Impaired activation of murine platelets lacking G alpha(i2).

The intracellular signaling pathways by which G protein-coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the G(i) pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet G alpha(i) subtype, G alpha(i2), has been disrupted. In intact platelets from G alpha(i2)-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin alpha IIb beta 3 (GPIIb-IIIa) were strongly reduced in platelets from G alpha(i2)-deficient mice. In addition, G alpha(i2)-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase--coupled platelet ADP receptor (P2Y(12)) on wild-type platelets with a selective antagonist. These observations suggest that G alpha(i2) is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the G alpha(i2)-linked ADP receptor.

Novel tricyclic benzothiazolo[2,3-c]thiadiazine antagonists of the platelet ADP receptor (P2Y(12)).

Novel non-nucleoside tricyclic platelet ADP receptor (P2Y(12)) antagonists have been discovered that bind reversibly and with high affinity to the platelet receptor. Condensation of various 2-aminobenzothiazoles with chlorosulfonylacetyl chloride affords these novel tricyclic heterocycles, which are novel and unpredicted products of this reaction.

Identification of the platelet ADP receptor targeted by antithrombotic drugs.

Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb-IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2Y(ADP), P2Y(AC), P2Ycyc or P2T(AC)) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the Gi-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.

Evidence for two distinct G-protein-coupled ADP receptors mediating platelet activation.

The identity of the receptors mediating platelet activation by ADP remains elusive. To distinguish between platelet ADP receptor subtypes, the effects of antagonists on platelet responses and the cloned P2Y1 receptor, a putative platelet ADP receptor, have been investigated. 2-methylthio-AMP (2MeSAMP), an inhibitor of ADP-dependent platelet aggregation, antagonized ADP-mediated inhibition of adenylyl cyclase, competed with binding of [3H]2-methylthio-ADP and inhibited the stimulation of [35S]GTPgammaS binding. 2MeSAMP did not inhibit platelet shape change and was only a weak antagonist of intracellular calcium mobilization in platelets or in cells expressing the cloned human P2YI receptor. By contrast, the P2Y1 receptor antagonist adenosine 3',5'-diphosphate (A3P5P) inhibited ADP-induced platelet aggregation, completely abolished shape change, but did not antagonize ADP effects on cyclic AMP generation or [3H]2-methylthio-ADP binding. However, A3P5P antagonized intracellular calcium mobilization in platelets and cells expressing the cloned P2Y1 receptor. Furthermore, using a specific monoclonal antibody and flow cytometry, P2Y1 receptor protein was detected on human platelets. These results support the existence of two G protein-coupled ADP receptors mediating platelet aggregation, one of which is coupled to Gi proteins and blocked by 2MeSAMP, whereas the second receptor is similar or identical to P2Y1 and coupled to Gq.

Cisplatin-DNA adducts inhibit ribosomal RNA synthesis by hijacking the transcription factor human upstream binding factor.

Several eukaryotic cellular proteins recognize DNA modified by the anticancer drug cisplatin (cis-diamminedichloroplatinum(II) or cis-DDP); among these proteins is a class of DNA-binding molecules containing the HMG (high-mobility group) box DNA recognition motif. We have previously reported the extraordinarily high binding activity to cisplatin adducts by human upstream binding factor (hUBF), an HMG box containing transcription factor that stimulates ribosomal RNA synthesis (Treiber et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5672-5676). In the present study, we discovered that (1) hUBF interacted selectively with DNA lesions formed by therapeutically effective platinum compounds [Pt(en)Cl2] and [Pt(dach)Cl2], in addition to the lesions formed by cis-DDP, suggesting a possible association with their anticancer effect; (2) multiple HMG boxes contributed additively to the hUBF-adduct interaction, providing a possible explanation for the unusually high affinity of hUBF for cis-DDP adducts as compared to the lower affinities of other HMG box proteins; and (3) ribosomal RNA transcription in a reconstituted system is specifically inhibited in the presence of cis-DDP adducts. In this third experiment, a ratio of adducts/promoter of approximately 4:1 completely abolished the transcription activated by hUBF. Taken together, these data lend support to the view that transcription factors involved in cellular growth regulation, such as ribosomal RNA transcription, may be hijacked by cis-DDP adducts resulting in functional inhibition.

Hydroperoxides mediate lithium effects on regeneration in Hydra.

Regeneration experiments in Hydra have shown that lithium long-term treatment apparently causes a transformation of prospective head into foot tissue. Although lithium ions are known to interfere with the PI-PKC signal-transduction system and evidence has been provided that this system plays a role in pattern formation in Hydra, its role in mediating the lithium effect on patterning is still obscure. The present study provides evidence that H2O2 and presumably also lipid hydroperoxides mediate the lithium effects. First, the perturbation of pattern formation is strikingly stronger in the strain Hydra vulgaris than in Hydra magnipapillata, and similar strain-specific differences are found in the long lasting accumulation of hydroperoxides following lithium treatment. Second, the antioxidant vitamins E and C, which suppress peroxide accumulation, and the H2O2-degrading enzyme catalase significantly protect H. vulgaris from lithium effects. Lithium treatment also negatively affects overall DNA synthesis in a similar strain-specific manner, which, however, cannot be rescued by antioxidant vitamins. The lithium-activated source of peroxide production differs from another source, which generates peroxide in untreated polyps of both strains. The results suggest that lithium treatment-induced peroxide accumulation in H. vulgaris provokes a cytotoxic response showing foot-like characteristics. Nevertheless, a role of peroxides as messengers in pattern forming processes can not be excluded.

Growth condition-induced precocious activation of p34cdc2 kinase inhibits the expression of developmental competence.

We examined the effect of growth conditions upon mitotic p34cdc2 kinase and developmental competence in Acanthamoeba castellanii. At G2/M of the cell cycle p34cdc2 kinase activity peaks in level, and p34cdc2 is transiently in a complex with newly synthesized cyclin B and phosphorylated on tyrosine (pre-MPF). Developmental competence peaks in level slightly preceding p34cdc2 kinase activation and pre-MPF accumulation. Under adverse growth conditions p34cdc2 kinase activation and transient pre-MPF accumulation occur at a reduced G2 phase length and smaller cell size. Developmental competence is not expressed during the shortened G2 phase. Cycloheximide inhibits the precocious p34cdc2 kinase activation and both the precocious accumulation of cyclin B and tyrosine phosphorylation on p34cdc2. No-codazole inhibits the precocious p34cdc2 kinase activation as well; however, it does not affect the precocious accumulation of pre-MPF. Developmental competence increases in level during artificially elongated G2 phases. The results indicate that, first p34cdc2 kinase activation requires cyclin B in a complex with p34cdc2 and the presence of intact microtubuli, second establishment of competence requires a certain length of G2, and third the regulation of pre-MPF accumulation, and thus of cyclin B expression, plays a role in the relationship among growth condition, cell cycle progression, and expression of the developmental program.

Cisplatin-DNA adducts are molecular decoys for the ribosomal RNA transcription factor hUBF (human upstream binding factor).

The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) approximately 60 pM) to the intrastrand cis-[Pt(NH3)2](2+-d(GpG) crosslink formed by the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin). When protein blots of human cell extracts are probed with cisplatin-modified DNA, 97- and 94-kDa proteins are detected, consistent with the known sites of hUBF species. A similar analysis of blots containing in vitro translated hUBF confirmed that the protein binds cisplatin adducts with high specificity. By contrast, DNA adducts of the clinically ineffective trans isomer of cisplatin, trans-diamminedichloroplatinum(II), are not recognized by hUBF. DNase I inhibition patterns of hUBF bound to a 100-base-pair DNA fragment containing a centrally located cis-[Pt(NH3)2](2+)-d(GpG) crosslink reveal specific protein-DNA interactions in a 14-base-pair region flanking the adduct. The affinity of hUBF for the rRNA promoter is similar (Kd(app) approximately 18 pM) to that measured for the cisplatin adduct. In addition, we observe that the hUBF-promoter interaction is highly sensitive to the antagonistic effects of cisplatin-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hijacking mechanisms could disrupt rRNA synthesis, which is stimulated in proliferating cells.

Multiple domains of the RNA polymerase I activator hUBF interact with the TATA-binding protein complex hSL1 to mediate transcription.

Recent evidence suggests that transcription initiation by all three eukaryotic RNA polymerases involves a complex of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, we map the functional domains of the nucleolar HMG box protein hUBF, which binds to the human rRNA promoter and stimulates transcription by RNA polymerase I through cooperative interactions with a distinct TBP-TAF complex, hSL1. DNase I footprint analysis of mutant hUBF proteins and of a synthetic peptide of 84 amino acids reveals that HMG box 1 is necessary and sufficient for DNA sequence specificity, whereas other HMG boxes and the amino terminus modulate the binding efficiency. hUBF contains multiple activation domains that include the acidic carboxyl terminus and three HMG boxes. HMG boxes 3 and 4 and the acidic tail contribute significantly to an extended footprinting pattern in the presence of hSL1, suggestive of specific protein-protein interactions. Moreover, the inability of xUBF from Xenopus laevis to form an initiation complex with hSL1 can be overcome by hybrid proteins containing human HMG box 4 and the acidic carboxyl terminus. These results strongly suggest an important role of transcription activation domains of hUBF in mediating interactions with the TBP-TAF complex hSL1.

Assembly of alternative multiprotein complexes directs rRNA promoter selectivity.

How can trans-activators with the same DNA binding specificity direct different transcriptional programs? The rRNA transcriptional apparatus offers a useful model system to address this question and to dissect the mechanisms that generate alternative transcription complexes. Here, we compare the mouse and human transcription factors that govern species-specific RNA polymerase I promoter recognition. We find that both human and mouse rRNA transcription is mediated by a specific multiprotein complex. One component of this complex is the DNA-binding transcription factor, UBF. Paradoxically, human and mouse UBF display identical DNA binding specificities even though transcription of rRNA is species specific. Promoter selectivity is conferred by a second essential factor, SL1, which, for humans, does not bind DNA independently but, instead, cooperates with UBF in the formation of high-affinity DNA-binding complexes. In contrast, mouse SL1 can selectively interact with DNA in the absence of UBF. Reconstituted transcription experiments establish that UBF and RNA polymerase I from the two species are functionally interchangeable, whereas mouse and human SL1 exhibit distinct DNA binding and transcription activities. Together, these results suggest a critical role for a specific multiprotein assembly in RNA polymerase I promoter recognition and reveal distinct mechanisms through which such complexes can generate functional diversity.

Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins.

The eukaryotic upstream binding factor (UBF), recognizes the ribosomal RNA gene promoter and activates transcription mediated by RNA polymerase I through cooperative interactions with the species-specific factor, SL1. Isolation of complementary DNA clones and sequence analysis reveals similarities between DNA binding domains of human UBF (hUBF) and high mobility group (HMG) protein 1. Expression, cellular localization and in vitro transcription studies establish that cloned hUBF encodes a nucleolar factor that binds specifically to the upstream control element and core of the rRNA gene promoter to activate transcription in a binding site-dependent manner.

[Lipoma of the uterus. Clinical aspects and pathology of a rare disease entity]. / Lipom des Uterus. Klinik und Pathologie einer seltenen Krankheitsentität.

Lipomatous lesions of the uterus are rare but may lead to problems in the differential diagnosis of uterine tumors. A typical case of uterine lipoma illustrates the characteristic clinical and morphological findings. Additional immuno-histochemical studies delineate the histogenesis of uterine lipomas.

Relationship between the timing of DNA replication and the developmental competence in Acanthamoeba castellanii.

In Acanthamoeba, two different cell types are known. Trophozoites are generated in the mitotic division cycle, whereas cells committed at late G2 phase of the cell cycle develop into cysts in response to starvation. In this paper we study the role of timing of DNA replication in regulating development. The investigation was performed with cultures growing in a non-defined medium (ND cells) that show a high encystation competence and with cultures that have been growing in a chemically defined medium (D cells) for several years and show a low encystation competence. Bivariate DNA/BrdUrd distributions show that ND cells progress through a cycle in which the short replication phase occurs immediately and exclusively after prior completion of mitosis. These cells arrest at late G2 phase of the cell cycle during the stationary stage. In D cells, DNA replication and mitosis seem to be uncoupled, since replication takes place before as well as after mitosis. These cells arrest within their replication phase during the stationary stage. These findings indicate that D cells do not progress into late G2 phase of the cell cycle and hence do not have the competence for commitment. The alternate timing of DNA replication and the low encystation competence of D cells can be reversed by cultivation of these cells in ND medium. Synchronization experiments reveal that late G2 phase ND cells exhibit a low capacity for BrdUrd incorporation and growth after transfer into D medium, whereas ND cells of earlier phases of the cell cycle show premitotic incorporation of BrdUrd into nuclear DNA and growth. These findings suggest on the one hand that premitotic DNA synthesis is a prerequisite for growth of cells in D medium, and that there is a dependence of the induction of premitotic DNA synthesis on the cell cycle, and on the other hand that a reciprocal relationship exists between the capacity of premitotic DNA synthesis and commitment to differentiation.

Functional cooperativity between transcription factors UBF1 and SL1 mediates human ribosomal RNA synthesis.

The human ribosomal RNA promoter contains two distinct control elements (UCE and core) both of which are recognized by the sequence-specific DNA binding protein UBF1, which has now been purified to apparent homogeneity. The purified factor activates RNA polymerase I (RNA pol I) transcription through direct interactions with either control element. A second RNA pol I transcription factor, designated SL1, participates in the promoter recognition process and is required to reconstitute transcription in vitro. Although SL1 alone has no sequence-specific DNA binding activity, deoxyribonuclease I footprinting experiments reveal that a cooperative interaction between UBF1 and SL1 leads to the formation of a new protein-DNA complex at the UCE and core elements. In vitro transcription experiments indicate that formation of the UBF1-SL1 complex is vital for transcriptional activation by UBF1. Thus, protein-protein interactions between UBF1 and SL1 are required for targeting of SL1 to cis-control sequences of the promoter.

Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene.

Two glucocorticoid response elements (GREs) located 2.5 kb upstream of the transcription initiation site of the tyrosine aminotransferase gene were identified by gene transfer experiments and shown to bind to purified glucocorticoid receptor. Although the proximal GRE has no inherent capacity by itself to stimulate transcription, when present in conjunction with the distal GRE, this element synergistically enhances glucocorticoid induction of gene expression. Cooperativity of the two GREs is maintained when they are transposed upstream of a heterologous promoter. An oligonucleotide of 22 bp representing the distal GRE is sufficient to confer glucocorticoid inducibility. As evidenced by the mapping of DNAase I hypersensitive sites, local alterations in the structure of chromatin at the GREs take place as a consequence of hormonal treatment.

Effect of essential amino acids on the phosphorylation of a 40S ribosomal protein and protein synthesis in Acanthamoeba castellanii.

Reversible and multiple phosphorylation of a 40S ribosomal protein is observed in a variety of eukaryotic cells. In the primitive eukaryote Acanthamoeba, one or three phosphorylated S3 derivatives are observed during growth phase in nondefined nutrient medium (ND cells) or in chemically defined nutrient medium (D cells), respectively. In both cases, stationary phase cells exhibit nonphosphorylated S3; however, transfer of these cells into the respective fresh nutrient media results in a transient accumulation of four phosphorylated S3 derivatives. Transfer of D cells into nutrient medium, deficient in all or any single essential amino acids, leads to reversible inhibition of S3 phosphorylation and growth arrest. The low level of phosphorylated S3 is not simply the consequence of growth arrest, since in cells where growth is arrested differently, the level of phosphorylated S3 can be high. In response to amino acid deficiency, a number of other changes can be observed. These include a 2-3-fold decrease of total protein synthesis, 13 changes in the cellular protein pattern, and specific alterations in the ribosome absorbance profiles and in the distribution of poly-A+ RNA within subribosomal and ribosomal fractions. While the rate of total protein synthesis seems to be associated with the level of phosphorylated S3, the level of the synthesis of at least 10 of the particular proteins can be dissociated from the level of S3 phosphorylation.

New cosmid vectors developed for eukaryotic DNA cloning.

A series of ColE1 and pSC101 cosmid vectors have been constructed suitable for cloning large stretches of DNA. All contain a single BamHI site allowing cloning of Sau3A, MboI, BglII, BclI , and BamHI-generated fragments. These vectors have the following characteristics: (i) they are relatively small (1.7-3.4 kb); (ii) the BamHI cloning site is flanked by restriction enzyme sites enabling direct cloning of unfractionated insert DNA without generating multiple insert or vector ligation products [ Ish - Horowitz and Burke, Nucl . Acids Res. 9 (1981) 2989-2998]; (iii) two vectors ( pHSG272 and pHSG274 ) contain a hybrid Tn5 KmR/ G418R gene which is selectable in both prokaryotic and eukaryotic cells, making them suitable for transferring DNA into eukaryotic cells, and (iv) the different prokaryotic selectable markers available in the other vectors described facilitate cosmid rescue of the transferred DNA sequences from the eukaryotic cell: CmR, ApR, KmR, ( pHSG429 ), CmR, ( pHSG439 ), colicin E1 immunity ( pHSG250 ), (v) the cosmid pHSG272 was used successfully to construct a shuttle vector based on the BPVI replicon [ Matthias et al., EMBO J. 2 (1983) 1487-1492].

Coordinate regulation of synthesis of ribosomal proteins during encystation of Acanthamoeba castellanii.

The synthesis of ribosomal proteins has been examined during growth and encystment of Acanthamoeba castellanii. Cells have been radiolabelled with [35S]methionine and ribosomal proteins have been extracted either from ribosomes or from total cell extracts. The results show that there is less synthesis of ribosomal proteins relative to total cell proteins soon after transfer of growing cells into non-nutrient medium, suggesting that the synthesis of all ribosomal proteins is under coordinate control. Furthermore, synthesis of ribosomal RNA and ribosomal proteins seems to be regulated coordinately, since within 1 h there is 70-80% decline in synthesis of both. Translation of total RNA in vitro indicates that the relative decrease in the synthesis of ribosomal proteins during development is not only due to a relative decrease in the content of ribosomal protein mRNA. However, a considerable (about 10-fold) and coordinate decrease in the relative content of ribosomal protein mRNA occurs during encystment. These data suggest a difference in the control of the synthesis of total proteins and ribosomal proteins as well as in the regulation of the content of total mRNA and ribosomal protein mRNA. The comparison of patterns of ribosomal proteins indicates that during development no qualitative changes occur. The content of one protein of the small ribosomal subunit is low in growing cells and increases during encystation. Since the differences in the concentration of this protein are neither due to changing levels of the corresponding mRNA nor to changing rates of synthesis, changing affinities of this protein to ribosomes in vivo are suggested.

Ribosomal phosphoproteins in Acanthamoeba castellanii.

Ribosomes and subunits from Acanthamoeba castellanii were purified. The pattern of ribosomal proteins was analysed by two-dimensional gel electrophoresis. The small subunit of cytoplasmic ribosomes contains 25 and the large 40 proteins. After incubation of exponentially growing cells with ortho[33P]phosphate for 1 h, one basic protein of the small subunit was radiolabelled. This phosphorylated protein has a molecular weight of about 45000 as estimated by dodecylsulfate/polyacrylamide gel electrophoresis. The level of phosphorylated ribosomal protein as estimated by staining of proteins separated by two-dimensional gel electrophoresis changes in response to growth conditions. The maximum level occurs after adding fresh nutrient medium to cells which have been allowed to grow to high density, whereas ribosomes of exponentially growing cells contain low amounts. Transfer of exponentially growing cells into a non-nutrient medium induces the formation of cysts containing ribosomes almost devoid of phosphorylated protein.