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Progressive encephalopathy and myopathy in transgenic mice expressing human foamy virus genes
(1991)
Transgenie mice carrying the bel region of human foamy retrovirus (HFV) under transcriptional control of its own long terminal repeat expressed tbe transgene in their centrat nervous systems and in smootb and striated muscle tissues. The animals developed a progressive degenerative disease of tbe centrat nervous system and of the striated muscle. Because expression of tbe transgene was dosely correlated witb the appearance of structural damage and inflammatory reactions were scanty, the disease is likely to be caused directly by tbe HFV proteins. These unexpected findings call for a reevaluation of tbe patbogenic potential of HFV in humans.
Dipole moments and various spectroscopic constants of some low-lying electronic states of the CaF molecule have been calculated using the multireference single· and double-excitation configuration-interaction (MRD-CI) method. The electronic structure of the highly ionic molecule in various excited states can be explained in tenns of different polarisations of the mainly Cacentered valence electron in the field of the F\(^-\) anion. Plots of natural orbitals occupied by the valence electron in the different states give a qualitative picture of the charge distribution and provide a visualisation of the different polarisations of the valence electron in the various states. Comparisons with the electrostatic polarisation model ofTörring, Ernstand Kändler (TEK model) are made. The unknown A' \(^2 \Delta\) state is predicted to lie about 21200 cm\(^{-1}\) above the ground state.
The goal of the present study was to determine whether 4- and 5-year-old kindergarten children could be trained to maintain an organizational strategy over 2- and 8 week periods through an elaborate training program. A second goal was to assess the effects of the training program on strategy awareness. Twenty-eight kindergarten children were pretested on two sort-recall tasks and their awareness of the use of the clustering strategy was assessed through a protocol type procedure. Half the children received seven half-hour sessions of individual training in the clustering strategy and half the children participated in a control group. Both groups were post-tested on two sort-recall tasks 2 weeks following training and again 8 weeks following training. Strategy awareness, as measured by verbal protocol, was assessed at both post-test points. The elaborate strategy training program was successful in inducing short- and long-term strategy maintenance of the clustering strategy. Trained children’s clustering during sorting and clustering during recall was consistently related to the amount of items correctly recalled. No differences in strategy awareness were found. These findings demonstrate that the elaborate training procedure used in this study can be a very effective memory technique for young kindergarten children.
A variety of muscarinic antagonists are currently used as tools to pharmacologically subclassify muscarinic receptors into M\(_1\), M\(_2\) and M\(_3\) subtypes. ln the present study I we have determined the affinity proflies of several of these antagonists at five cloned human muscarinic receptors (m1-m5) stably expressed in Chinesehamster ovary cells (CHO-K1). At all five receptorsl the (R)-enantiomers of trihexyphenidyl and hexbutinol displayed considerably higher affinities (up to 525-fold) than their corresponding (S)-isomers. The stereoselectivity ratios [inhibition constant( S)/inhibition constant(R)] for both pairs of enantiomers were lowest at m2 receptors, suggesting that less stringent configurational demands are made by this receptor subtype. The "M\(_1\)-selective" antagonist (R)-trihexyphenidyl displayed high affinities for m1 and m4 receptors. The "M\(_2\)-selective" antagonists himbacinel (±}-5, 11-dihydro-11-1[(2-[(dipropylamino)methyl]-1- piperidinyllethyl)amino]carbonyii-6H-pyrido(213-b)(1 ~4)benzodiazepine- 6-one (AF-DX 384)1 11-(14-[4-(diethylamino)butyl)-1-piperidinyll acetyl)-5~ 11-dihydro-6H-pyrido(2~3-b) (1~4)benzodiazepine-6-one (AQ-RA 741) and (+K11-(12-[(diethylamino)methyl]-1-piperidinyll acetyl)-5~ 11-di-hydro-6H-pyrido(2~3-b)(1,4)benzodiazepine-6-one (AF-OX 250; the (+)-enantiomer of AF-DX 116] exhibited high affinities for m2 and m41 intermediate affinities for m1 and m3 and low affinities for m5 receptors. This selectivity profile was most prominent for AQ-RA 7 41 I which displayed 195- and 129-fold higher affinities for m2 and m4 receptors than for mS receptors. The "M\(_3\)-selective" antagonist (±)-p-fluoro-hexahydro-sila-difenidol hydrochloride (pFHHsiD) exhibited high affinity for m1 I m3 and m4 receptors. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) bound with up to 7 -fold higher affinities to m1 I m31 m4 and m5 receptors than to m2 receptors. Although none of the tested antagonists showed more than 2-fold selectivity for one subtype over all other subtypes, each receptor displayed a unique antagonist binding profile.
Reliable prediction of the isotropic hyperfine coupling constant, a\(_{iso}\), is still a difficult task for ab initio calculations. Strong dependence on the method used for its calculation is found. Within a truncated multi-referencc ansatz a\(_{iso}\) is strongly affected by the size ofthe reference space and the nurober of terms in the truncated Cl expansion. In the present paperdifferent effects of the neglected Cl space are discussed. Modified B\(_K\) and A\(_K\) methods are used to estimate the contributions ofthe neglected configurations. lt can be shown that a combination of both methods is able to recover about 90-9 S% of the total error in a\(_{iso}\)· Furthermore, it was found that to obtain about 90% of the B\(_K\) correction only about I 0-20% ofthe configurations within H0 have to be corrected.
A panel of simple repetitive oligonucleotide probes has been designed and tested for multilocus DNA fingerprinting in some 200 fungal, plant and animal species as well as man. To date at least one of the probes has been found to be informative in each species. The human genome, however, has been the major target of many fingerprintins studies. Using the probe (CAC)5 or (GTG)5, individualization of all humans is possible except for monozygotic twins. Paternity analyses are now perfonned on a routine basis by the use of multilocus fingerprints, inctuding also cases of deficiency, i.e. where one of the parents is not available for analysis. In forensie science stain analysis is feasible in all tissue remains containing nuc)eated cells. Depending on the degree of DNA degradation a variety of oligonucleotides are informative, and they have been proven useful in actual case work. Advantages in comparison to other methods including enzymatic DNA amplification techniques (PCR) are evident. Fingerprint patterns of tumors may be changed due to the gain or loss of chromosomes and/or intrachromosomal deletion and amplification events. Locus-specific probes were isolated from the human (CAC)5/( GTG)5 fingerprint with a varying degree of informativeness (monomorphic versus truly hypervariable markers). The feasibility of three different approaches. for the isolation of hypervariable mono-locus probes was evaluated. Finally, one particular mixed simple (gt)n(ga)m repeat locus in the second intron of the HLA-DRB genes has been scrutinized to allow comparison of the extent of exon-encoded (protein-) polymorphisms versus intronie bypervariability of simple repeats: adjacent to a single gene sequence (e.g. HLA-DRB1*0401) many different length alleles were found. Group-specific structures of basic repeats were identified within the evolutionarily related DRB alleles. As a further application it is suggested here that due to the ubiquitous interspersion of their targets, short probes for simple repeat sequences are especially useful tools for ordering genomic cosmid, yeast artificial chromosome and phage banks.
A method was developed to detennine the affinities of antimuscarinic drugs at M\(_1\) receptors. [\(^3\)H](±)-Telenzepine served as radioligand in crude preparations of calf superior cervical ganglia and showed high affinity for a single receptor population. consisting of M1 receptors (K\(_D\) = 1.12 nM). Kinetic experiments showed monophasic association (k\(_1\) =0.017 min\(^{-1}\) nM\(^{-1}\) ) and dissociation (k\(_1\) = 0.017 min\(^{-1}\) ) kinetics, the half-life of dissociation being 41 min at 37°C. The kinetie K\(_D\) value amounted to 1.00 nM. M\(_1\) affinities for pirenzepine, methoctramine. hexahydro-sila-difenidol and p-fluoro-hexahydro-sila-difenidol detennined in competition experiments were similar to those found in functional studies with MI receptors in rabbit isolated vas deferens. The binding assay was used to deterriline the affinities of the (R) and (S) enantiomers of tertiary (trihexyphenidyl, hexahydro-difenidol. hexbutinol, p-fluoro-hexbutinol) and quatemary musearlnie antagonists (trihexyphenidyl methiodide. hexbutinol methiodide). Comparison of results obtained with the rabbit vas deferens suggested that the ionic environment may influence the affinities.
CARDIOVASCULAR and vasopressin (A VP) responses to hcmorrhagc wcrc studicd in rats with lesions of the hypothalamic supraoptic nuclei (SONL). Bleeding caused hypotension and increase in heart rate (HR) and A VP. SONL rats failed to fully recover from bleeding as compared to normal rats. Plasma A VP in SONL rats was in the normal in basal conditions, but failed to increase to levels attained in normal rats throughout the post-hemorrhage period. These data suggcst that the supraoptic nuclei are the primary regulatory sitcs for A VP release in rcsponse to hemorrhage and that lack of adequate A VP release significantly retards blood pressure recovery after bleeding.
So me species of the paleotropical tree genus Macaranga (Euphorbiaceae) live in elose association with ants. Thc genus comprises the full range of species from those not regularly inhabited by ants to obligate myrmecophytes. In Malaysia (peninsular and Borneo) 23 ofthe 52 species areknown to be ant-associated (44%). The simplest structural adaptation of plants to attract ants are extrafloral nectaries. We studied the distribution of extraflural nectaries in the genus Macaranga to assess the significance of this character as a possible predisposition for the evolution of obligate myrmecophytism. All species have marginal glands on the leaves. However, only the glands of nonmyrmecophytic species function as nectaries, whereas liquids secreted by these glands in myrmecophytic species did not contain sugar. Some non-myrmecophytic Macaranga and transitional Macaranga species in addition have extrafloral nectaries on the leaf blade near the petiole insertion. All obligatorily myrmecophytic Macaranga species, however, lack additional glands on the lamina. The non-myrmecophytic species are visited by a variety of different ant species, whereas myrmecophytic Macaranga are associated only with one specific ant-partner. Since these ants keep scale insects in the hollow sterns, reduction of nectary production in ant-inhabited Macaranga seems to be biologically significant. We interpret this as a means of (a) saving the assimilates and (b) stabilization of maintenance of the association's specificity. Competition with other ant species for food rewards is avoided and thereby danger ofweakening the protective function ofthe obligate antpartner for the plant is reduced. A comparison with other euphorb species living in the same habitats as Macaranga showed that in genera in which extrafloral nectaries are widespread, no myrmecophytes have evolved. Possession of extrafloral nectaries does not appear to be essential for the development of symbiotic ant-plant interactions. Other predispositions such as nesting space might have played a more important role.
Nucleoli provide the fascinating possibility of linking morphologically distinct structures such as those seen in the electron microscope with biochemical f eatures of the formation and step wise maturation of ribosomes. Localization of proteins by immunocytochemistry and of rRNA genes and their transcripts by in situ hybridization has greatly improved our understanding of the structural-functional relationships of the nucleolus. The present review describes some recent results obtained by electron microscopic in situ hybridization and argues that this approach has the potential to correlate each step of the complex pre-rRNA maturation pathway with nucleolar structures. Evidence is accumulating that the nucleolus-specific U3 snRNPs (small nuclear ribonucleoprotein particles) participate in rRNA processing events, similar to the role played by the nucleoplasmic snRNPs in mRNA maturation. The intranucleolar distribution of U3 snRNA is consistent with the view that it is involved in both early and late stages of pre-rRNA processing.
Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecu/ar identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confinn that ES 1 is a distant marker for R ( approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity Ievels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markersthat bridge the large distance between ESl and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding Iandmarks on a physical map of the R-containing chromosomal region.
A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the grain-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DHSa as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately SO times that of the combined E. coli catalases. The nucleutide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of tbe L. seeligeri catalase gene.
Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as weil as within protozoan organisms. Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human.embryo lung fibro:blastic cells, it is demonstrated that Legionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival of Legionella a genomic library of strain Legionella pneumophila Philadelphia I was established in Escherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further sturlies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella. Zusammenfassung: Intrazelluläres Überleben
Legionella pneumophila generares exotoxins, cytolysins, proteases oc hemolysins that darnage host cells llke erythrocytes or rissue cu lrure cells. The gene for a new L. pneumophila hemolysin withour a proteolytic activiry was idemified, cloned in E. coli and sequenced. The gene producr was analysed by SDS-Polyacrylamide-gel-electrophoresis.
The src-gene family in mammals and birds consists of 9 closely related protein tyrosine kinases. We have cloned the c-yes and fyn bomologues of the src-family from the teleost fish Xiphophorus helleri. Both genes show a high degree of sequence conservation and exhibit all structural motifs diagnostic for functional src-like protein tyrosine kinases. Sequence comparisons revealed three domains (exon 2, exons 3--6, exons 7-12) which evolve at different rates. Both genes exhibit an identical expression pattern, with preferential expression in neural tissues. No transcripts of c-yes were found in liver wbich is contrary to the situation in higher vertebrales. In malignant melanoma, elevated Ieveis of c-yes andfyn were detected indicating a possible function during secondary steps of tumor progression for src-related tyrosine kinases.
A rapid method for macroscopic and microscopic investigation of human CNS is proposed. After fonnalin fixation, gelatin or agarose embedding, and cryoprotective treatment, frozen human spinal cords, brainstems, or hemispheres can be serially cut into 0.7 mm thick slices. Stained with gallocyanin-chromalum, these slices facilitate cytoarchitectonic, neuropathologic, and quantitative examination. Regions of interest from parallel fonnalin-stored unstained slices can be embedded into paraffin and stained by any irnrnunocytologic and histologic stain compatible with fonnalin fixation and paraffin embedding.
Racemic dimethylphenyl(l-(phenylacetamido)ethyl)silane [rac-5) has been made by a four-step synthesis starting from (chloromethyl)dimethylphenylsilane [PhMe\(_2\)SiCH2Cl (1) ~ PhMe\(_2\)SiCH(Cl)Me (rac-2) - PhMe\(_2\)SiCH(l)Me (rac-3) - PhMe2SiCH(NH2)Me (rac-4) ~ PhMe\(_2\)SiCH[N(H)C(O)CH\(_2\)Ph]Me ( rac-5); total yield 41% ). Enantioselective enzymatic hydrolysis of rac-5, catalyzed by immobilized penicillin G acylase (E.C. 3.5.1.11) from Escherichia coli 5K (pHM 12), gave (R)-(1- aminoethyl)dimethylphenylsilane [( R )-4] in 40% yield with an enantiomeric purity of 92% ee.
Studies were performed in the rabbit aortic rings, precontracted with norepinephrine, to determine the subtype(s) of muscarinic receptors involved in endothelium-dependent relaxation and contraction in the absence of endothelium elicited by cholinergic stimuli. Acetylcholine (ACh) and arecaidine propargyl ester (APE), a M2 and M3 agonist, produced a dose-dependent relaxation and contraction in endothelium-intact and endothelium-denuded rabbit aortic rings, respectively. Both of these responses were blocked by the muscarinic receptor antagonist atropine. M1 selective agonist McN-A-343 [4-[N-(3-chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium+ ++ chloride] did not produce any effect on the tone of precontracted aortic rings. ACh- and APE-induced relaxation in aortic rings with intact endothelium was selectively blocked by M3 receptor antagonists hexahydrosila-difenidol and p-fluoro-hexahydro-sila-difenidol (pA2 of 7.84 and 7.18) but not by M1 antagonist pirenzepine or M2 receptor antagonists AF-DX 116 [11-(2-[(diethylamino)methyl]- 1-piperidinyl]acetyl)-5, 11-dihydro-6H-pyrido-[2,3-b][1,4]-benzo-diazepin-6-one] and methoctramine. ACh- and APE-induced contraction was inhibited by M2 receptor antagonists AF-DX 116 and methoctramine (pA2 of 7.11 and 6.71) but not by pirenzepine, hexahydro-sila-difenidol or p-fluoro-hexahydro-sila-difenidol. ACh- and APE-induced relaxation or contraction were not altered by nicotinic receptor antagonist hexamethonium or cyclooxygenase inhibitor indomethacin. These data suggest that relaxation elicited by cholinergic stimulin in endothelium-intact aortic rings is mediated via release of endothelium-derived relaxing factor consequent to activation of M3 receptors located on endothelial cells, whereas the contraction in aortic rings denuded of their endothelium is mediated via stimulation of M2 receptors located on smooth muscle cells.
The cDNA for ciliary neurotrophic factor (CNTF), a polypeptide involved in the survival of motoneurons in mammals, has recently been cloned (Stöckli et al., Nature, 342, 920 - 923, 1989; Lin et al. Science, 246, 1023 - 1025, 1989). We have now localized the corresponding gene Cntf to chromosome 19 in the mouse, using an interspecific cross between Mus spretus and Mus musculus domesticus. The latter was carrying the gene wobbler (wr) for spinal muscular atrophy. DNA was prepared from backcross individuals and typed for the segregation of species-specific Cntf restriction fragments in relation to DNA markers of known chromosomal location. The M.spretus allele of Cntf cosegregated with chromosome 19 markers and mapped closely to Ly-1, to a region of mouse chromosome 19 with conserved synteny to human chromosome 11q. Cntf is not linked to wr, and the expression of CNTF mRNA and protein appears close to normal in facial and sciatic nerves, of affected (wr/wr) mice, suggesting that motoneuron degeneration of wobbler mice has its origin in defects other than reduced CNTF expression.
Quasirelativistic and nonrelativistic lo-valence-electronp seudopotentialsf or Ca, Sr, and Ba are presented. Results of calculations with 6s6p5d basis sets for MH, MH\(^+\) , and MH\(_2\), are compared with all-electron and 2-valence-electron pseudopotential calculations with and , without core-polarization potentials. The lo-valence-electron pseudopotential approach agrees well with all-electron calculations. It circumvents problems for the 2-valence-electron pseudopotentials arising from an incomplete separation of valence and subvalence shells in polar molecular systems due to strongly contracted occupied (n - 1 )-d orbitals. All higherlevel calculations show SrH\(_2\) and BaII\(_2\), to be bent with angles of - 140° and 120°, respectively, while CaH\(_2\) is linear with a flat potential-energy surface for the bending motion. The use of a core-polarization potential together with the 2-valence-electronp seudopotentiala pproach allows an investigation of the relative importance of core-polarization vs direct d-orbital bonding participation as reasons for the bent structures. The calculations strongly suggest that both contribute to the bending in SrH\(_2\) and BaII\(_2\). Even at the Hartree-Fock level of theory lovalence- electronp seudopotentialc alculations given reasonablea nglesw hen the potentialenergy surface is not exceedingly flat, and only moderately contracted basis sets including both compact d functions and diffuse p functions are used. The effect of core-valence correlation and the importance off functions also are discussed.
Radioligand binding to A\(_1\) adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1 ,3-[\(^3\)H]dipropylxanthine ([\(^3\)H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [\(^3\)H]DPCPX binding showed that the most potent compounds were DPCPX with K\(_i\) values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with K\(_i\) values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radio Iigand 2-chloro-N\(^6\) -[\(^3\)H]cyclopen tyladenosine ([\(^3\)H]CCP A) were less pronounced, rauging from a K\(_D\) value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [\(^3\)H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoadenosine (NECA) and S-N\(^6\)-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different K\(_D\) values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the "classical" A\(_1\) receptor profile (R-PIA > -NECA > S-PIA) is not sufficient to speculate that A\(_1\) receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences.
Mutant proteins (muteins) of human lnterleukin-4 (llA) were constructed by means of in vitro mutagenesis. The muteins were expressed in E. co/1, submitted to a renaturation and purification protocol and analysed for biological activity. Exchange of the cysteines at either position 46 or 99 which form one of the three disulfide bridges resulted. in a nearly co•mplete loss · of biological actiyity and an unstable protein. The exchange of tyrosine 124 also inactivated the protein, while a mutation of tyrosine 56 left some residual activity. Exchange of the other four cysteines or of · the single tryptophane had smaller etTects.
To decrease immunogenicity of the rat kidney, grafts were perfused with an anti-MHC class li monoclonal antibody (mAb ). How effectively this procedure blocked dass li-positive cells, which were mainly dendritic in appearance, was checked by immunostaining renal sections after perfusion and comparing them with in vitro stained sections. Optimum conditions were applied for graft pretreatment before transplantation. This procedure prolonged graft survival, though not satisfactorily from the biological point ofview (9.6 ± 0.8 versus 7.7 ± 0.5 days in the control group; P < 0.02). The dendritic cells were not killed but blocked. Several hours after transplantation, the mAb dissociated from these dass li-positive cells. It was also shown that donor cells migrate into the recipient's spieen early after transplantation. The number of these cells was smaller when the transplanted organ was perfused with the mAb. Further studies are suggested to deplete the graft of donor dendritic cells more adequately. They should also combine graft perfusion with antidass II mAb and recipient immunosuppression at reduced doses.
Cell kinetic studies of T cells stimulated with the interleukin 2 (11-2), D-4, or both lymphokines were performed with conventional [3H] thymidine incorporation and with the bivariate BrdU/Hoechst technique. 11-2 and 11-4 are able to drive phytohemagglutininactivated T cells through more than one cell cycle. Neither synergistic nor inhibitory efl'ect on T -cell proliferationwas seen for the stimulation with both 11-2 and 11-4 as compared with the effect ofll-2 alone. The quantitative data ofthe cell cycle distribution ofphytohemagglutininactivated T cells suggestthat the population ofll-4-responsive cells is at least an overlapping population, if not a real subset of the ·population of the 11-2-responsive cells.
It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxyphenytisopropyladenosine [(R)-AHPIA] into the A, adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of celular cAMP Ieveis [Mol. Pharmacol. 30:403-409 (1986)]. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenytate cyclase Stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies in-dicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase Stimulation of up to SOOk of the control value. Similarly, the activation via these 10-20% of receptors occurs with a halflife that is only 2 times Ionger than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenytate cyclase Stimulation. These observations require a modification of the models of receptor-adenytate cyclase coupling, which is described in the accompanying paper [Mol. Pharmacol. 39:524-530 (1991)].
A genomic library of Legionello pneumophihz, the causative agent of Legionnaires disease in humans, was constructed in Escherichill coli K-12, and the recombinant clones were screened by immuno-colony blots with im antiserum raised against heat-killed L. pneumophilo. Twenty-three clones coding for a LegioneUa-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found tobe associated with the peptidoglycan layer bothin L. pneumophilo andin the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions witb a monospecific polyclonal 19-kDa protein-specific antiserum. Tbe protein was termed peptidoglycan-associated protein of L. pneumophilo (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb Clol fragment. DNA sequence studies revealed that two open reading frames, pplA and pplB, coding for putative proteins of 18~9 and 16.8 kDa, respectively, were located on the Clol fragment. Exonuclease 111 digestion studies confirmed tbat pplA is the gene coding for the peptidoglycan.;.associated 19-kDa protein of L. pneumophilo. The amino acid sequence of PpiA exhibits a high degree of homology to the sequences of the Pal Iipoproteins of E. coli K-12 and liaemophilus injluenvze.
Mechanistic possibilitles responsible for nonlinear shapes of the dose-response relationship in chemical carcinogenesis are discussed. (i) Induction and saturation of enzymatic activation and detoxification processes and of DNA repair affect the relationship between dose and steady-state DNA adduct Ievel; (ii) The fixation of DNA adducts in the form of mutations is accelerated by stimulation of the cell division, for Jnstance due to regenerative hyperplasia at cytotoxic dose Ievels; (iii) The rate of tumor formation results from a superposition of the rates of the individual steps. It can become exponential with dose if more than one step is accelerated by the DNA damage exerted by the genotoxic carcinogen. The strongly sigmoidal shapes often observed for dose-tumor incidence relationships in animal bioassays supports this analysis. A power of four for the dose in the su~linear part of the curve is the maximum observed (formaldehyde). In contrast to animal experiments, epidemiological data ln humans rarely show a slgnificant deviation from linearity. The discrepancy might be explained by the fact that a I arge nu mber of genes contribute to the overall sensitivity of an individual and to the respective heterogeneity within the human population. Mechanistic nonlinearities are flattened out in the presence of genetic and life-style factors which affect the sensitivity for the development of cancer. For a risk assessment, linear extrapolation from the high-dose lncidence to the spontaneaus rate can therefore be approprlate in a heterogeneous population even if the mechanism of action would result in a nonlinear shape of the dose-response curve in a homogeneaus population.
Rtgulatory aclio11s Iaken to reduu tht risk of harmfultffects of exposure to chemieals ofltn arenot commensurDtt with the toxicologicDf risk SJsstS&ment. A numbtr of factors relating to psychology, sociology, economics Dntl politics rather than science and medicine afftct tht final decision. Wemer Lutz and colleagues illustratt the situation using tht feuktmia-indudng chtmiCJJI benzene as an examplt.
Over a period of 3 years, Legionella pneumophila serogroup 6 strains were isolated from warm water outlets and dental units in the Dental Faculty and from the Surgery and Internal Medicine Clinics at the University of Dresden, Dresden, Germany. In the bacteriological unit of the above-mentioned facility, L. pneumophila serogroups 3 and 12 were grown frl,)m warm water specimens. The medical facilities are located in separate buildings connected with a ring pipe warm water system. All L. pneumophila serogroup 6 strains isolated from the warm water supply reacted with a serogroup-specific monoclonal antibody, but not with two other monoclonal antibodies which are subgroup specific, reacting with other serogroup 6 strains. The NolI genomic profiles obtained by pulsed-field gel electrophoresis of 25 serogroup 6 strains isolated from the Dental Faculty over a 3-year period, 1 isolate from the Internal Medicine Clinic, and 4 strains from the Surgery Clinic were identical. Furthermore, all these strains hybridized with a 3OO-kb NolI fragment when a legiolysin (lIy)-specific DNA probe was used. The NolI pattern, however, differed from those of six serogroup 6 strains of other origins, one serogroup 12 strain from the bacteriological unit, and another six unrelated strains of serogroups other than serogroup 6. L. pneumophila serogroup 6 strains which can be divided into only two subgroups by the use of monoclonal antibodies are differentiated in at least six Noli cleavage types obtained by pulsed-field electrophoresis.