Hmx homeobox gene function in inner ear and nervous system cell-type specification and development

The Hmx homeobox gene family is comprised of three members in mammals, Hmx1, Hmx2, and Hmx3, which are conserved across the animal kingdom and are part of the larger NKL clustered family of homeobox genes. Expression domains of Hmx genes in distantly related species such as Drosophila and mouse suggest an ancestral function in rostral central nervous system development. During vertebrate evolution, the Hmx genes appear to have been recruited into additional roles in inner ear morphogenesis and specification of vestibular inner ear sensory and supporting cell types. Being derived from a common ancestor, the vertebrate Hmx gene family is thus a strong candidate to investigate functional overlap versus the unique roles played by multiple genes belonging to the same family. The functions of Hmx2 and Hmx3 were investigated via directed gene mutagenesis and the primary regions where Hmx2 and Hmx3 exert their individual functions are consistent with their expression domains, such as the vestibule and uterus. Meanwhile, it is notable that some tissues where both Hmx2 and Hmx3 are extensively expressed were not severely affected in either of the Hmx2 or Hmx3 single mutant mice, suggesting a possible functional overlap existing between these two genes. Compound Hmx2 and Hmx3 double mutant mice showed more severe defects in the inner ear than those displayed by either single knockout. Furthermore, novel abnormalities in the hypothalamic-neuroendocrine system, which were never observed in either of the single mutant mice, confirmed a hypothesis that Hmx2 and Hmx3 also function redundantly to control embryonic development of the central nervous system.     

In vivo tracing of canonical Wnt signaling in Xenopus tadpoles by means of an inducible transgenic reporter tool

The canonical Wnt pathway is recurrently used during embryogenesis and adult life. To track the cellular output of Wnt signaling in a living organism, we designed a hormone-inducible Wnt responsive system, capable to dynamically and specifically report Wnt pathway activities through eGFP expression. In contrast to previous in vivo reporters, our system essentially avoids interference of consecutive signals by remaining dormant until addition of hormone, which makes it a valuable tool to map canonical Wnt signaling in post-embryonic stages. Transgenic Xenopus laevis embryos were analyzed revealing at tadpole stage in specific tissues and organs cell populations with high Wnt pathway activity.     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

Differential effects of corticotropin-releasing hormone on central dopaminergic and noradrenergic neurons

Corticotropin-releasing hormone (CRH) has been shown to be a central mediator for most, if not all, stress-induced responses. Since stressful stimuli may decrease hypothalamic tuberoinfundibular and tuberohypophysial dopaminergic neuronal activities, we aimed to determine whether CRH is involved. Using central administration of various doses of ovine CRH (oCRH; 1, 3 and 10 µg/rat) into the lateral cerebroventricle of either male or female rats, the neurochemical changes in various parts of the central nervous system, including the hypothalamus, were determined by high-performance liquid chromatography at various times after the injection (30, 60, 120 and 240 min). The concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG), two major metabolites of dopamine and norepinephrine, respectively, in discrete brain regions were used as indices for catecholaminergic neuron activity. Plasma corticosterone levels increased significantly after all doses of oCRH and at all time points studied. oCRH also exerted significant stimulatory effects on noradrenergic neuron terminals in the frontal cortex, and on dopaminergic neuron terminals in the nucleus accumbens, hypothalamic paraventricular and periventricular nuclei, and intermediate pituitary lobe. Dopaminergic neuron terminals in the median eminence and the neural lobe of the pituitary, however, were not affected. There was no major difference in the responses between male and female rats. We conclude that CRH has a differential effect on central catecholaminergic neurons.     

PRIMARY STRUCTURE OF ALLATOSTATIN 4, A NEVROPEPTIDE INHIBITOR OF JUVENILE HORMONE SYNTHESIS

Abstract Isolation and identification of allatostatin 4 (AST 4) from crude brain extracts of female Diploptera punctata are described. Synthetic analogues of allatostatin 4 truncated from the N-terminal were evaluated to gain some knowledge of structure-activity correlation. AST 4 reversibly inhibits the biosynthesis of JH in vitro . AST 4 has the following sequence: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂. This neurohormone (AST 4) has no sequence similarity with any known neuropeptide from other organisms. Synthetic AST 4 as well as truncation fragments, including two with the five and six amino acids terminal residues deleted showed in vitro activity distinguishable from that of the native allatostatin.     

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor

The thyroid hormone receptors (TRs) mediate thyroid hormone (T₃)-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRβ1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRβ1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRβ1 population were significantly greater in MED1^−/− cells, compared with MED1^+/+ cells. Exposure to T₃ and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRβ1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.     

Requirement for matrix metalloproteinase stromelysin-3 in cell migration and apoptosis during tissue remodeling in Xenopus laevis

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) was originally discovered as a gene whose expression was associated with human breast cancer carcinomas and with apoptosis during organogenesis and tissue remodeling. It has been shown previously, in our studies as well as those by others, that ST3 mRNA is highly upregulated during apoptotic tissue remodeling during Xenopus laevis metamorphosis. Using a function-blocking antibody against the catalytic domain of Xenopus ST3, we demonstrate here that ST3 protein is specifically expressed in the cells adjacent to the remodeling extracellular matrix (ECM) that lies beneath the apoptotic larval intestinal epithelium in X. laevis in vivo, and during thyroid hormone-induced intestinal remodeling in organ cultures. More importantly, addition of this antibody, but not the preimmune antiserum or unrelated antibodies, to the medium of intestinal organ cultures leads to an inhibition of thyroid hormone-induced ECM remodeling, apoptosis of the larval epithelium, and the invasion of the adult intestinal primodia into the connective tissue, a process critical for adult epithelial morphogenesis. On the other hand, the antibody has little effect on adult epithelial cell proliferation. Furthermore, a known MMP inhibitor can also inhibit epithelial transformation in vitro. These results indicate that ST3 is required for cell fate determination and cell migration during morphogenesis, most likely through ECM remodeling.     

樟叶越桔组培苗生根和移栽技术研究

为解决樟叶越桔(Vaccinium dunalianum)组培苗生根质量不佳、移栽成活率低的问题,该研究以樟叶越桔继代苗为材料,采用单因子试验从激素类型及浓度、培养基类型和蔗糖质量浓度对其生根的适宜条件进行筛选,进一步研究了不同基质配比对樟叶越桔移栽苗存活率的影响。结果表明:激素类型和浓度、培养基类型对樟叶越桔生根率的影响最大,其次为蔗糖质量浓度;最适合樟叶越桔生根的激素及浓度为IBA2.0 mg·L~(-1)、基本培养基类型为1/4MS、蔗糖质量浓度为15 g·L~(-1),樟叶越桔组培苗最佳生根培养基为1/4MS+IBA 2.0 mg·L~(-1)+活性炭0.1 g·L~(-1)+蔗糖15 g·L~(-1),生根率达100%,平均生根数为每株7.67条;根系呈辐射状、基部无愈伤组织,组培苗生长健壮、叶色浓绿;樟叶越桔组培苗移栽时以全腐殖土基质为佳,成活率为83.7%,植株叶片舒展,生长状况良好。该研究建立的优化体系有效地提高了樟叶越桔组培生根苗的生根率和生根质量,解决了后期移栽成活困难的问题,为优良的樟叶越桔植株规模化生产提供了科学依据和技术支持。     

Fatty acyl-CoA binding activity of the nuclear thyroid hormone receptor

Long-chain fatty acids and their acyl-CoA esters are potent inhibitors of nuclear thyroid hormone (T₃) receptor in vitro. In the present study, we obtained evidence for acyl-CoA binding activity in the nuclear extract from rat liver. The activity sedimented at a position (3.5 S) identical with that of the T₃ receptor, and the two activities sedimented together. Similarly, they coeluted on DEAE-Sephadex. After partial purification of the receptor, it was again inhibited strongly by acyl-CoAs. Heat stability and a partial trypsin digestion of the receptor both suggested that the action site of oleoyl-CoA overlapped the T₃-binding domain of the receptor. In addition, thyroid hormone receptor β1, synthesized in vitro, bound oleoyl-CoA specifically and its T₃-binding activity was inhibited. The dissociation constant for oleoyl-CoA binding to the partially purified receptor was 1.2 × 10^?7 M. This value as well as its molecular size distinguished the nuclear binding sites from the cytoplasmic fatty acid/acyl-CoA binding proteins. Oleoyl-CoA had no effect on the glucocorticoid receptor, another member of the nuclear hormone-receptor superfamily. From these results, we propose that thyroid hormone receptor is a specific acyl-CoA binding protein of the cell nucleus.     

Urinary and serum electrolyte changes in athletes during periodic and continuous hypokinetic and ambulatory conditions

Hypokinesia (HK) (diminished movement) induces significant electrolyte changes, but little is known about the effect of periodic hypokinesia (PHK) on minerals. The aim of this study was to measure the effect of PHK and continuous hypokinesia (CHK) on urinary and serum electrolytes. Studies were done during a 30-d period of prehypokinesia (HK) and during 364 d of PHK and CHK periods. Thirty male athletes aged 24.6±7.7 yr were chosen as subjects. They were equally divided into three groups: unrestricted ambulatory control subjects (UACS), continuously hypokinetic subjects (CHKS), and periodically hypokinetic subjects (PHKS). The UACS group experienced no changes in the daily activities and regular training and they were maintained under an average running distance of 11.7 km/d. The CHKS group was limited to an average walking distance of 0.7 km/d; and the PHKS group was limited to an average walking distance of 0.7 and running distance of 11.7 km/d for 5 d and 2 d/wk, respectively, for a period of 364 d. Urinary and serum phosphate (P), calcium (Ca), sodium (Na) and potassium (K), serum intact parathyroid hormone (iPTH), calcitonin (CT), plasma renin activity (PRA) and aldosterone (PA) levels, food and water intakes, and physical characteristics were measured. Urinary P, Ca, Na, and K loss, serum Ca, P, Na, and K, and PRA and PA values increased significantly (p≤0.01), whereas serum iPTH and CT levels decreased significantly (p≤0.01) in the PHKS and CHKS groups when compared with the UACS group. However, significant (p≤0.01) differences were observed between PHKS and CHKS groups regarding urinary and serum electrolytes, serum and plasma hormones. Food and water intakes, body weight, body fat, and peak oxygen uptake decreased significantly (p ≤ 0.01) in the CHKS group when compared with PHKS and UACS groups. Food and fluid intakes, body fat, and body weight increased significantly (p≤0.01), whereas peak oxygen uptake remained significantly (p≤0.01) higher in the PHKS group when compared with the CHKS group. Serum and urinary minerals, serum hormones, food and fluid intakes, and physical characteristics did not change significantly (p>0.01) in the UACS group when compared with their baseline control values. It was shown that both PHK and CHK induce significant serum and urinary electrolyte changes. However, urinary and serum electrolyte changes were significantly (p≤0.01) greater during PHK than CHK. It was concluded that the greater the stability of muscular activity, the smaller the serum and urinary electrolyte changes during prolonged HK.