Stanniocalcin: a novel protein regulating calcium and phosphate transport across mammalian intestine.

Stanniocalcin (STC) is an anti-hypercalcemic glycoprotein hormone previously identified in the corpuscles of Stannius in bony fish and recently in the human genome. This study undertook to express human STC in Chinese hamster ovary (CHO) cells and to determine its effects on calcium and phosphate absorption in swine and rat intestine. Unidirectional mucosal-to-serosal (Jm-->s) and serosal-to-mucosal (Js-->m) 45Ca and 32P fluxes were measured in vitro in duodenal tissue in voltage-clamped Ussing chambers. Addition of STC (10-100 ng/ml) to the serosal surface of the duodenum resulted in a simultaneous increase in calcium Jm-->s and Js-->m fluxes, with a subsequent reduction in net calcium absorption. This was coupled with an STC-stimulated increase in phosphate absorption. Intestinal conductance was increased at the highest dose of STC (100 ng/ml) in swine tissue. The addition of STC to the mucosal surface had no effect on calcium and phosphate fluxes. STC at doses of 10-1,000 ng/ml had no effect on short-circuit current in any region of the rat intestine. In conclusion, human recombinant STC decreases the absorption of calcium and stimulates the absorption of phosphate in both swine and rat duodenum. STC is a novel regulatory protein that regulates mammalian intestinal calcium and phosphate transport.

Pre- and posttreatment comparison of the kinematics of the fluent speech of persons who stutter.

This study reports changes in acoustic, respiratory, laryngeal, and articulatory kinematics of 3 males who stutter, following participation in a version of the Hollins Precision Fluency Shaping Program. Two nonstuttering controls received no treatment. Subjects repeated phrases of the form "He see CVC again" at self-selected slow, normal, and fast speaking rates. For experimental subjects, acoustic duration of the phrases increased significantly in 7 out of 9 comparisons of before- and after-treatment conditions, whereas controls decreased the duration of the phrases in 4 out of 6 comparisons of measurements made over approximately the time interval during which the experimental group received treatment. The experimental group increased inspiratory volume for 7 out of 9 conditions and average expiratory flow significantly for all conditions, whereas the controls decreased both. The experimental group prolonged laryngeal opening in 6 of 7 comparisons, but only 3 of the increases were significant. Lip and jaw movements for consonants were significantly reduced in amplitude for the experimental group for 30 of 36 measures. The direction of change for laryngeal and upper articulator measures was mixed for controls. These results show that behavioral treatment can produce significant changes in the fluent speech of persons who stutter with respect to respiration, laryngeal valving, and articulation. Possible relationships between the observed changes in speech production and the increased fluency of the subjects are discussed.

Characterization of transgenic mice with an increased content of chromosomal protein HMG-14 in their chromatin.

Chromosomal protein HMG-14 is a ubiquitous nuclear protein that may modulate the chromatin structure of transcriptionally active genes. To gain insights into the cellular function of the HMG-14 protein, we generated two transgenic mouse lines carrying either two or six copies of the human HMG-14 gene. The transgenic mice express human HMG-14 mRNA and protein in all tissues examined at a level reflecting the increased gene dosage, suggesting that the HMG14 transgene contains all the control regions necessary for regulated gene expression. Expression of the human HMG-14 protein does not alter the expression of the endogenous mouse HMG-14 protein or its close homolog, protein HMG-17. The intracellular distribution of the exogenous human protein is indistinguishable from that of the endogenous mouse protein, resulting in a three-fold increase in the level of the chromatin-bound HMG-14. The transgenic mice had a higher incidence of epithelial cysts in their thymus than did control animals. We conclude that the cellular levels of HMG-14/-17 are determined by gene copy number, that the DNA fragment containing the gene and about 1,000 bp flanking its 5' and 3' ends contain most of the elements necessary for gene expression, that the upper limits of HMG-14 in chromatin are not stringently regulated, and that a three-fold increase in chromatin-bound protein cause only mild phenotypic changes in the transgenic mice.

Incorporation of chromosomal proteins HMG-14/HMG-17 into nascent nucleosomes induces an extended chromatin conformation and enhances the utilization of active transcription complexes.

The role of chromosomal proteins HMG-14 and HMG-17 in the generation of transcriptionally active chromatin was studied in a Xenopus laevis egg extract which supports complementary DNA strand synthesis and chromatin assembly. Chromosomal proteins HMG-14/HMG-17 enhanced transcription from a chromatin template carrying a 5S rRNA gene, but not from a DNA template. The transcriptional potential of chromatin was enhanced only when these proteins were incorporated into the template during, but not after, chromatin assembly. HMG-14 and HMG-17 stimulate transcription by increasing the activity, and not the number, of transcribed templates. They unfold the chromatin template without affecting the nucleosomal repeat or decreasing the content of histone B4. We suggest that HMG-14/HMG-17 enhance transcription by inducing an extended conformation in the chromatin fiber, perhaps due to interactions with histone tails in nucleosomes. By disrupting the higher order chromatin structure HMG-14/HMG-17 increase the accessibility of target sequences to components of the transcriptional apparatus.

The footprint of chromosomal proteins HMG-14 and HMG-17 on chromatin subunits.

The position of chromosomal proteins HMG-14 and HMG-17 in nucleosome cores and in chromatosomes lacking linker histones has been mapped by hydroxyl radical footprinting. Both the nucleosome core and the H1/H5 depleted chromatosome can specifically bind two molecules of HMG-14/-17. The path of HMG-14 on the surface of chromatin subunits is indistinguishable from that of HMG-17. The bound HMGs protect the DNA from hydroxyl radical cleavage 25 base-pairs from the end of the DNA in nucleosome cores and in each of the two major grooves of the DNA flanking the nucleosomal dyad axis. Thus, in both cores and H1/H5-depleted chromatosomes the proteins bridge two adjacent DNA strands on the surface of the particles. The sites occupied by HMG near the end of the chromatosome-length particles are distinct from those occupied by the H1/H5 linker histones. In the region of the dyad axis the binding sites of HMGs overlap those of the linker histones. The placement of HMG-14/-17 near the nucleosomal dyad axis raises the possibility that interactions between histone H1 and HMGs may affect the transcriptional potential of chromatin.

Deposition of chromosomal protein HMG-17 during replication affects the nucleosomal ladder and transcriptional potential of nascent chromatin.

A cell-free system from Xenopus eggs was used to study the role of chromosomal protein HMG-17 in the generation of the chromatin structure of transcriptionally active genes. Addition of HMG-17 protein to the extracts, which do not contain structural homologs of the HMG-14/-17 protein family, indicates the protein is incorporated into the nascent template during replication, prior to completion of chromatin assembly. The protein binds to and stabilizes the structure of the nucleosomal core thereby improving the apparent periodicity of the nucleosomal spacing of nascent chromatin. Assembly of HMG-17 into the nascent chromatin structure significantly increased the transcription potential of the 5S RNA gene and satellite I chromatin. Kinetic studies indicate that the increase in transcriptional potential is observed only when HMG-17 is incorporated into nucleosomes during chromatin assembly.

Aberrant expression of high mobility group chromosomal protein 14 affects cellular differentiation.

High mobility group (HMG) 14 is a ubiquitous chromosomal protein that binds specifically to nucleosomal DNA and may be involved in a process that confers distinct properties to the chromatin structure of transcriptionally active genes. To explore the involvement of this protein in regulation of gene expression, we studied the effect of aberrant expression of HMG-14 protein on cellular differentiation. We produced stably transfected C2C12 mouse myoblasts expressing the human HMG-14 protein under the control of the mouse mammary tumor virus promoter. Transformed colonies retained their potential do differentiate into myotubes. Induction of human HMG-14 expression by dexamethasone inhibited the myogenic process. Revertant colonies, which lost the ability to express human HMG-14, regained the ability to differentiate into myotubes. Inhibition of myoblast differentiation by aberrantly expressed HMG-14 correlated with down-regulation of myogenic determination factors. The results suggest that proper cellular differentiation requires regulated expression of HMG-14 protein and are consistent with the possibility that this protein may be involved in gene regulation.

Immunohistochemical localization of spermatid nuclear transition protein 2 in the testes of rats and mice.

Transition protein 2 (TP2) of the rat was isolated by differential precipitation with trichloroacetic acid, chromatography over Bio-Rex 70, and preparative gel electrophoresis. A polyclonal rabbit antiserum was raised that did not cross-react with unrelated acid-soluble proteins from liver or testes. The antiserum was used to identify TP2-related proteins obtained from testes of mice, hamsters, guinea pigs, rabbits, and boars by Western blotting. Immunohistochemical techniques were used to localize TP2 in paraffin-embedded testis sections from mice and rats. In both species, TP2 was first detected in spermatids that had essentially completed the morphological change from a round to an elongate nucleus and that were undergoing chromosomal condensation (spermatids of step 13 in rat and step 12 in mouse). TP2 was retained in spermatid nuclei until early step 16 in the rat and step 14 in the mouse. Serial sections of rat testis exposed separately to antisera to TP1 and TP2 showed that the great majority of labeled tubules were reactive to both antisera. However, in occasional tubules, TP1 reactivity was retained in relatively late spermatids that were negative for TP2. Thus both TP1 and TP2 appear in the nucleus essentially simultaneously, in association with the beginning of chromatin condensation and at a point well after much of the nuclear shaping has occurred.

Nucleosome core binding region of chromosomal protein HMG-17 acts as an independent functional domain.

Chromosomal proteins HMG-14 and HMG-17 have a modular structure. Here we examine whether the putative nucleosome-binding domain in these proteins can function as an independent module. Mobility shift assays with recombinant HMG-17 indicate that synthetic molecules can be used to analyze the interaction of this protein with the nucleosome core. Peptides corresponding to various regions of the protein have been synthesized and their interaction with nucleosome cores analyzed by mobility shift, thermal denaturation and DNase I digestion. A 30 amino acid long peptide, corresponding to the putative nucleosome-binding domain of HMG-17, specifically shifts the mobility of cores as compared to free DNA, elevates the tm of both the premelt and main melt of the cores and protects from DNase I digestion the same nucleosomal DNA sites as the intact protein. The binding of both the peptide and the intact protein is lost upon digestion of the histone tails by trypsin. The nucleosomal binding sites of the peptide appear identical to those of the intact protein. Thus, a region of the protein can acts as an independent functional domain. This supports the notion that HMG-14 and HMG-17 are modular proteins. This finding is relevant to the understanding of the function and evolution of HMG-14/-17, the only nucleosome core particle binding proteins known to date.

Speech segment duration measurements in adult speakers with repaired cleft palate and hypernasality.

Consonant closure duration, vowel duration preceding word-final stop consonants, and total VCVC utterance duration were measured in order to investigate if normal durations in these variables were exhibited in subjects with cleft palate and hypernasality. The data revealed no significant differences among three adult subject groups (noncleft, repaired cleft with mild hypernasality, and repaired cleft with severe hypernasality) on any of the duration measurements investigated, suggesting that speech timing for these features were not significantly altered by the presence of the nasal air leak. Speakers in the two hypernasal groups, however, showed smaller differences between vowel durations in voiced and voiceless stop environments than did speakers without cleft palate. There were significant differences among groups in durational cues between vowel types and between voicing features of stop consonants, both of which were in agreement with previous research.

Physiological bases of acoustic LRT in nonstutterers, mild stutterers, and severe stutterers.

The simple reaction time paradigm, incorporating a variable foreperiod, was used to investigate relative contributions of the respiratory and laryngeal systems to mild and severe stutterers' prolonged acoustic laryngeal reaction time (LRT) values. Prephonatory kinematic data were analyzed in terms of frequency of initiation, timing, and organization of events executed to attain the functional physiological targets of respiratory inflation during foreperiods and phonation onset after foreperiods. Acoustic data replicated a previously observed composite stuttering severity and foreperiod effect on stutterers' acoustic LRT values. Kinematic data revealed that, in general, the mild stutterers demonstrated delayed initiation of respiratory events and appropriate organization of respiratory and laryngeal events while the severe stutterers demonstrated delayed initiation of laryngeal events and inappropriate organization of respiratory and laryngeal events. That is, kinematic data both account for group differences in acoustic LRT values as a function of foreperiod and support the notion that differential respiratory and laryngeal deficits underly mild and severe stutterers' prolonged acoustic LRT values.

Multiple-phoneme-misarticulating children's perception and production of voice onset time.

6 language-impaired misarticulating and 6 normal kindergarten children produced and perceived differences in word-initial stop consonant voicing. Individuals' productive and perceptual phonemic boundaries were similar. No statistically reliable differences were noted between the groups' mean productive or perceptual boundaries. Individual exceptions suggest that some misarticulating , language-impaired children may be inordinately challenged by synthetic speech stimuli or may pass through a developmental stage in which perceptual ability outstrips productive ability.

Productive voice onset time characteristics of esophageal speech.

The voice onset times (VOT) of a large number of stop-consonant initiated syllables produced by esophageal and normal speakers were measured. Esophageal speakers systematically varied VOT during the production of speech-sound categories with the same manner of production. Average voice onset times associated with the production of prevocalic voiceless stops of esophageal speakers were significantly shorter than those of normal speakers, while takler-group comparisons associated with the production of voiced prevocalic stops were nonsignificant. Voice onset times of both esophageal and normal speakers were differentially sensitive to place of articulation. Findings are discussed in terms of furthering current understanding of how effectively esophageal speakers achieve important phonological contrasts.