Assessment of voice, speech and communication changes associated with cervical spinal cord injury.

BACKGROUND: Respiratory muscle impairment following cervical spinal cord injury (CSCI) may lead to reduced voice function, although the individual variation is large. Voice problems in this population may not always receive attention since individuals with CSCI face other, more acute and life-threatening issues that need/receive attention. Currently there is no consensus on the tasks suitable to identify the specific voice impairments and functional voice changes experienced by individuals with CSCI. AIMS: To examine which voice/speech tasks identify the specific voice and communication changes associated with CSCI, habitual and maximum speech performance of a group with CSCI was compared with that of a healthy control group (CG), and the findings were related to respiratory function and to self-reported voice problems. METHODS & PROCEDURES: Respiratory, aerodynamic, acoustic and self-reported voice data from 19 individuals (nine women and 10 men, aged 23-59 years, heights = 153-192 cm) with CSCI (levels C3-C7) were compared with data from a CG consisting of 19 carefully matched non-injured people (nine women and 10 men, aged 19-59 years, heights = 152-187 cm). OUTCOMES & RESULTS: Despite considerable variability of performance, highly significant differences between the group with CSCI and the CG were found in maximum phonation time, maximum duration of breath phrases, maximum sound pressure level and maximum voice area in voice-range profiles (all p = .000). Subglottal pressure was lower and phonatory stability was reduced in some of the individuals with CSCI, but differences between the groups were not statistically significant. Six of 19 had voice handicap index (VHI) scores above 20 (the cut-off for voice disorder). Individuals with a vital capacity below 50% of the expected for an equivalent reference individual performed significantly worse than participants with more normal vital capacity. Completeness and level of injury seemed to impact vocal function in some individuals. CONCLUSIONS & IMPLICATIONS: A combination of maximum performance speech tasks, respiratory tasks and self-reported information on voice problems help to identify individuals with reduced voice function following CSCI. Early identification of individuals with voice changes post-CSCI, and introducing appropriate rehabilitation strategies, may help to minimize development of maladaptive voice behaviours such as vocal strain, which can lead to further impairments and limitations to communication participation.

WUSCHEL-RELATED HOMEOBOX4 (WOX4)-like genes regulate cambial cell division activity and secondary growth in Populus trees.

Plant secondary growth derives from the meristematic activity of the vascular cambium. In Arabidopsis thaliana, cell divisions in the cambium are regulated by the transcription factor WOX4, a key target of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-RELATED 41 (CLE41) signaling pathway. However, function of the WOX4-like genes in plants that are dependent on a much more prolific secondary growth, such as trees, remains unclear. Here, we investigate the role of WOX4 and CLE41 homologs for stem secondary growth in Populus trees. In Populus, PttWOX4 genes are specifically expressed in the cambial region during vegetative growth, but not after growth cessation and during dormancy, possibly involving a regulation by auxin. In PttWOX4a/b RNAi trees, primary growth was not affected whereas the width of the vascular cambium was severely reduced and secondary growth was greatly diminished. Our data show that in Populus trees, PttWOX4 genes control cell division activity in the vascular cambium, and hence growth in stem girth. This activity involves the positive regulation of PttWOX4a/b through PttCLE41-related genes. Finally, expression profiling suggests that the CLE41 signaling pathway is an evolutionarily conserved program for the regulation of vascular cambium activity between angiosperm and gymnosperm tree species.

Postprandial Levels of Branch Chained and Aromatic Amino Acids Associate with Fasting Glycaemia.

High fasting plasma concentrations of isoleucine, phenylalanine, and tyrosine have been associated with increased risk of hyperglycaemia and incidence of type 2 diabetes. Whether these associations are diet or metabolism driven is unknown. We examined how the dietary protein source affects the postprandial circulating profile of these three diabetes associated amino acids (DMAAs) and tested whether the postprandial DMAA profiles are associated with fasting glycaemia. We used a crossover design with twenty-one healthy individuals and four different isocaloric test meals, containing proteins from different dietary sources (dairy, fish, meat, and plants). Analysis of the postprandial DMAAs concentrations was performed using targeted mass spectrometry. A DMAA score was defined as the sum of all the three amino acid concentrations. The postprandial area under the curve (AUC) of all the three amino acids and the DMAA score was significantly greater after intake of the meal with dairy protein compared to intake of the three other meals. The postprandial AUC for the DMAA score and all the three amino acids strongly associated with fasting glucose level and insulin resistance. This indicates the importance of the postprandial kinetics and metabolism of DMAAs in understanding the overall association between DMAAs and glycaemia.

Expression patterns and subcellular localization of carbonic anhydrases are developmentally regulated during tooth formation.

Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.

A contracted DNA repeat in LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs.

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

Nuclear Janus-activated kinase 2/nuclear factor 1-C2 suppresses tumorigenesis and epithelial-to-mesenchymal transition by repressing Forkhead box F1.

Progression to metastasis is the proximal cause of most cancer-related mortality. Yet much remains to be understood about what determines the spread of tumor cells. This paper describes a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness. We identify two transcription factors, nuclear factor 1-C2 (NF1-C2) and Forkhead box F1 (FoxF1), downstream of prolactin/nuclear Janus-activated kinase 2, with opposite effects on these processes. We show that NF1-C2 is lost during mammary tumor progression and is almost invariably absent from lymph node metastases. NF1-C2 levels in primary tumors correlate with better patient survival. Manipulation of NF1-C2 levels by expression of a stabilized version or using small interfering RNA showed that NF1-C2 counteracts EMT, motility, invasiveness, and tumor growth. FoxF1 was found to be a direct repressed target of NF1-C2. We provide the first evidence for a role of FoxF1 in cancer and in the regulation of EMT in cells of epithelial origin. Overexpression of FoxF1 was associated with a mesenchymal phenotype, increased invasiveness in vitro, and enhanced growth of breast carcinoma xenografts in nude mice. The relevance of these findings is strengthened by the correlation between FoxF1 expression and a mesenchymal phenoype in breast cancer cell isolates, consistent with the interpretation that FoxF1 promotes invasion and metastasis.

Dissecting the molecular basis of the regulation of wood formation by auxin in hybrid aspen.

Indole acetic acid (auxin) is a key regulator of wood formation, and an observed overlap between auxin concentration gradient and developing secondary xylem cells has led to the hypothesis that auxin regulates wood formation by acting as a morphogen. We dissected the role of auxin in wood formation by identifying the auxin-responsive transcriptome in wood-forming tissues and investigating alterations in wood formation in transgenic hybrid aspen plants (Populus tremula x Populus tremuloides) with perturbed auxin signaling. We showed that auxin-responsive genes in wood-forming tissues respond dynamically to changes in cellular auxin levels. However, the expression patterns of most of the auxin-responsive genes displayed limited correlation with the auxin concentration across this developmental zone. Perturbing auxin signaling by reducing auxin responsiveness reduced the cambial cell division activity, caused spatial deregulation of cell division of the cambial initials, and led to reductions in not only radial but also axial dimensions of fibers and vessels. We propose that, instead of acting as a morphogen, changes in auxin concentration in developing secondary xylem cells may provide important regulatory cues that modulate the expression of a few key regulators; these, in turn, may control the global gene expression patterns that are essential for normal secondary xylem development.

Abnormal hair development and apparent follicular transformation to mammary gland in the absence of hedgehog signaling.

We show that removing the Shh signal tranducer Smoothened from skin epithelium secondarily results in excess Shh levels in the mesenchyme. Moreover, the phenotypes we observe reflect decreased epithelial Shh signaling, yet increased mesenchymal Shh signaling. For example, the latter contributes to exuberant hair follicle (HF) induction, while the former depletes the resulting follicular stem cell niches. This disruption of the niche apparently also allows the remaining stem cells to initiate hair formation at inappropriate times. Thus, the temporal structure of the hair cycle may depend on the physical structure of the niche. Finally, we find that the ablation of epithelial Shh signaling results in unexpected transformations: the follicular outer root sheath takes on an epidermal character, and certain HFs disappear altogether, having adopted a strikingly mammary gland-like fate. Overall, our study uncovers a multifaceted function for Shh in sculpting and maintaining the integrity and identity of the developing HF.

Expression of Pit2 sodium-phosphate cotransporter during murine odontogenesis is developmentally regulated.

Different sodium-dependent inorganic phosphate (P(i)) uptake mechanisms play a major role in cellular P(i) homeostasis. The function and detailed distribution patterns of the type III Na(+)-phosphate cotransporter, PiT-2, in different organs during development are still largely unknown. We therefore examined the temporospatial expression patterns of Pit2 during murine odontogenesis. Odontoblasts were always devoid of Pit2 expression, whereas a transient, but strong, expression was detected in young secretory ameloblasts. However, the stratum intermedium and, later on, the papillary layer and cells of the subodontoblastic layer, exhibited high levels of Pit2 mRNA, which increased gradually as the tooth matured. Hormonal treatment or P(i) starvation of tooth germs in vitro did not alter Pit2 levels or patterns of expression, indicating mechanisms of regulation different from those of PiT-1 or other cell types. PiT-2 also functions as a retroviral receptor, and functional membrane-localized protein was confirmed throughout the dental papilla/pulp by demonstrating cellular permissiveness to infection by a gammaretrovirus that uses PiT-2 as a receptor. The distinct pattern of Pit2 expression during odontogenesis suggests that its P(i)-transporter function may be important for homeostasis of dental cells and not specifically for mineralization of the dental extracellular matrices. The expression of viral receptors in enamel-forming cells and the dental pulp may be of pathological significance.

Nuclear Jak2 and transcription factor NF1-C2: a novel mechanism of prolactin signaling in mammary epithelial cells.

The classical mechanism by which prolactin transduces its signal in mammary epithelial cells is by activation of cytosolic signal transducer and activator of transcription 5 (Stat5) via a plasma membrane-associated prolactin receptor-Janus kinase 2 (Jak2) complex. Here we describe an alternative pathway through which prolactin via Jak2 localized in the nucleus activates the transcription factor nuclear factor 1-C2 (NF1-C2). Previous reports have demonstrated a nuclear localization of Jak2, but the physiologic importance of nuclear Jak2 has not been clear. We demonstrate that nuclear Jak2 regulates the amount of active NF1-C2 through tyrosine phosphorylation and proteasomal degradation. Our data also demonstrate a link between prolactin and p53 as well as the milk gene carboxyl ester lipase through nuclear Jak2 and NF1-C2. Hence, we describe a novel pathway through which nuclear Jak2 is subject to regulation by prolactin in mammary epithelial cells.

Nuclear factor 1-C2 is regulated by prolactin and shows a distinct expression pattern in the mouse mammary epithelial cells during development.

We have previously demonstrated that the transcription factor nuclear factor (NF)1-C2 plays an important role in the mammary gland for the activation of the tumor suppressor gene p53. It also activates the milk genes carboxyl ester lipase and whey acidic protein, implying that NF1-C2 participates both in the establishment of a functional gland and in protection of the gland against tumorigenesis during proliferation. In this study, we have developed a new sensitive NF1-C2-specific antiserum for immunohistochemical analyses of the NF1-C2 distribution during mammary gland development. We show that the NF1-C2 protein is present in the epithelial compartment at the virgin stage and throughout mammary gland development. However, in the lactation stage the NF1-C2 protein levels strongly decreased, and many epithelial nuclei stained negative. In situ hybridization shows that NF1-C2 transcripts are expressed in the whole epithelium at pregnancy as well as the lactation stage, indicating that the reduction in protein levels is posttranscriptionally regulated. At involution, the NF1-C2 proteins are back to high levels. Based on studies using NMuMG cells and mammary tissue from heterozygous prolactin receptor knockout mice, we also demonstrate that prolactin has a direct effect in the maintenance of the NF1-C2 protein levels in the mammary epithelial nuclei at the virgin stage and during pregnancy. Hence, we have identified another transcription factor in the mammary gland, besides signal transducer and activator of transcription 5, through which prolactin may control mammary gland development. Furthermore, our data suggest a link between prolactin and p53 in the mammary gland, through NF1-C2.

A high-resolution transcript profile across the wood-forming meristem of poplar identifies potential regulators of cambial stem cell identity.

Plant growth is the result of cell proliferation in meristems, which requires a careful balance between the formation of new tissue and the maintenance of a set of undifferentiated stem cells. Recent studies have provided important information on several genetic networks responsible for stem cell maintenance and regulation of cell differentiation in the apical meristems of shoots and roots. Nothing, however, is known about the regulatory networks in secondary meristems like the vascular cambium of trees. We have made use of the large size and highly regular layered organization of the cambial meristem to create a high-resolution transcriptional map covering 220 microm of the cambial region of aspen (Populus tremula). Clusters of differentially expressed genes revealed substantial differences in the transcriptomes of the six anatomically homogenous cell layers in the meristem zone. Based on transcriptional and anatomical data, we present a model for the position of the stem cells and the proliferating mother cells in the cambial zone. We also provide sets of marker genes for different stages of xylem and phloem differentiation and identify potential regulators of cambial meristem activity. Interestingly, analysis of known regulators of apical meristem development indicates substantial similarity in regulatory networks between primary and secondary meristems.

Association between a polymorphism in the carboxyl ester lipase gene and serum cholesterol profile.

Carboxyl ester lipase (CEL) is involved in the hydrolysis and absorption of dietary lipids, but it is largely unknown to what extent CEL could be involved in determining the serum lipid levels. The C-terminal part of CEL consists of a unique structure with proline-rich O-glycosylated repeats of 11 amino-acid residues each. The common variant of the human CEL gene contains 16 proline-rich repeats, but there is a high degree of polymorphism in the repeated region. While the biological function of the polymorphic repeat region is unknown, it has been suggested that it may be important for protein stability and/or secretion of the enzyme. Given that the polymorphism in the repeated region may affect the functionality of the protein, this study aimed to investigate whether the number of repeated units is correlated to serum lipid phenotype. Comparison of CEL repeat genotype and serum lipid phenotype revealed an association between the number of repeats and serum cholesterol profile. Individuals carrying at least one allele with fewer than the common 16 repeats had significantly lower total and low-density lipoprotein (LDL) cholesterol levels compared to individuals carrying two common alleles. This gives support to the notion that CEL may be involved in determining the plasma lipid composition.

The p53 tumor suppressor gene is regulated in vivo by nuclear factor 1-C2 in the mouse mammary gland during pregnancy.

The p53 tumor suppressor protein plays an important role in preventing cancer development by arresting or killing potential tumor cells. Downregulated p53 levels, or mutations within the p53 gene, leading to the loss of p53 activity, are found in many breast carcinomas. Here we demonstrate that the p53 gene is transcriptionally upregulated in the normal mouse mammary gland at midpregnancy. We show that the specific isoform nuclear factor 1-C2 (NF1-C2) plays an important role in this activation. Functional mutation of the NF1-binding site in the mouse p53 promoter resulted in a reduction of the gene expression to less than 30% in mammary epithelial cells. By the use of two powerful techniques, chromatin immunoprecipitation and oligonucleotide decoy, we verify the importance of NF1-C2 in p53 gene activation in vivo. These findings demonstrate a broader role for NF1-C2 in the mammary gland at midpregnancy, beyond its earlier reported activation of milk protein genes. We also demonstrate that NF1-A1 proteins are produced in the mouse mammary gland. However, due to their lower affinity to the NF1-binding site, these proteins are not involved in the transcriptional upregulation of p53 at midpregnancy. This paper constitutes the first report demonstrating the importance of NF1 proteins in the p53 gene activation in the mouse mammary gland. It is also the first time that p53 gene activation is coupled to a specific, endogenously expressed NF1 isoform.

Transcriptional regulation of the human carboxyl ester lipase gene in THP-1 monocytes: an E-box required for activation binds upstream stimulatory factors 1 and 2.

The bile salt-stimulated carboxyl ester lipase (CEL) is important for the digestion and absorption of dietary lipids, and is expressed at high levels by the exocrine pancreas and the lactating mammary gland. However, the presence of CEL in human plasma suggests that the role of CEL in lipid metabolism may stretch beyond its function in the intestinal lumen, and possibly include interactions with cholesterol and oxidized lipoproteins to modulate the progression of atherosclerosis. We have used the CEL-expressing human monocytic cell line THP-1 to investigate the transcriptional regulation of the human CEL in monocytes. Analyses of the promoter region revealed that an E-box located at -47/-52 is necessary for CEL expression. Point mutations in the E-box almost completely abolish the transcriptional activity. Electrophoretic mobility-shift assay analyses reveal that the E-box binds the upstream stimulatory factors 1 and 2, and the binding of an upstream stimulatory factor-containing complex in THP-1 cells also requires the presence of a putative nuclear receptor-binding site at -60/-66. Furthermore, we demonstrate that the E-box is also necessary for CEL expression in the pancreas and the mammary gland, although there are tissue-specific requirements for additional activating elements.

Nuclear factor 1-C2 contributes to the tissue-specific activation of a milk protein gene in the differentiating mammary gland.

Members of the nuclear factor 1 (NF1) transcription factor family have been postulated to be involved in the regulation of milk genes. In this work we have been able to identify the splice variant NF1-C2 as an important member of a tissue-specific activating complex that regulates the milk gene encoding carboxyl ester lipase (CEL). Mutation of the NF1-binding site in the CEL gene promoter results in a drastic reduction of the gene expression to about 15% in mammary epithelial cells. Furthermore, we demonstrate that the NF1-C2 protein interacts with a higher affinity to the NF1-binding site in the CEL gene promoter than other NF1 family members do and that NF1-C2 in the mouse mammary gland is a phosphorylated protein. During development of the mouse mammary gland, binding of NF1-C2 to the CEL gene promoter is induced at midpregnancy, in correlation with the induction of CEL gene expression. The fact that the NF1-C2 involving complex remains throughout the lactation period and decreases during the weaning period, when the CEL gene is down-regulated, supports its importance in the regulation of CEL gene expression. To our knowledge, this is the first report identifying a specific, endogenously expressed NF1 isoform to be involved in the tissue-specific activation of a gene.