Not all voxels are created equal: Reducing estimation bias in regional NODDI metrics using tissue-weighted means.

Neurite orientation dispersion and density imaging (NODDI) estimates microstructural properties of brain tissue relating to the organisation and processing capacity of neurites, which are essential elements for neuronal communication. Descriptive statistics of NODDI tissue metrics are commonly analyzed in regions-of-interest (ROI) to identify brain-phenotype associations. Here, the conventional method to calculate the ROI mean weights all voxels equally. However, this produces biased estimates in the presence of CSF partial volume. This study introduces the tissue-weighted mean, which calculates the mean NODDI metric across the tissue within an ROI, utilising the tissue fraction estimate from NODDI to reduce estimation bias. We demonstrate the proposed mean in a study of white matter abnormalities in young onset Alzheimer's disease (YOAD). Results show the conventional mean induces significant bias that correlates with CSF partial volume, primarily affecting periventricular regions and more so in YOAD subjects than in healthy controls. Due to the differential extent of bias between healthy controls and YOAD subjects, the conventional mean under- or over-estimated the effect size for group differences in many ROIs. This demonstrates the importance of using the correct estimation procedure when inferring group differences in studies where the extent of CSF partial volume differs between groups. These findings are robust across different acquisition and processing conditions. Bias persists in ROIs at higher image resolution, as demonstrated using data obtained from the third phase of the Alzheimer's disease neuroimaging initiative (ADNI); and when performing ROI analysis in template space. This suggests that conventional ROI means of NODDI metrics are biased estimates under most contemporary experimental conditions, the correction of which requires the proposed tissue-weighted mean. The tissue-weighted mean produces accurate estimates of ROI means and group differences when ROIs contain voxels with CSF partial volume. In addition to NODDI, the technique can be applied to other multi-compartment models that account for CSF partial volume, such as the free water elimination method. We expect the technique to help generate new insights into normal and abnormal variation in tissue microstructure of regions typically confounded by CSF partial volume, such as those in individuals with larger ventricles due to atrophy associated with neurodegenerative disease.

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

Yeast strains in which the Ras-cyclic AMP (cAMP) pathway is constitutively active are sensitive to heat shock, whereas mutants in which the activity of this pathway is low are hyperresistant to heat shock. To determine the molecular basis for these differences, we examined the transcriptional induction of heat shock genes in various yeast strains. Activation of heat shock genes was attenuated in the strains in which the Ras-cAMP pathway is constitutively active. In contrast, in a strain deficient in cAMP production, several heat shock genes were induced by removal of cAMP from the medium. These results indicate that the Ras-cAMP pathway affects the induction of heat shock genes. In all of the mutants, heat shock transcription factor expression and activity were identical to those in wild-type cells. The response to heat shock in Ha-ras-transformed rat fibroblasts was also studied. While no induction of Hsp68 was observed in Ha-ras-transformed cells, proper regulation of heat shock transcription factor was found. Therefore, in mammals, as in Saccharomyces cerevisiae, the Ras pathway controls the transcription of heat shock genes via a mechanism not involving the heat shock transcription factor.

The essential yeast NLT1 gene encodes the 64 kDa glycoprotein subunit of the oligosaccharyl transferase.

The yeast oligosaccharyl transferase catalyzes the glycosylation of asparagine residues in secreted, vesicular, and membrane proteins. A complex of at least four membrane-bound polypeptides is responsible for oligosaccharyl transferase activity. Amino acid sequences from the 64 kDa glycoprotein subunit of the complex were used to clone the essential NLT1 (N-linked oligosaccharyl transferase) gene. The Nlt1p gene product is a processed, multiply glycosylated type I membrane protein; it has an extensive amino-terminal soluble domain, a potential hydrophobic transmembrane domain, and a short carboxy-terminal soluble domain. The Nlt1p is significantly similar than the mammalian ribophorin I, a component of the mammalian oligosaccharyl transferase complex, and the enzyme is conserved throughout eukaryotic evolution.

Allied health faculty attitudes toward rural clinical practice.

Research findings of allied health practitioners' attitudes toward rural practice are limited. The purpose of this study was to identify attitudes of faculty members in a school of allied health toward rural vs. urban living, clinical education, and practice. A survey consisting of demographic and attitudinal questions was mailed to 233 faculty representing five professions. The response rate was 63.5%. The majority viewed rural living as having both positive and negative aspects. Placement of clinical students in rural areas was seen as enhancing rural recruitment. Rural professional issues were viewed as mixed with the most positive aspect being greater intellectual challenge. There were a few significant attitude differences by gender, age, years of experience, profession, hometown location, and practice location site. The findings of this study generally support previous research and contribute additional knowledge regarding attitudes toward rural practice. Further studies of allied health professionals appear warranted.

Charting by exception.

Documentation takes up a significant portion of a home care nurse's daily workload. As cost containment measures reduce the amount of time nurses can spend with each patient, they need to find ways to make documentation more efficient to avoid compromising patient care. One agency found that charting by exception reduces documentation time so that nurses can devote more hours to hands-on care.

A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity.

Drosophila RNA polymerase II requires at least two chromatographically distinct transcription factors (designated A and B) to initiate transcription accurately in vitro. We describe the partial purification and concentration of one of these transcription factors, the B factor. Footprint analysis of the B fraction demonstrated the presence of a sequence-specific DNA-binding component in the transcription factor preparation. This component binds specifically to a 65 bp region of DNA surrounding the start point of transcription of the histone H3, H4, and actin 5C genes. Included in this binding region is the TATA box, the start point of transcription, and a portion of the leader region. The pattern of protection from DNAase I cleavage on the coding strand of the histone H3 gene is asymmetric with regard to the complementary noncoding strand. Sequence-specific binding of the B fraction occurs in the apparent absence of RNA polymerase II. The potential function of the binding component in the initiation of transcription by RNA polymerase II is discussed.

The 3' end of drosophila histone H3 mRNA is produced by a processing activity in vitro.

We have examined the process by which the 3' terminus of the Drosophila histone H3 mRNA is produced in vitro. When a template containing a portion of DNA that flanks the normal 3' end of the histone H3 gene and an oligo dC tail on the template strand is transcribed in vitro by Drosophila RNA polymerase II, transcription continues beyond the 3' end of the H3 gene. A processing activity was identified that cleaves the precursor transcript generating an RNA species with the same 3' end as the mature H3 mRNA. The processing activity was partially purified by ion exchange chromatography and sucrose gradient sedimentation. The isolated activity was found to require Mg++ but did not require addition of a nucleoside triphosphate for activity. The activity sedimented with a molecular weight of approximately 140,000 daltons. Transcription of the template and processing of the RNA can be uncoupled in vitro.

A Drosophila RNA polymerase II transcription factor binds to the regulatory site of an hsp 70 gene.

A Drosophila RNA polymerase II transcription factor that is specific for at least one of the heat-shock genes has been isolated (designated HSTF for heat-shock transcription factor). This factor is required for active transcription of an hsp 70 gene in addition to RNA polymerase II and another general transcription factor, the A factor. Footprint analysis of the HSTF on the hsp 70 gene reveals that it binds specifically to a 55 bp region upstream from the TATA box. Both coding and noncoding DNA strands are completely protected from DNAase I cleavage by the HSTF . HSTF binding occurs in the apparent absence of RNA polymerase II. The HSTF is present in both heat-shocked and nonshocked cells, although it is more transcriptionally active when isolated from heat-shocked cells. The previously described B factor (an RNA polymerase II transcription factor that binds to the TATA box), isolated from nonshocked cells, is significantly reduced in both binding and transcriptional activity in heat-shocked cells. The potential role of the HSTF and the B factor in the activation of heat-shock gene transcription is discussed.

Binding of Drosophila heat-shock gene transcription factor to the hsp 70 promoter. Evidence for symmetric and dynamic interactions.

A Drosophila heat-shock gene transcription factor (HSTF) has been shown to bind to three domains upstream from the TATA homology on a hsp 70 gene. The domain closest to the TATA homology consists of two contiguous binding sites with different binding affinities. Occupancy of the TATA homology proximal site (site 1) coordinates HSTF binding to the neighboring site (site 2) in a cooperative manner (Topol, J., Ruden, D. M., and Parker, C. S. (1985) Cell 42, 527-537). We have used alkylation interference and protection experiments to determine which residues within the binding sites are closely contacted by the HSTF. The contacts inferred from these studies included the residues present in the consensus sequence found in all HSTF binding sites and exhibit rotational symmetry, suggestive of a multimeric HSTF. By employing a gel electrophoresis separation technique we were able to resolve two protein-DNA complexes consisting of site 1 occupancy (complex A) and sites 1 and 2 occupancy (complex B). Analysis of these discrete species reveals that a subset of contacts within site 1 change upon HSTF binding to site 2, suggesting that a conformational change in the protein-DNA complex occurs. Implications for the activation of heat-shock gene transcription are discussed.

Bending of promoter DNA on binding of heat shock transcription factor.

The Drosophila heat-shock transcription factor (HSTF) has been shown to bind to three domains of the heat shock protein 70 gene (hsp 70) control region. The most critical of these for transcriptional activation appears to be the one closest to the TATA-homology region. This domain, spanning sequences from -40 to -95, consists of two contiguous HSTF binding sites (sites 1 and 2) that are occupied in a cooperative manner (see Fig. 1). Recent alkylation interference and protection studies suggest a conformational change occurs in the protein-DNA complex at site 1 upon sequential HSTF binding at site 2 (ref. 5). We report here that HSTF binding to a single site or to both contiguous sites results in the introduction of a specific DNA bend within this domain of the hsp 70 promoter.

Selective and accurate transcription of the Xenopus laevis 5S RNA genes in isolated chromatin by purified RNA polymerase III.

Chromatin isolated from immature oocytes was found to contain an endogenous RNA polymerase activity (RNA nucleotidyltransferase; nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) that synthesizes predominately 5S RNA. However, the levels of total RNA synthesis and 5S RNA synthesis in chromatin were each stimulated 10- to 50-fold by an exogenous RNA polymerase III purified from X. laevis oocytes. The 5S genes in chromatin were transcribed by the exogenous enzyme in a highly selective (3000-fold above random) and predominately asymmetric fashion. A significant fraction of 5S RNA sequences were also found in a discrete transcript, approximately 5S in size. Total RNA synthesis was significantly stimulated when chromatin was transcribed by oocyte RNA polymerase I, murine RNA polymerase II, and low levels of Escherichia coli RNA polymerase. However, these enzymes did not significantly stimulate 5S RNA synthesis above the endogenous levels. Both homologous oocyte RNA polymerase I and III and E. coli RNA polymerase transcribed the 5S genes in deproteinized DNA to approximately the same extent (severalfold above random) and both the sense and anti-sense strands of the gene were transcribed. It appears, therefore, that both chromatin-associated components and a purified RNA polymerase III are necessary and sufficient for the selective and accurate transcription of the 5S RNA genes in vitro.

Isolation of the gene encoding the S. cerevisiae heat shock transcription factor.

The yeast heat shock transcription factor gene, HSF1, has been isolated from an S. cerevisiae genomic expression library (in lambda gt11). The sequenced gene encodes an 833 amino acid protein having a mass of 93,218 daltons. Expression of specific DNA-binding activity in E. coli and of transcriptional activity in yeast confirmed the identity of the cloned gene. Southern analysis and gene-disruption experiments indicate that the heat shock transcription factor is encoded by a single-copy, essential gene. The DNA-binding domain was localized to a 118 amino acid region in the amino-terminal third of the protein. Inspection of the DNA-binding domain reveals no resemblance to any currently known secondary structural motifs implicated in DNA recognition and binding.

Transcriptional control of Drosophila fushi tarazu zebra stripe expression.

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a characteristic pattern of seven stripes during early embryogenesis. We have used ftz-lacZ fusion genes to determine the effects of deleting relatively small segments of the ftz promoter region necessary for this expression. We find that this regulatory region contains multiple activator and repressor elements. The deletion of one particular activator element results in a preferential loss of expression in the posterior stripes, whereas the deletion of other activator elements causes a general reduction in expression throughout the germ band. The removal of repressor elements results in a loss of repression in the odd-numbered parasegments. We also find that the ftz upstream enhancer element functions primarily in epidermal cells. Our results indicate that ftz transcription is activated in each parasegment through the 'zebra stripe' promoter region and is then inhibited selectively in the odd-numbered parasegments by repressors that bind directly to elements within this promoter region.

The caudal gene product is a direct activator of fushi tarazu transcription during Drosophila embryogenesis.

A drosophila pair-rule segmentation gene, fushi tarazu (ftz), encodes a protein which is expressed in a characteristic seven-stripe pattern. The promoter sequences that are sufficient for generating this spatially restricted pattern of expression are located within 669 base pairs upstream of the transcription start site. Multiple transcriptional activators and repressors interact with this 'zebra-stripe' promoter unit to bring about the positional specificity of ftz transcription. Here we report that the homoeodomain-containing protein encoded by caudal (cad) is one such regulator. The cad gene product can increase the level of ftz transcription in the posterior half of the embryo by interacting with multiple copies of a TTTATG consensus sequence located in the zebra-stripe unit. This result demonstrates one pathway by which the product of a maternally expressed segmentation gene, expressed in an antero-posterior concentration gradient, can directly regulate the expression of a pair-rule gene.

Transcription of cloned Xenopus 5S RNA genes by X. laevis RNA polymerase III in reconstituted systems.

When incubated with a soluble extract from large oocytes of Xenopus laevis, recombinant DNA plasmids containing either X. laevis oocyte 5S DNA or X. borealis oocyte 5S DNA direct the synthesis of discrete 5S RNAs, which by size and sequence analysis are similar or identical to the corresponding 5S RNAs synthesized in vivo. Synthesis of the 5S RNAs is mediated by a soluble endogenous RNA polymerase III (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6), which presumably recognizes specific initiation and termination sites in the 5S genes. Optimal conditions for accurate synthesis and the kinetics of the reactions have been determined. A soluble postchromatin supernatant fraction has also been isolated from immature oocytes. Although devoid of a functional endogenous RNA polymerase III, this extract contains a component(s) that effects the accurate transcription of 5S genes (in a plasmid) by a purified RNA polymerase III.

Sequences required for in vitro transcriptional activation of a Drosophila hsp 70 gene.

The hsp 70 gene of Drosophila contains three domains to which a heat shock gene specific transcription factor (HSTF) binds. In addition to the previously described 55 bp binding domain proximal to the TAT homology, there are two 25 bp binding sites farther upstream. Footprinting studies with 5' and 3' deletion mutations show two contiguous HSTF binding sites of different intrinsic affinities within the 55 bp binding domain. Determinations made with an agarose-acrylamide gel assay suggest that the HSTF possesses a 12.5-fold higher intrinsic affinity for the site closest to the TATA homology than for the more distal site. Binding of HSTF to the distal site thus appears cooperative, requiring occupancy of the first site. Transcription studies in vitro on the 5' deletions with nuclear extracts and reconstitution experiments show that the TATA proximal site alone, is insufficient for maximal transcriptional activation of the hsp 70 gene.

Developmental regulation of the heat shock response by nuclear transport factor karyopherin-alpha3.

During early stages of Drosophila development the heat-shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a Drosophila nuclear transport protein karyopherin-alpha3 (dKap-alpha3). Biochemical methods demonstrate that the dKap-alpha3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-alpha3 protein does not associate with NLSs that contain point mutations, which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-alpha3. Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-alpha3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

Transcriptional regulation of the Drosophila segmentation gene fushi tarazu (ftz)

ftz is one of the 'pair rule' segmentation genes of Drosophila melanogaster, and is an important component of the segmentation process in the fruit fly. We discuss the transcriptional mechanism which causes ftz to be expressed in a seven stripe pattern during embryogenesis.