Maintenance of NF-kappa B activity is dependent on protein synthesis and the continuous presence of external stimuli.

The activation of NF-kappa B-like activities (called NF-kappa B) by tumor necrosis factor alpha (TNF alpha) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) were compared. High levels of NF-kappa B activity were found 2 to 4 min after TNF alpha addition to human HL60 cells and lasted for at least 3 h, although the half-life of active NF-kappa B was less than 30 min. Inactive NF-kappa B, however, was relatively stable. NF-kappa B activation by TNF alpha was initially cycloheximide insensitive, but maintenance of NF-kappa B activity required ongoing protein synthesis and continuous stimulation by TNF alpha. Thus, the cells did not remain in an activated state without stimulation. In HL60 cells, NF-kappa B induction by PMA required 30 to 45 min and was completely dependent on de novo protein synthesis, while PMA (and interleukin-1) induced NF-kappa B activity rapidly in mouse 70Z/3 cells via a protein synthesis-independent mechanism. The NF-kappa B-like activities obtained under each condition behaved identically in methylation interference and native proteolytic fingerprinting assays. The NF-kappa B-like factors induced are thus all very similar or identical. We suggest that cell-specific differences in the protein kinase C-dependent activation of NF-kappa B may exist and that TNF alpha and PMA may induce expression of the gene(s) encoding NF-kappa B.

Cyclic AMP-independent activation of transcription factor NF-kappa B in HL60 cells by tumor necrosis factors alpha and beta.

No correlation exists in HL60 cells between NF-kappa B activation by tumor necrosis factor (TNF alpha) and TNF beta and intracellular levels of cyclic AMP. Cyclic AMP levels did not increase upon treatment of cells with each of these cytokines, although NF-kappa B was activated. Forskolin or 1-isobutyl-3-methylxanthine drastically increased intracellular levels of cyclic AMP, but neither activated NF-kappa B nor influenced TNF-induced NF-kappa B activation.

Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534.

The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster.

Relationship of intestinal parasites to the environment and to behavioral factors in children in the Bolikhamxay Province of Lao PDR.

From March to July 1998 the infection rates of 732 children aged below 15 years were assessed. The investigation was conducted in selected villages of the Bolikhamxai Province in Lao PDR. Socio-economic conditions and behavioral pattern were studied. The three soil-transmitted helminths, Ascaris lumbricoides, Trichuris trichiura and hookworm were found with prevalence rates of 67.14, 17.49 and 12.83%, respectively. Infection rates with other intestinal parasites were negligible. Of the children investigated, 56.7% harbored one and 20.45% more than one parasite. Except for hookworms, no statistically significant differences were found between genders. The probability of being infected with A. lumbricoides is associated with living in mountainous areas. For hookworms, infection is associated with staying in the plains. A river in the vicinity of the village is linked with the probability of being infested with Trichuris trichiura. Not to belong to a family with the ability to own expensive items increases the probability by almost two times of getting infested with A. lumbricoides. Unhygienic behavioral factors were important in increasing the probability of suffering from A. lumbricoides and T trichiura infection. Behavioral factors did not seem to be related to hookworm infections. It was concluded that after mass treatment, besides promoting the construction of toilets, it is also important to improve personal hygiene so that a lasting impact on the infection rate of the most prevalent parasites in Lao PDR could be achieved. Measures to control parasitic infections do not have to be postponed until a marked improvement of the economic situation has occurred.

[Tetrachlorethene pollution--results of follow-up of neighbors of chemical dry cleaning establishments and current legal developments]. / Tetrachlorethen(TCE)-Belastung--Ergebnisse einer Nachuntersuchung bei Anwohnern Chemischer Reinigungen und aktuelle gesetzliche Entwicklungen.

New legal provisions set a limiting value of 0.1 mg TCE/m3 air in flats. Follow-up examinations by the Public Health Office Bremen show again that so far this limit can not be complied with in flats adjacent to dry cleaners. Interviews with affected residents give no hint for a change in utilisation habits. The Upper House of the German Federal Parliament has taken the legislative initiative for a complete prohibition of TCE in consequence of the fundamental problem of limiting values.

[The tetrachloroethylene burden of residents adjacent to chemical dry cleaning establishments]. / Tetrachlorethenbelastung bei Anwohnern von chemischen Reinigungen.

The paper describes the concentration of tetrachlorethen (TCE) in the air of flats in Bremen adjacent to drycleaners and the resulting TCE concentration in the food and blood of the residents. The results indicate that--regardless of the cleaning system--a high TCE pollution must be expected with nearly everybody living next to a TCE dry cleaning shop. The currently propagated guiding value of 0.1 mg TCE per m3 of air or 0.1 mg TCE per kg of food can hardly be complied with--even after reorganisation in the drycleaning shop. The problem of guiding and limit values of TCE and possible alternatives to dry cleaning with TCE are discussed. The paper shows that Public Health Offices--in cooperation with other institutions--can make relevant contributions to investigations in the field of environmental health.

[Development of public health regulations for tattooing and piercing and their realization]. / Entwicklung von Hygieneregeln für das Tätowieren und Piercing und ihre Umsetzung.

In the course of prevention of infectious diseases the Public Health office of the city of Bremen has made increase efforts to improve hygienic conditions in tattoo and piercing studios. Defined and practicable hygienic standards have been developed and formulated for and in cooperation with the studios. Supported by intense personal counselling of the studios the hygienic standards are now--after 6-9 months--widely accepted and increasingly observed in practice. This programme has been--as a crucial point--supplemented by intense information for the general public and the customers of the studios in order to support (and control) the practice of hygienic standards in tattooing and piercing also from this point of view.

Staphylococcus aureus and derived exotoxins induce nuclear factor kappa B-like activity in murine bone marrow macrophages.

Heat-killed gram-positive Staphylococcus aureus as well as S. aureus-derived exotoxins B and toxic shock syndrome toxin 1 can induce nuclear factor kappa B (NF-kappa B)-like activity in murine bone marrow macrophages. The induction of NF-kappa B-like activity in murine macrophages by S. aureus was as effective as induction by tumor necrosis factor alpha (TNF-alpha) or lipopolysaccharides (LPS) and was observed in macrophages derived from LPS-sensitive and LPS-resistant mice. Stimulation of macrophages with S. aureus but not with the exotoxins resulted in the accumulation of TNF-alpha in the culture medium. The induction of NF-kappa B-like activity by S. aureus, however, clearly preceded TNF-alpha secretion and was not inhibited by a neutralizing serum against TNF-alpha. In addition, pretreatment of macrophages with the protein synthesis inhibitor cycloheximide or dexamethasone, which prevented the secretion of TNF-alpha from macrophages, did not interfere with the induction of NF-kappa B-like activity by S. aureus. This findings reveal the existence of bacterial components other than LPS which can induce NF-kappa B-like activity in susceptible cells.

Alcohol-induced brain pathology and behavioral dysfunction: using an animal model to examine sex differences.

BACKGROUND: Human epidemiological studies suggest that the female brain may be more susceptible to the toxic effects of alcohol and that this is the reason why women show greater behavioral dysfunction after chronic alcohol exposure. This hypothesis was tested by using a rat model of chronic alcoholism [chronic ethanol treatment (CET)]. The investigation assessed sex differences in neuropathology and behavior after chronic exposure and subsequent withdrawal from alcohol. METHODS: Young male and female rats (approximately 3 months old) were assigned to either a CET group, which received a 20% ethanol drinking solution for 20 weeks, or a pair-fed control group, which received ad libitum tap water and a restricted diet for 20 weeks. After the CET groups were phased off the 20% alcohol solution, learning and memory abilities were examined by using matching-to-position and nonmatching-to-position tasks. Neuropathology was assessed in the frontal cortex and medial septal region. RESULTS: CET was shown to cause behavioral deficits. The behavioral dysfunction was sex, task, and process dependent; i.e., CET-female rats displayed a delay-dependent impairment on delayed matching-to-position, whereas CET-male rats displayed a delay-independent impairment on delayed nonmatching-to-position. CET resulted in a significant reduction in the frontal cortical (FR1) and collosal thickness, as well as a decrease in cells staining immunopositive for choline acetyltransferase in the medial septal region. However, relative to male rats exposed to CET, female rats did not show any accelerated neuropathology after CET. CONCLUSIONS: Chronic exposure to ethanol does result in both brain and behavior dysfunction in male and female rats. The results demonstrate that different cognitive processes are altered by chronic ethanol exposure in male and female rats. However, the neurobiological mechanisms responsible for these differences remain to be determined.

Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC.

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.

Significance of kin relations and individual preferences in the social behaviour of Cebus apella.

Captive tufted capuchins (Cebus apella) formed a group characterized by greater affiliative behaviour within matrilines. Affiliation within the matriline is dependent upon social intimacy during infancy with matriline members. Courtship and mating are not identically distributed: subordinate and subadult males participate in a larger proportion of matings than of courtships. In all these aspects of social behaviour, variability across individuals contributes to the complex dynamics of the social group.

Social relations in groups of the black-capped capuchin (Cebus apella) in captivity. Interactions of group-born infants during their second half-year of life.

During the second half-year of life, capuchin monkey infants maintain close contacts to kin-related animals. Apart from these contacts, they frequently interact with other infants and juveniles 1 year older. During this period of life the 'peer phase' begins in the life of the infants. Furthermore, gender differences, especially in respect to social play, become obvious.

Two different cell types have different major receptors for human tumor necrosis factor (TNF alpha).

The receptors for tumor necrosis factor alpha (TNF alpha) were analyzed on myeloid cells (HL60, U937, K562, and freshly isolated blood monocytes) and on cells of epithelial origin (MCF7, HEp2 and HeLa cells), by use of radiolabeled TNF alpha and cross-linking experiments. Both cell types had high but slightly different affinities for TNF alpha. The myeloid cells had major cross-linked products of 98-100 kDa, which were similar in their N-linked glycosylation, whereas the cells of epithelial origin contained a major cross-linked product of 75 kDa, a second product of 95 kDa. The major receptors of both cell types (studied mostly with HL60 and HEp2 cells) are different proteins because (a) their apparent molecular masses were different and no evidence was obtained for cell-specific proteases, which could generate the differently sized receptors from one common receptor molecule; (b) anti-receptor antibodies, which precipitated the 95- and 75-kDa products, did not precipitate the 100-kDa cross-linked complex; (c) the native TNF alpha-receptor complexes had different proteolytic fingerprints; (d) the tryptic fragments differed in their association with the cell membrane vesicles; (e) the receptors differed in their degree of N-linked glycosylation; and (f) O-linked glycosylation was found on the major receptor of HL60 but not of HEp2 cells. In addition, myeloid cells may also contain a small amount of the HEp2-type of TNF alpha receptor. We suggest that at least two different receptors for TNF alpha exist.

Protein kinases negatively affect nuclear factor-kappa B activation by tumor necrosis factor-alpha at two different stages in promyelocytic HL60 cells.

HL60 and EL4 cells incubated with tumor necrosis factor-alpha (TNF-alpha) plus staurosporin, a potent inhibitor of protein kinases, showed at least 2-fold increased levels of nuclear factor-kappa B (NF-kappa B) activity compared with TNF-alpha alone both during rapid NF-kappa B activation from the cytosolic pool and protein synthesis-dependent NF-kappa B activation. NF-kappa B activation by phorbol 12-myristate 13-acetate (PMA) and interleukin-1 was inhibited by staurosporin. Staurosporin treatment hardly affected the TNF-alpha-induced increase in mRNA for the p51 subunit of NF-kappa B but interfered with any phorbol ester (PMA)-induced increase in p51 mRNA. Thus, induction of NF-kappa B and p51 mRNA by TNF-alpha was not mediated by a staurosporin-sensitive factor, but NF-kappa B activation by TNF-alpha was even reduced by action of a staurosporin-sensitive factor. Decreased levels of phosphorylation of TNF-R alpha (TNF receptor type alpha) after staurosporin-treatment correlated with increased induction of NF-kappa B by TNF-alpha. Staurosporin-treatment did not affect TNF-R levels. Although protein kinase C stimulation by PMA inhibited NF-kappa B activation by TNF-alpha, its action mechanism may be different from that of the staurosporin-sensitive factor. PMA induced disappearance of TNF-R alpha by shedding into the surrounding medium, with kinetics similar to those of its inhibition of NF-kappa B activation by TNF-alpha. Phosphorylation may not mediate receptor shedding, since PMA treatment did not detectably affect TNF-R alpha phosphorylation.

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface.

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.

Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties.

The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine. Ei and the activating enzyme were purified to homogeneity and structurally characterized. Ei has an alpha 2 oligomeric structure (2 X 85 kDa) and contains no cofactor. The amino acid composition has been determined. Out of a total of six cysteinyl residues per subunit, one shows an unusually fast reaction with iodoacetate (k2 = 7 (M-1 s-1) at pH 6.8, 30 degrees C), which is accompanied by loss of the activatability of the enzyme. The 1500-fold purified activating enzyme is a monomeric protein of 30 kDa. It contains a covalently bound, as yet unidentified chromophoric factor which has an optical absorption peak at 388 nm. Further studies of the in situ state of pyruvate formate-lyase detected a reversible backconversion of the active form Ea into Ei when anaerobic cells become nutrient-depleted.

Applicability of four new antibodies for the detection of fetal nucleated cells out of maternal blood by FISH analysis.

The availability of fetal cells from the maternal peripheral blood opens up the possibility for a noninvasive prenatal diagnosis. Until now this prospect has been hampered by the fact that there are no highly specific antibodies to fetal cells. In the present study, four as yet untested monoclonal antibodies - 2G5, 3F9, EPO-R and Flk1 - were analyzed in view of their effectiveness to stain fetal nucleated cells. Therefore, 20 ml of peripheral blood from 40 women carrying a male fetus were collected in the 16th to 28th weeks of pregnancy. Mononucleated cells, enriched by density gradient, were stained with one of the four antibodies and isolated with magnetic cell sorting. Subsequently, fluorescence in situ hybridization with chromosome X and Y centromere probes was performed which allowed us to analyze the maternal or fetal origin of the enriched cells. For evaluation, 500 nuclei per case were examined. The antibody 3F9 detected 0.2 cells on average (n = 5), 2G5 1.2 (n = 11), EPO-R 1.6 (n = 10) and Flk1 1.8 cells (n = 8) with fetal specific Y-positive hybridization signals. The antibody against CD71 (n = 6) used as control gave no Y-specific signals. Considering these results, the tested antibodies, especially Flk1, appear to be more successful in detecting fetal cells than the commonly used CD71 antibody.

Tumor necrosis factors-alpha and -beta bind to the same two types of tumor necrosis factor receptors and maximally activate the transcription factor NF-kappa B at low receptor occupancy and within minutes after receptor binding.

HL60 cells have types A and B tumor necrosis factor (TNF) receptors whereas HEp2 cells have only the type B receptor (Hohmann, H.-P., Remy, R., Brockhaus, M., and van Loon, A.P.G.M. (1989) J. Biol. Chem. 264, 14927-14934). TNF-beta can be cross-linked to each of these receptors and competes with TNF-alpha for binding to both receptors. TNF-alpha and TNF-beta activate the transcription factor NF-kappa B in HL60 and HEp2 cells. Maximal activation of NF-kappa B required binding of TNF-alpha or TNF-beta to 20-25% of the total number of TNF receptors and was achieved within minutes after the addition of TNF-alpha to HL60 cells. Both TNF-alpha and TNF-beta activate NF-kappa B at 5-10-fold lower concentrations in HL60 cells compared with HEp2 cells, and this correlates well with their different affinities for binding to these cells. Thus, TNF-alpha and TNF-beta are indistinguishable with respect to the correlation between degrees of receptor binding and activation of NF-kappa B.

Cell-free sulfation of the contact site A glycoprotein of Dictyostelium discoideum and of a partially glycosylated precursor.

An 80-kDa glycoprotein of Dictyostelium discoideum, designated contact site A, has been implicated in EDTA-stable cell adhesion. This protein is known to be the major sulfated protein of aggregation-competent cells and has been shown to contain two types of carbohydrate, sulfated type 1 and unsulfated type 2 carbohydrate moieties. Here we investigate the cell-free sulfation of this protein. In the homogenate of developing cells, [35S]sulfate was transferred by endogenous sulfotransferase from [35S]3'-phosphoadenosine-5'-phosphosulfate to the contact site A glycoprotein and to various other endogenous proteins. The sulfate was transferred to carbohydrate rather than to tyrosine residues. After differential centrifugation of the homogenate, the capacity for sulfation of the contact site A glycoprotein was barely detected in the plasma membrane-enriched 10,000 X g pellet fraction which contained the bulk of this glycoprotein, but was largely recovered in the 100,000 X g pellet fraction which contained only a small portion of this glycoprotein. After sucrose gradient centrifugation, the membranes containing the sulfation capacity were found to have a density characteristic for Golgi membranes. In immunoblots, monoclonal antibodies raised against the contact site A glycoprotein recognized not only this 80-kDa protein, but also a sulfatable 68-kDa protein found in the 100,000 X g pellet fraction. The 68-kDa protein did not react with monoclonal antibodies against type 2 carbohydrate but was converted by endoglycosidases F and H into a 53-kDa protein, indicating that it was a partially glycosylated form of the 80-kDa glycoprotein containing only type 1 carbohydrate. Isoelectric focusing showed that a substantial portion of the 68-kDa glycoprotein was unsulfated, even after cell-free sulfation. The 68-kDa glycoprotein was not found in the plasma membrane-enriched 10,000 X g pellet fraction and did not accumulate in parallel with the 80-kDa contact site A glycoprotein during cell development. We conclude that the 68-kDa glycoprotein is a precursor that is converted by attachment of type 2 carbohydrate and sulfation of type 1 carbohydrate into the mature 80-kDa glycoprotein. The precursor nature of the 68-kDa glycoprotein was supported by results obtained with mutant HL220 which is defective in glycosylation (Murray, B. A., Wheeler, S., Jongens, T., and Loomis, W. F. (1984) Mol. Cell. Biol. 4, 514-519). This mutant specifically lacks type 2 carbohydrate and produces a 68-Kda glycoprotein instead of the 80-kDa contact site A glycoprotein (Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2663-2670).(ABSTRACT TRUNCATED AT 400 WORDS)