reductases for selenate and nitrate respiration in Thauera selenatis are two distinct enzymes.

A number of approaches have been used to show that a recently isolated selenate-respiring bacterium, Thauera selenatis, is able to synthesize both a selenate reductase (SR) and a nitrate reductase (NR). (i) The pH optimum of the SR was found to be 6.0; that of the NR was 7.0. (ii) The presence of nitrate did not inhibit selenate reduction in selenate-grown cells. (iii) In cell extracts, the highest SR or NR activity was observed in cells grown with the respective electron acceptor. (iv) Mutants that were unable to grow with nitrate as the terminal electron acceptor and lacked NR activity were isolated; these mutants grew normally with selenate and synthesized SR. (v) The SR was found in the periplasmic space of the cell, whereas the NR was present in the cytoplasmic membrane. A hypothetical electron transport system involving the SR is described.


The gene encoding a Prevotella loescheii lectin-like adhesin contains an interrupted sequence which causes a frameshift.

We cloned and sequenced the Prevotella loescheii gene plaA, which encodes a lectin-like adhesin that mediates the coaggregation of P. loescheii 1295 with Streptococcus oralis 34. A probe derived from the N-terminal amino acid sequence of the purified adhesin was used to identify the plaA gene from a P. loescheii genomic library constructed in lambda GEM-11. Sequence analysis of plaA indicates that the initial translation product contains a 22-amino-acid leader. The reading frame of the plaA gene is interrupted after amino acid 28 of the mature protein by a TAA termination codon. Amplification of the P. loescheii genomic DNA in the region surrounding this codon by the polymerase chain reaction followed by DNA sequencing of the cloned DNA fragment established that this stop codon was not an experimental artifact. A frameshift beginning 29 bp downstream of the ochre terminator was required to access the only large open reading frame in the gene. Amino acid sequences of six purified peptides derived by limited proteolysis of adhesin with endoproteinase Lys-C matched the downstream amino acid sequence derived by translation of the large open reading frame. The gene coding sequence of 2.4 kb contains sufficient information for the synthesis of an 89-kDa protein. A putative rho-independent terminator (delta G = -25.5 kcal/mol [ca. -107 kJ/mol]) was detected 38 bp downstream from the plaA stop codon.


The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus.

Several genes (cfx genes) encoding Calvin cycle enzymes in Alcaligenes eutrophus are organized in two highly homologous operons comprising at least 11 kb. One cfx operon is located on the chromosome; the other is located on megaplasmid pHG1 of the organism (B. Bowien, U. Windhövel, J.-G. Yoo, R. Bednarski, and B. Kusian, FEMS Microbiol. Rev. 87:445-450, 1990). Corresponding regions of about 2.7 kb from within the operons were sequenced. Three open reading frames, designated cfxX (954 bp), cfxY (765 bp), and cfxE (726 bp), were detected at equivalent positions in the two sequences. The nucleotide identity of the sequences amounted to 94%. Heterologous expression of the subcloned pHG1-encoded open reading frames in Escherichia coli suggested that they were functional genes. The observed sizes of the gene products CfxX (35 kDa), CfxY (27 kDa), and CfxE (25.5 kDa) closely corresponded to the values calculated on the basis of the sequence information. E. coli clones harboring the cfxE gene showed up to about 19-fold-higher activities of pentose-5-phosphate 3-epimerase (PPE; EC 5.1.3.1) than did reference clones, suggesting that cfxE encodes PPE, another Calvin cycle enzyme. These data agree with the finding that in A. eutrophus, PPE activity is significantly enhanced under autotrophic growth conditions which lead to a derepression of the cfx operons. No functions could be assigned to CfxX and CfxY.


Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa.

PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and cysteine for phenylalanine show that Km values remain close to that measured for wild-type substrate, while kcat and kcat/Km values were significantly decreased. This indicates that while the affinity of enzyme for substrate is relatively unaffected by the substitutions, the maximum rate of catalysis favors a phenylalanine at this position. Interesting, PilD cleavage of one mutated pillin (asparagine) resulted in a lower Km value of 52.5 microM, which indicates a higher affinity for the enzyme, as well as a lower kcat value of 6.1 min m(-1). This suggests that it may be feasible to design peptide inhibitors of PilD.