Multiple roles and interactions of tbx4 and tbx5 in development of the respiratory system

Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung and trachea; and, although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. In this study, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes Bmp4 and Spry2, in the epithelium is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal/bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea; but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal/bronchial cartilage development.     

Heterogeneous cellular expression of creatine kinase isoenzyme during normal rat heart development

The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.     

Downregulation of N1 gene expression inhibits the initial heartbeating and heart development in axolotls

Recessive mutant gene c in the axolotl results in a failure of affected embryos to develop contracting hearts. This abnormality can be corrected by treating the mutant heart with RNA isolated from normal anterior endoderm or from endoderm conditioned medium. A cDNA library was constructed from the total conditioned medium RNA using a random priming technique in a pcDNAII vector. We have previously identified a clone (designated as N1) from the constructed axolotl cDNA library, which has a unique nucleotide sequence. We have also discovered that the N1 gene product is related to heart development in the Mexican axolotl [Cell Mol. Biol. Res. 41 (1995) 117]. In the present studies, we further investigate the role of N1 on heartbeating and heart development in axolotls. N1 mRNA expression has been determined by using semi-quantitative RT-PCR with specifically designed primers. Normal embryonic hearts (at stages 30-31) have been transfected with anti-sense oligonucleotides against N1 to determine if downregulation of N1 gene expression has any effect on normal heart development. Our results show that cardiac N1 mRNA expression is partially blocked in the hearts transfected with anti-sense nucleotides and the downregulation of N1 gene expression results in a decrease of heartbeating in normal embryos, although the hearts remain alive as indicated by calcium spike movement throughout the hearts. Confocal microscopy data indicate some myofibril disorganization in the hearts transfected with the anti-sense N1 oligonucleotides. Interestingly, we also find that N1 gene expression is significantly decreased in the mutant axolotl hearts. Our results suggest that N1 is a novel gene in Mexican axolotls and it probably plays an important role in myofibrillogenesis and in the initiation of heartbeating during heart development.     

Cloning and expression analysis of the mouse T-box gene tbx20

T-box genes constitute a conserved multi-gene family with important roles in many developmental processes. In this report, we describe the cloning and expression analysis of a novel mouse T-box gene, Tbx20. Expression is prominent in the extraembryonic mesoderm, in the developing heart, the eye anlage and motor neurons of hindbrain and spinal cord.     

WNT5A selectively inhibits mouse ventral prostate development

The establishment of prostatic budding patterns occurs early in prostate development but mechanisms responsible for this event are poorly understood. We investigated the role of WNT5A in patterning prostatic buds as they emerge from the fetal mouse urogenital sinus (UGS). Wnt5a mRNA was expressed in UGS mesenchyme during budding and was focally up-regulated as buds emerged from the anterior, dorsolateral, and ventral UGS regions. We observed abnormal UGS morphology and prostatic bud patterns in Wnt5a null male fetuses, demonstrated that prostatic bud number was decreased by recombinant mouse WNT5A protein during wild type UGS morphogenesis in vitro, and showed that ventral prostate development was selectively impaired when these WNT5A-treated UGSs were grafted under under kidney capsules of immunodeficient mice and grown for 28 d. Moreover, a WNT5A inhibitory antibody, added to UGS organ culture media, rescued prostatic budding from inhibition by a ventral prostatic bud inhibitor, 2,3,8,7-tetrachlorodibenzo-p-dioxin, and restored ventral prostate morphogenesis when these tissues were grafted under immunodeficient mouse kidney capsules and grown for 28 d. These results suggest that WNT5A participates in prostatic bud patterning by restricting mouse ventral prostate development.     

Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.     

Ventricular myosin light chain-2 gene expression in developing heart of chicken embryos

Recent gene knock-out studies in mice have suggested that ventricular myosin light chain-2 (vMLC2) has a role in the regulation of cardiogenic development and that perturbation in expression of vMLC2 is linked to the onset of dilated cardiomyopathy. In an attempt to develop an avian model for such studies, we examined the expression pattern of vMLC2 in chicken embryos at various stages and analyzed the effect of antisense oligonucleotide-mediated interference of vMLC2 function in cultures of whole embryos. Our results showed vMLC2 to be a specific marker for ventricular chamber throughout chicken embryonic development and antisense vMLC2 treatment of primitive streak stage (stage 4) embryos to produce pronounced dilation of heart tube with severe deficiency in formation of striated myofibrils. Further studies with antisense mRNA techniques of whole embryo cultures should, therefore, be useful to evaluate the role of vMLC2 and other putative regulatory factors in cardiac myofibrillogenesis.     

Resveratrol inhibits Cdk5 activity through regulation of p35 expression

Background

We have previously reported that cyclin-dependent kinase 5 (Cdk5) participates in the regulation of nociceptive signaling. Through activation of the ERK1/2 pathway, Tumor Necrosis Factor-α (TNF-α) induces expression of Egr-1. This results in the sustained and robust expression of p35, a coactivator of Cdk5, in PC12 cells, thereby increasing Cdk5 kinase activity. The aim of our present study was to test whether resveratrol, a polyphenolic compound with known analgesic activity, can regulate Cdk5/p35 activity.

Results

Here we used a cell-based assay in which a p35 promoter-luciferase construct was stably transfected in PC12 cells. Our studies demonstrate that resveratrol inhibits p35 promoter activity and also blocks the TNF-α mediated increase in Cdk5 activity in PC12 cells. Resveratrol also inhibits p35 expression and blocks the TNF-α mediated increase in Cdk5 activity in DRG neurons. In the presence of resveratrol, the MEK inhibitor decreased p35 promoter activity, whereas the inhibitors of p38 MAPK, JNK and NF-κB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The TNF-α-mediated increase in Egr-1 expression was decreased by resveratrol treatment with a concomitant reduction in p35 expression and protein levels, resulting in reduced Cdk5 kinase activity.

Conclusions

We demonstrate here that resveratrol regulates p35 promoter activity in PC12 cells and DRG neurons. Most importantly, resveratrol blocks the TNF-α-mediated increase in p35 promoter activity, thereby reducing p35 expression and subsequent Cdk5 kinase activity. This new molecular mechanism adds to the known analgesic effects of resveratrol and confirms the need for identifying new analgesics based on their ability to inhibit Cdk5 activity for effective treatment of pain.

     

Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. Here, we provide the first evidence that hypoxia inhibits CCR5 chemokine receptor expression in mouse macrophages. CCR5 was constitutively expressed in macrophages and upregulated by IFNgamma. Hypoxia downregulated both constitutive and IFNgamma-induced CCR5 mRNA and protein. Reoxygenation of hypoxic cells reverted CCR5 inhibition. CCR5 upregulation by IL-10, LPS, and IL-4 was also antagonized by hypoxia. CCR5 inhibition may be a way to retain/concentrate recruited macrophages at hypoxic sites or a feedback mechanism to control the autocrine activation of macrophages which produce CCR5 ligands.     

Berberine inhibits TPA-induced MMP-9 and IL-6 expression in normal human keratinocytes

Berberine is a plant ingredient that has anti-inflammatory and anti-oxidative effects. Matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) are known to be highly induced by ultraviolet (UV) light and may play important roles in UV-induced skin inflammation and the skin aging process. In this study, we investigated the effects of berberine on MMP-9 and IL-6 expression in normal human keratinocytes (NHK). Our results demonstrated that berberine dose-dependently inhibited basal and TPA-induced expression and activity of MMP-9, and also suppressed TPA-induced IL-6 expression. Berberine prevented TPA-induced ERK activation and AP-1 DNA binding activity. Therefore, berberine may be used as an effective ingredient for anti-skin aging products, which can prevent skin inflammation and the degradation of extracellular matrix proteins, including collagen, by MMPs.     

Signal transduction in early heart development (II): ventricular chamber specification, trabeculation, and heart valve formation

The formation of a four-chambered heart with ventricular chambers aligned in a left-right orientation begins with the rightward looping of the linear heart tube in accordance with the left-right embryonic axis. The functional specification of the ventricular chambers in the looped heart occurs with the formation of a trabeculated myocardium along the outer curvature of the realigned heart tube. Two major signal transduction pathways are involved in this process, the retinoic acid and neuregulin signaling pathways, with the retinoic acid pathway also participating in rightward heart tube looping. With the establishment of the atrial and ventricular chambers, maintenance of a unidirectional flow of blood between the two chambers must be ensured. To achieve this, heart valves develop at the atrioventricular juncture. This process begins with formation of endocardial cushions, the primordia of heart valves, and ends with formation of heart valve leaflets. Underlying this process is a complex network of signal transduction pathways that mediate communication between the endocardial and myocardial cell layers to form the endocardial cushions and nascent heart valve. Some of the signaling molecules involved are vascular endothelial growth factor, Wnts, bone morphogenetic proteins, epidermal growth factor, hyaluronic acid, neurofibromin, and calcium.