A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region.

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli.

Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing. In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon. The resultant suppressor tRNA, Su79, is a very strong amber suppressor. Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing. These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon. The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor. The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification. The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.

Comparative studies of Drosophila Antennapedia genes.

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster controls cell fates and pattern formation in the epidermis, nervous system and mesoderm of thoracic segments. Its expression is controlled at the levels of transcription, alternative RNA splicing, polyadenylation and translation. Two nested Antp transcription units extend over 103 kb and produce sixteen different transcripts. We have compared the Antp genes of Drosophila virilis, Drosophila subobscura and D. melanogaster to determine which structural features are conserved and therefore may be important to the gene's function. The overall gene structures are similar. There are many conserved sequence blocks throughout the large introns, at least 15 kb upstream of the first promoter, and at least 3 kb downstream of the last polyadenylation site. Intron and exon sequence conservation around alternative splice sites indicates that alternative protein coding forms may also be conserved. Protein coding potential is perfectly conserved around the C-terminal homeodomain, well conserved in the N-terminal region, and more variable in the middle. The large size of the Antp gene may reflect a large number of control elements necessary for appropriate Antp protein expression. The conservation of transcript complexity suggests functional requirements for the different protein forms.

POU domain factors in neural development.

Transcription factors serve critical roles in the progressive development of general body plan, organ commitment, and finally, specific cell types. Comparison of the biological roles of a series of individual members within a family permits some generalizations to be made regarding the developmental events that are likely to be regulated by a particular class of transcription factors. Here, we evidence that the developmental functions of the family of transcription factors characterized by the POU DNA binding motif exerts roles in mammalian development. The POU domain family of transcription factors was defined following the observation that the products of three mammalian genes, Pit-1, Oct-1, and Oct-2, and the protein encoded by the C. elegans gene unc-86, shared a region of homology, known as the POU domain. The POU domain is a bipartite DNA binding domain, consisting of two highly conserved regions, tethered by a variable linker. The approximately 75 amino acid N-terminal region was called the POU-specific domain and the C-terminal 60 amino acid region, the POU-homeodomain. High-affinity site-specific DNA binding by POU domain transcription factors requires both the POU-specific and the POU-homeodomain. Resolution of the crystal structures of Oct-1 and Pit-1 POU domains bound to DNA as a monomer and homodimer, respectively, confirmed several of the in vitro findings regarding interactions of this bipartite DNA binding domain with DNA and has provided important information regarding the flexibility and versatility of POU domain proteins. Overall the crystal structure of a monomer of the Oct-1 POU domain bound to the octamer element was similar to that predicted by the NMR solution structures of the POU-specific domain and the POU-homeodomain in isolation, with the POU-specific domain consists of four alpha helices, with the second and third helices forming a structure similar to the helix-turn-helix motif of the lambda and 434 repressors; several of the DNA base contacts are also conserved. A homodimer of the Pit-1 POU domain was crystallized bound to a Pit-1 dimer DNA element that is closely related to a site in the proximal promoter of the prolactin gene. The structure of the Pit-1 POU domain on DNA is very similar to that of Oct-1, and the Pit-1 POU-homeodomain/DNA structure is strikingly similar to that of other homeodomains, including the Oct-1 POU-homeodomain. The DNA contacts made by the Pit-1 POU-specific domain are also similar to those of Oct-1 and conserved with many made by the prokaryotic repressors. In the Oct-1 crystal, the POU-specific domain recognizes a GCAT half-site, while the corresponding sequence recognized by the Pit-1 POU-specific domain, GTAT, is on the opposing strand. As a result, the orientation of the Pit-1 POU-specific domain relative to the POU-homeodomain is flipped, as compared to the Oct-1 crystal structure, indicating the remarkable flexibility of the POU-specific domain in adapting to variations in sequence within the site. Also in contrast to the Oct-1 monomer structure is the observation that the POU-specific and POU-homeodomain of each Pit-1 molecule make major groove contacts on the same face of the DNA, consistent with the constraints imposed by its 15 amino acid linker. As a result, the Pit-1 POU domain homodimer essentially surrounds its DNA binding site. In the Pit-1 POU domain homodimer the dimerization interface is formed between the C-terminal end of helix 3 of the POU-homeodomain of one Pit-1 molecule and the N-terminus of helix 1 and the loop between helices 3 and 4 of the POU-specific domain of the other Pit-1 molecule. In contrast to other homeodomain crystal structures, the C-terminus of helix 3 in the Pit-1 POU-homeo-domain has an extended structure. (ABSTRACT TRUNCATED)

Hyperthyroidism with an FSH-and TSH-secreting pituitary adenoma.

A 34-year-old man was found to have elevated thyroxine (T4), triiodothyronine (T3), calculated free T4, thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and alpha subunits of TSH and FSH. A computed tomography scan of the head showed a 16-mm macroadenoma of the pituitary gland. There was no evidence of loss or excess secretion of other pituitary hormones. The large chromophobe adenoma was removed via a transphenoidal approach. The patient has been taken off all medication. Thyroid function has returned to normal and there has been no loss of pituitary secretory capacity of other pituitary hormones. The occurrence of a combined TSH- and FSH-secreting pituitary adenoma is rare; to the authors' knowledge, only one case has been documented in the literature.

Structure-function relations in the human placenta.

The human haemochorial placenta is a complex and dynamic interface between embryonic and maternal tissues. A myriad array of compounds has been identified at this interface, some of which exert local effects which might be important in maintaining the integrity of the organ. These compounds are diverse in nature and function; they include enzymes, hormones and bioactive peptides. Successful nidation requires the synchronization of endometrial maturation and embryonic development. The complex nature of this interface requires the application of sound sampling strategies. The new stereological methods have thrown fresh light on the growth and development of the human placenta. These methods permit the objective, quantitative description of morphology by efficient design-based methods. This approach has permitted a better definition of the functional morphology of the placenta. Applications of these methodologies are providing a spatial and temporal framework on which to lay the new physiological and molecular information. Here we review the essential features of the stereological approach, identify useful structural quantities and provide some examples of their application. The problems associated with the quantification of immunocytochemistry are illustrated with the use of immunoreactivity to insulin-like growth factor I receptor in normal placentae and in pre-eclampsia. Although stereology can provide useful quantitative information about the structure of this dynamic tissue, other anatomical methods that could be applied to better define the relationships between structure and function will be discussed. These include confocal microscopy, to examine the dynamic physiological interactions of the different tissue compartments, and low-temperature electron microscopy techniques such as cryosubstitution, to allow better access to the biochemical information resident in the tissue. The complex and dynamic nature of the tissue requires a multidisciplinary approach; central to these investigations is a comprehensive understanding of its fine structure.

Developmentally regulated alternative splicing of transcripts from the Drosophila homeotic gene Antennapedia can produce four different proteins.

Antennapedia (Antp) is a Drosophila homeotic gene that controls differentiation of the thoracic segments. Antp transcripts are produced from either of two promoters that are independently regulated in temporally and spatially distinct patterns. In addition, Antp transcripts utilize either of two major polyadenylation sites. Antp primary transcripts contain the same protein coding sequences. Alternative RNA splicing at two positions within the primary transcripts produces mRNAs that can encode four slightly different Antp proteins. Different classes of alternatively spliced transcript predominate early and late in Drosophila development, indicating that the Antp gene is regulated by the processing of its transcripts as well as by controlling their transcription. Alternative splicing appears to be independent of which promoter and which polyadenylation site is used.

Genetic analysis of insulin-like growth factor II and HLA-G in pre-eclampsia.

Pre-eclampsia (PE) is uniquely a disease of pregnancy and is the major cause of foetal and maternal morbidity and mortality. Epidemiological studies show that PE is highly heritable, with a high incidence in all populations. The underlying pathology indicates that absent or shallow invasion of foetal trophoblasts into maternal arteries is a feature of true PE. The objective of this study was to determine the genetic factors influencing PE. A large number of mother-father-baby trios were collected in which the first pregnancy was complicated by severe PE. After careful examination of the epidemiology and pathology of the disease, two plausible candidate genes, namely insulin-like growth factor II (IGF-II) and HLA-G, were analysed for association with PE. No association was found between a commonly occurring polymorphism in IGF-II and PE. Three polymorphisms in HLA-G were analysed in the sample cohorts. No association was found between three polymorphisms in HLA-G and PE. However, the frequency of the HLA-G insertion/deletion polymorphism in exon 8 deviated significantly from Hardy-Weinberg expectations in PE off-spring, reflecting an excess of heterozygotes for these polymorphisms in PE offspring. The significance of this deviation is not clear and further genetic analysis will be necessary to confirm this finding and to explore further the candidacy of HLA-G in PE.

Expression of the gene for the POU domain transcription factor Tst-1/Oct6 is regulated by an estrogen-dependent enhancer.

Expression of the POU domain protein Tst-1/Oct6 during development of glia and neurons is subject to a tight multifactorial control. Here we show that 17beta-estradiol increases the level of endogenous Tst-1/Oct6 in glial cells. This effect was mediated at the level of gene expression by an enhancer present in the 5' flanking region of the mouse gene for Tst-1/Oct6, approximately 5 kb upstream of the transcriptional start site. The enhancer contained as the functional element a sequence motif that closely resembled a classical estrogen response element. It consisted of an imperfect palindrome with a spacing of 3 bp, and was bound in vitro by activated estrogen receptor. Furthermore, this element was able to confer estrogen responsiveness when introduced into a heterologous promoter. In the Tst-1/Oct6 gene enhancer, a TPA response element was found in close proximity to the estrogen receptor binding site. As a consequence, TPA and estrogen activated transcription of the Tst-1/Oct6 gene in a synergistic manner.

Different patterns of transcription from the two Antennapedia promoters during Drosophila embryogenesis.

The homeotic genes of Drosophila control the differentiation of segments during development. Mutations in these genes cause one or more segments to develop structures normally found elsewhere in the organism. Several studies have shown that the spatial patterns of homeotic gene transcription are highly complex, and that these precise patterns of transcription are critical to normal development. The homeotic gene Antennapedia (Antp), a member of the Antennapedia Complex, is required for the correct differentiation of thoracic segments in both embryos and adults. The patterns of total Antp transcript and protein accumulation have been described in detail, but the contribution of each promoter to the overall pattern in embryos has not been reported. We have examined in detail the spatial distribution of transcripts from each of the Antp promoters in both embryo sections and whole embryos by in situ hybridization using promoter-specific probes. We show that the transcripts from each of the two promoters accumulate in distinct, but overlapping patterns during embryogenesis. The results demonstrate that the two Antp promoters are differentially regulated in embryos and provide a basis for examining the regulation of the two promoters and characterizing more fully the function of Antp during embryogenesis. In addition, we have examined the regulation of each of the Antp promoters by genes of the bithorax complex (BX-C). We show that in BX-C- embryos both promoters are derepressed in the abdomen.

Promyelinating Schwann cells express Tst-1/SCIP/Oct-6.

Tst-1/SCIP/Oct-6, a POU domain transcription factor, is transiently expressed by developing Schwann cells and is required for their normal development into a myelinating phenotype. In tst-1/scip/oct-6-null sciatic nerves, Schwann cells are transiently arrested at the "promyelinating" stage, when they have a one-to-one relationship with an axon but before they have elaborated a myelin sheath. To determine when Schwann cells express Tst-1/SCIP/Oct-6, we examined beta-galactosidase (beta-gal) expression in heterozygous tst-1/scip/oct-6 mice, in which one copy of the tst-1/scip/oct-6 gene has been replaced with the LacZ gene. beta-Gal expression from the LacZ gene seems to parallel Tst-1/SCIP/Oct-6 expression from the endogenous tst-1/scip/oct-6 gene in developing and regenerating sciatic nerves. Furthermore, electron microscopic examination of 5bromo-4-chloro-3-indolyl-beta-D-galactopyranoside- (X-gal) and halogenated indolyl-beta-D-galactoside- (Bluo-gal) stained nerves showed that promyelinating Schwann cells express the highest levels of beta-gal, both in developing and in regenerating nerves. Thus, the expression of beta-gal, a surrogate marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell development at which development is arrested in tst-1/scip/oct-6-null mice, indicating that Tst-1/SCIP/Oct-6 has a critical role in promyelinating Schwann cells.

Modification of representational difference analysis applied to the isolation of forskolin-regulated genes from Schwann cells.

Many aspects of the response of Schwann cells to axonal cues can be induced in vitro by the adenylyl cyclase activator forskolin, yet the role of cAMP signaling in regulating Schwann cell differentiation remains unclear. To define better the relationship between cAMP signaling and Schwann cell differentiation, we used a modification of cDNA representational difference analysis (RDA) that permits the analysis of small amounts of mRNA and identified additional genes that are differentially expressed by forskolin-treated and untreated Schwann cells. The genes that we have identified, including MKP3, a regulator of ERK signaling, and the sphingosine-1-phosphate receptor edg3/lp(B3), may play important roles in mediating Schwann cell differentiation.

SC35 plays a role in T cell development and alternative splicing of CD45.

Molecular diversity via alternative splicing is important for cellular function and development. SR proteins are strong candidate regulators of alternative splicing because they can modulate splice site selection. However, endogenous substrates for SR proteins are largely unknown, and their roles as splicing regulators in vertebrate development are unclear. Here we report that Cre-mediated conditional deletion of the prototypical SR protein SC35 in the thymus causes a defect in T cell maturation. Deletion of SC35 alters alternative splicing of CD45, a receptor tyrosine phosphatase known to be regulated by differential splicing during thymocyte development and activation. This study establishes a model to address the function of SR proteins in physiological settings and reveals a critical role of SC35 in a T cell-specific regulated splicing pathway.

Sequence of the human factor VIII-associated gene is conserved in mouse.

cDNA and genomic clones corresponding to the human factor VIII-associated gene (F8A) were isolated from mouse cDNA and F8A-enriched genomic libraries. The sequences of these clones revealed an intronless gene coding for 380 amino acids, with 85% identity to the predicted human sequence. The single murine gene copy is genetically linked to factor VIII, but appears to lie outside the factor VIII gene by physical mapping. Like the human gene, the mouse F8A gene is highly expressed in a wide variety of tissues. This evolutionary comparison has helped to clarify the derived amino acid sequence in the human and strongly supports the hypothesis that the F8A gene encodes a protein.

Reduced fertility in mice deficient for the POU protein sperm-1.

Members of the POU-homeodomain gene family encode transcriptional regulatory molecules that play important roles in terminal differentiation of many organ systems. Sperm-1 (Sprm-1) is a POU domain factor that is exclusively expressed in the differentiating male germ cell. We show here that the Sprm-1 protein is expressed in the haploid spermatid and that 129/Sv Sprm-1(-/-) mice are subfertile when compared with wild-type or heterozygous littermates yet exhibit normal testicular morphology and produce normal numbers of mobile spermatozoa. Our data suggest that the Sprm-1 protein plays a discrete regulatory function in the haploid spermatid, which is required for the optimal function, but not the terminal differentiation, of the male germ cell.

The accuracy of the enzyme-linked immunosorbent assay D-dimer test in the diagnosis of pulmonary embolism: a meta-analysis.

STUDY OBJECTIVE: We sought to determine the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) D -dimer test in the diagnosis of pulmonary embolism (PE) in the adult emergency department population. METHODS: A search of MEDLINE, EMBASE, and bibliographies of previous systematic reviews was conducted, with no language restriction. Experts in the field of PE research were contacted to identify unpublished studies. Prospective investigations involving a predominately outpatient population suspected of PE that used ELISA D -dimer tests were included. Two authors extracted data independently and assessed study quality on the basis of the patient spectrum and reference standard. Consensus was reached by means of conference. The analysis was based on a summary receiver operating characteristic curve and pooled estimates for sensitivity and specificity by using a random-effects model. RESULTS: The search yielded 52 publications. No unpublished studies were found. Eleven studies met the inclusion criteria and provided a sample of 2,126 patients. The summary receiver operating characteristic curve analysis found significant heterogeneity among the 11 studies. Subgroup analysis of the 9 studies that used traditional ELISA D -dimer methods yielded the most valid pooled estimates, with a sensitivity of 0.94 (95% confidence interval [CI] 0.88 to 0.97) and a specificity of 0.45 (95% CI 0.36 to 0.55). Advanced age resulted in a lower specificity. A prolonged duration of symptoms decreased the sensitivity and specificity. CONCLUSION: The ELISA D -dimer test is highly sensitive but nonspecific for the detection of PE in the clinical setting. This test might help clinicians safely rule out PE, especially in the face of low and low-to-moderate pretest probabilities.

Chromosomal localization of mouse and human genes encoding the splicing factors ASF/SF2 (SFRS1) and SC-35 (SFRS2).

The mammalian SR-type splicing factors ASF/SF2 and SC-35 play crucial roles in pre-mRNA splicing and have been shown to shift splice site choice in vitro. We have mapped the ASF/SF2 gene in mice and humans and the SC-35 gene in mice. Somatic cell hybrid mapping of the human ASF/SF2 gene (SFRS1 locus) reveals that it resides on chromosome 17, and fluorescence in situ hybridization refines this localization to 17q21.3-q22. Recombinant inbred mapping of the mouse ASF/SF2 gene (Sfrs1 locus) and the mouse SC-35 gene (Sfrs2 locus) demonstrates that both genes are located in a part of mouse chromosome 11 that is homologous to human chromosome 17. Mapping of Sfrs1 using F1 hybrid backcross mice between the strains C57BL/6 and DDK places Sfrs1 very near the marker D11Mit38 and indicates that the ASF/SF2 gene is closely linked to the Ovum mutant locus.

Use of capnography for assessment of the adequacy of alveolar ventilation during weaning from mechanical ventilation.

A prospective study was conducted to determine the reliability of noninvasive end-tidal CO2 (PETCO2) monitoring as a reflection of arterial CO2 tension (PaCO2) during weaning from mechanical ventilation (MV). Simultaneous PaCO2 and PETCO2 determinations were compared during MV and again during a spontaneous breathing trial just before returning the patient to MV. Three groups of patients recovering from acute respiratory failure were evaluated. Group 1 consisted of 16 patients (28 observations) without parenchymal lung disease. Group 2 consisted of 22 patients (31 observations) with alveolar filling diseases. Group 3 was composed of 13 patients (22 observations) with emphysema. Significant Pearson correlation coefficients were demonstrated between PaCO2 and PETCO2 during both MV and spontaneous breathing in all three groups. Significant correlation was also demonstrated between the change in PaCO2 and the change in PETCO2 associated with weaning for each group; however, the degree of correlation varied between groups. Our data suggest that capnography offers a reasonable estimate of PaCO2 and changes in PaCO2 during weaning in patients without parenchymal lung disease. However, PETCO2 is less sensitive to changes in PaCO2 for patients with parenchymal lung disease, particularly patients with emphysema. Interpretation of capnographic data requires a full understanding of its limitations. An approach to capnographic monitoring during weaning is discussed.

Tst-1/Oct-6/SCIP regulates a unique step in peripheral myelination and is required for normal respiration.

The terminal differentiation of myelinating glia involves complex interactions that culminate in the formation of myelin. The POU domain transcription factor Tst-1/Oct-6/SCIP is expressed transiently during myelination, and we report here that it has a critical role in this developmental process. Deletion of the Tst-1/Oct-6/SCIP gene produces a severe defect in peripheral myelination by arresting Schwann cell maturation before axonal wrapping. Unexpectedly, the activation of major myelin-specific genes appears to be unaffected by the Tst-1/Oct-6/SCIP mutation, demonstrating that multiple, independently regulated events are required for terminal differentiation of Schwann cells. In addition, aberrant differentiation and migration of specific neurons in Tst-1/Oct-6/SCIP mutant homozygotes is associated with a fatal breathing defect, providing a model for investigating the regulation of pulmonary homeostasis.