Spermatozoa characteristics in six psittacine species using light microscopy.

Even though breeding of companion birds has increased continuously for years, the fecundity assessment of birds has hardly been acknowledged. Knowledge of the structure of spermatozoa is crucial for evaluation of the basic reproductive biology of any species as well as for phylogenetic research and cladistic analyses of internal relationships. Spermatozoa of six different psittacine species (Nymphicus hollandicus, Myiopsitta monachus, Agapornis roseicollis, Melopsittacus undulatus, Tanygnathus lucionensis, Guarouba guarouba) were examined using light microscopy. Head length (nucleus including acrosome), head width, midpiece length and tail length were measured and documented. Significant differences were obvious among almost all of the species for almost all four parameters. However, in all the six species a significant moderate correlation between spermatozoa midpiece lengths and tail lengths (r=0.535, p

A prospective trial on the safety of long-term intensive plasmapheresis in donors.

BACKGROUND AND OBJECTIVES: There are still concerns about the safety of long-term intensive donor plasmapheresis, because the reasons that donors drop out of plasmapheresis programmes have not been determined in prospective studies. MATERIALS AND METHODS: Seventy-two donors were switched from a moderate plasmapheresis programme to an intensive plasmapheresis programme and observed over a 3-year period. In addition to measuring total serum protein (TSP), albumin, immunoglobulin G (IgG) and haemoglobin (Hb) levels, parameters of iron metabolism and blood coagulation, and biochemical cardiovascular risk markers, were determined at baseline and at every 15th donation. We also collected statements from donors who dropped out of the plasmapheresis programme about their reasons for withdrawal. RESULTS: Dropouts were predominantly related to socioeconomic (n = 34) or medical reasons not related to plasma donations (n = 8). Three donors had to drop out when their TSP levels fell below threshold for the third time within a 5-week period. At baseline, donors had significantly lower TSP, albumin, IgG, Hb and ferritin levels than gender-matched and age-matched non-donor controls. However, subsequent intensive plasmapheresis over 3 years did not impair the individuals' ability to donate plasma. TSP, IgG, Hb, ferritin, transferrin, cardiovascular risk markers and parameters of blood coagulation did not change significantly during the observation period. CONCLUSIONS: The reasons why donors cease to participate in intensive plasmapheresis programmes are predominantly not directly related to the plasma donation itself.

Crystal structure of negative cofactor 2 recognizing the TBP-DNA transcription complex.

The X-ray structure of a ternary complex of Negative Cofactor 2 (NC2), the TATA box binding protein (TBP), and DNA has been determined at 2.6 A resolution. The N termini of NC2 alpha and beta resemble histones H2A and H2B, respectively, and form a heterodimer that binds to the bent DNA double helix on the underside of the preformed TBP-DNA complex via electrostatic interactions. NC2beta contributes to inhibition of TATA-dependent transcription through interactions of its C-terminal alpha helix with a conserved hydrophobic feature on the upper surface of TBP, which in turn positions the penultimate alpha helix of NC2beta to block recognition of the TBP-DNA complex by transcription factor IIB. Further regulatory implications of the NC2 heterodimer structure are discussed.

Isolation and characterization of a novel gene from the DiGeorge chromosomal region that encodes for a mediator subunit.

Hemizygous deletions on chromosome 22q11.2 result in developmental disorders referred to as DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS). We report the isolation of a novel gene, PCQAP (PC2 glutamine/Q-rich-associated protein), that maps to the DiGeorge typically deleted region and encodes a protein identified as a subunit of the large multiprotein complex PC2. PC2 belongs to the family of the human Mediator complexes, which exhibit coactivator function in RNA polymerase II transcription. Furthermore, we cloned the homologous mouse Pcqap cDNA. There is 83% amino acid identity between the human and the mouse predicted protein sequences, with 96% similarity at the amino- and carboxy-terminal ends. To assess the potential involvement of PCQAP in DGS/VCFS, its developmental expression pattern was analyzed. In situ hybridization of mouse embryos at different developmental stages revealed that Pcqap is ubiquitously expressed. However, higher expression was detected in the frontonasal region, pharyngeal arches, and limb buds. Moreover, analysis of subjects carrying a typical 22q11 deletion revealed that the human PCQAP gene was deleted in all patients. Many of the structures affected in DGS/VCFS evolve from Pcqap-expressing cells. Together with the observed haploinsufficiency of PCQAP in DGS/VCFS patients, this finding is consistent with a possible role for this novel Mediator subunit in the development of some of the structures affected in DGS/VCFS.

The chromatin structure of the dual c-myc promoter P1/P2 is regulated by separate elements.

The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.

Contributions of flow cytometry to the analysis of the myelodysplastic syndrome.

As the population of North America ages, the incidence of MDS is likely to rise. Epidemiologic survey instruments need to be put in place to document changes in the incidence. The basic mechanism of disease in MDS is largely unknown. No unifying, testable hypothesis is yet available, but apoptosis, with mitochondria playing a key role, are central to any discussion of MDS. Cytogenetic abnormalities have not provided an explanation of MDS but are of diagnostic and prognostic significance. The emergence of immunologic factors is of major importance and emphasizes the need for early detection. Flow cytometry can be used diagnostically to exclude other causes of cytopenias, document the phenotypic manifestations of myeloid dysmaturation, and provide blast enumeration. The distinctions between MDS and acute leukemia are arbitrary, and the process should be conceptualized as a continuum. There is a need for continued work to establish minimal diagnostic criteria for MDS. The current prognostic scoring systems do not incorporate findings from the newer technologies.

A single point mutation in TFIIA suppresses NC2 requirement in vivo.

Negative cofactor 2 (NC2) is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TATA-box-binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB. Here we study molecular mechanisms of repression by human NC2 in vivo in yeast. Yeast NC2 genes are essential and can be exchanged with human NC2. The physiologically relevant regions of NC2 have been determined and shown to match the histone-fold dimerization motif. A suppressor screen based upon limiting concentrations of NC2beta yielded a cold-sensitive mutant in the yeast TFIIA subunit Toa1. The single point mutation in Toa1 alleviates the requirement for both subunits of NC2. Biochemical characterization indicated that mutant (mt)-Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP-TFIIA-DNA complexes. Wild-type but not the mt-Toa1 can relieve NC2 effects in purified transcription systems. These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes.

Multi-platform, multi-site instrumentation and reagent standardization.

As flow cytometry laboratories involve themselves in more multi-site domestic and international clinical trial and research studies, it becomes imperative that they develop and adopt qualitative and quantitative standardization. This standardization does not need to be at the instrument-design level but it may evolve from a general consensus on instrument setup, internationally accepted standardized procedures, and quantitative fluorescence intensity units. Instrument condition, age, and setup as well as model and manufacturer all affect the overall instrument performance and quantitative characteristics. Therefore, when working with multiple instruments, platforms, or sites, a standard window of analysis is essential. Furthermore, we should strive to characterize instrument performance and quantitative indices so that data can be compared directly. The same thoughts and ideals hold true for standardizing procedures and reagents. Clones, conjugation, incubation times, pH, temperature, and other environmental conditions all combine to affect the qualitative and quantitative cellular indices that we are attempting to measure. Data are presented that illustrates why standardization is needed and how we have attempted to achieve it in our laboratories.

Interaction of PC4 with melted DNA inhibits transcription.

PC4 is a nuclear DNA-binding protein that stimulates activator-dependent class II gene transcription in vitro. Recent biochemical and X-ray analyses have revealed a unique structure within the C-terminal domain of PC4 that binds tightly to unpaired double-stranded (ds)DNA. The cellular function of this evolutionarily conserved dimeric DNA-binding fold is unknown. Here we demonstrate that PC4 represses transcription through this motif. Interaction with melted promoters is not required for activator-dependent transcription in vitro. The inhibitory activity is attenuated on bona fide promoters by (i) transcription factor TFIIH and (ii) phosphorylation of PC4. PC4 remains a potent inhibitor of transcription in regions containing unpaired ds DNA, in single-stranded DNA that can fold into two antiparallel strands, and on DNA ends. Our observations are consistent with a novel inhibitory function of PC4.

Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6.

The transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2 zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2alpha/DRAP1 and NC2beta/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2alpha in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

A conserved tissue-specific structure at a human T-cell receptor beta-chain core promoter.

The T-cell receptor (TCR) beta-chain promoters have been characterized as nonstructured basal promoters that carry a single conserved ubiquitous cyclic AMP-responsive element. Our investigation of the human TCR beta gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vbeta 8.1 promoter. The core of the promoter (positions -42 to +11) is recognized by the lymphoid cell-specific transcription factors Ets-1, LEF1, and AML1 as well as by CREB/ATF-1, as is demonstrated in gel shift and footprinting experiments. With the exception of LEF1, these factors activate transcription in T cells. Binding sites at the core region show little conservation with consensus sites. Nonetheless, CREB, Ets-1, and AML1 bind and activate cooperatively and very efficiently through the nonconsensus binding sites at the core promoter region. Moderate ubiquitous activation is further induced by CREB/ATF and Sp1 factors through proximal upstream elements. The tissue-specific core promoter structure is apparently conserved in other T-cell-specifically expressed genes such as the CD4 gene. Our observations suggest that both the enhancer and the promoter have a complex tissue-specific structure whose functional interplay potentiates T-cell-specific transcription.

Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro.

Mammalian cells contain activities that amplify the effects of activators on class II gene transcription in vitro. The molecular identity of several of these cofactor activities is still unknown. Here we identify poly(ADP-ribose) polymerase (PARP) as one functional component of the positive cofactor 1 activity. PARP enhances transcription by acting during preinitiation complex formation, but at a step after binding of transcription factor IID. This transcriptional activation requires the amino-terminal DNA-binding domain, but not the carboxyl-terminal catalytic region. In purified systems, coactivator function requires a large molar excess of PARP over the number of templates, as reported for other DNA-binding cofactors such as topoisomerase I. PARP effects on supercoiled templates are DNA concentration-dependent and do not depend on damaged DNA. The PARP coactivator function is suppressed by NAD+, probably as a result of auto-ADP-ribosylation. These observations provide another example of the potentiation of trancription by certain DNA-binding cofactors and may point to interactions of PARP with RNA polymerase II-associated factors in special situations.

A mechanism for repression of class II gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains.

Negative co-factor 2 (NC2) regulates transcription of the class II genes through binding to TFIID and inhibition of pre-initiation complex formation. We have isolated and cloned NC2, and investigated the molecular mechanism underlying repression of transcription. NC2 consists of two subunits, termed NC2alpha and NC2beta, the latter of which is identical to Dr1. The NC2 subunits dimerize and bind to TATA binding protein (TBP)-promoter complexes via histone fold domains of the H2A-H2B type. Repression of basal transcription requires the histone fold and carboxy-terminal domains of the NC2 subunits. Several mechanisms probably contribute to transcriptional repression. Binding of NC2 inhibits association of TFIIB with TBP-promoter complexes. NC2 binds directly to DNA, and binding of NC2 to TBP-promoter complexes affects the conformation of DNA, which could be one cause for the inhibition of TFIIB. In addition, multimerization of repressor-TBP complexes on DNA might inhibit the assembly of the pre-initiation complex. We suggest that binding of the repressor to TRP-promoter complexes establishes a mechanism that controls the rate of transcription by RNA polymerase II.

[Does piggy-back liver transplantation have a detrimental effect on venous drainage?--A comparative duplex ultrasound study]. / Hat die Piggy-back Lebertransplantation einen nachteiligen Einfluss auf den venösen Abfluss?--Eine duplexsonographische Vergleichsstudie.

Venous drainage in "Piggy-back" Liver Transplantation "Piggy-back" orthotopic liver transplant (OLT) may offer some advantages (hemodynamic stability without veno-venous bypass) over standard OLT, but there is concern about the risk of venous outflow obstruction associated with this technique. In this study (n = 19 piggy-back, n = 12 standard OLT) it was possible to show, by means of duplex scanning, that flow velocity, resistance and flow of hepatic veins do not differ significantly.

Activation of transcription by recombinant upstream stimulatory factor 1 is mediated by a novel positive cofactor.

The transcription factor USF1 belongs to the family of basic helix-loop-helix proteins that are involved in the regulation of various important cellular processes. Here we characterized the factors involved in the activation of transcription by upstream stimulatory factor 1 (USF1) in a reconstituted class II gene transcription system. Activation of transcription by both wild type USF1 and a GAL-USF (amino acids 1-94 of the yeast activator protein GAL4 fused to amino acids 17-196 of USF) fusion protein required the presence of at least one positive cofactor. A novel positive cofactor (PC5) that functions specifically through the activation domain of USF1 was partially purified and biochemicaly distinguished from previously described positive cofactors. The mechanism by which PC5 mediates activation of transcription through USF1 was investigated in order-of-addition experiments. PC5 had to be present during binding of transcription factor (TF) IID to the TATA box to observe transcriptional activation. However, this event alone did not result in transcriptional activation, which also required the presence of the activator and of PC5 after binding of TFIID. Hence, PC5 may enter transcription during binding of TFIID to function in concert with the activator during subsequent steps in transcription.

The coactivator p15 (PC4) initiates transcriptional activation during TFIIA-TFIID-promoter complex formation.

We have analyzed the mechanisms underlying stimulation of transcription by the activator GAL4-AH and the recombinant coactivator p15 (PC4). We show that p15 binds to both double-stranded and single-stranded DNA. Analyses of deletion mutants correlates binding to double-stranded DNA with the ability to mediate activator-dependent transcription. Consistent with this finding, phosphorylation of p15 by casein kinase II inhibits binding to double-stranded DNA and the activity of p15. The functional characterization suggests interactions of p15 with both DNA and components of the TFIID complex. GAL4-AH functions in concert with p15 during formation of TFIIA-TFIID-promoter (DA) complexes, as concluded from order-of-addition experiments. At limiting TFIID concentrations, the number of DA complexes is enhanced. The activator also stimulates transcription moderately after DA complex formation, independently of the concentrations of general transcription factors.