Adrenomedullin induces matrix metalloproteinase-2 activity in rat aortic adventitial fibroblasts

Background

The delicate balance of the extracellular matrix (ECM) determines the stiffness of the vascular wall, and adventitial fibroblasts are involved in ECM formation by synthesizing and degrading matrix proteins. In the present study, we examined the effect of the bioactive peptide adrenomedullin (AM) on activity and expression of matrix metalloproteinases (MMPs) in cultured aortic adventitial fibroblasts.

Methods and results

In cultured adventitial fibroblasts isolated from aorta of adult Wistar rats, 10⁻⁶ mol/L angiotensin II (Ang II) significantly (p < 0.05) down-regulated MMP-2 activity as determined by in vitro gelatin zymography. In contrast, 10⁻⁷ mol/L synthetic rat AM significantly (p < 0.05) stimulated zymographic MMP-2 activity by 23%, increasing intracellular cAMP, and AM abolished the action of Ang II, augmenting the MMP-2 activity. Similarly, Ang II down-regulated MMP-2 protein expression assessed by Western blotting, whereas AM increased it. Furthermore, 8-bromo-cAMP, an analogue of cAMP, mimicked the effect of AM, and H-89, an inhibitor for protein kinase A (PKA), significantly decreased the basal and AM-induced MMP-2 activity.

Conclusion

This study provides a new insight into the biological action of AM and its intracellular signaling system of cAMP/PKA stimulating the matrix degrading enzyme MMP-2, suggesting an important role for this molecule in modulating ECM deposition in the adventitial layer.     

Using model substrates to study the dependence of focal adhesion formation on the affinity of integrin-ligand complexes

The adhesion of mammalian cells is mediated by the binding of cell-surface integrin receptors to peptide ligands from the extracellular matrix and the clustering of these receptors into focal adhesion complexes. This paper examines the effect of one mechanistic variable, ligand affinity, on the assembly of focal adhesions (FAs) in order to gain mechanistic insight into this process. This study uses self-assembled monolayers of alkanethiolates on gold as a substrate to present either a linear or cyclic Arg-Gly-Asp peptide at identical densities. Inhibition assays showed that the immobilized cyclic RGD is a higher affinity ligand than linear RGD. 3T3 Swiss fibroblasts attached to substrates presenting the cyclic peptide at twice the rate they attached to substrates presenting the linear peptide. Quantitation of focal adhesions revealed that cells on cyclic RGD had twice the number of FAs as did cells on linear RGD and that these focal adhesions were on average smaller. These findings show that affinity affects the assembly of integrins into focal adhesions and support a model based on competing rates of nucleation and growth of FAs to explain the change in distribution of FAs with ligand affinity. This study is important because it provides a model system that is well-suited for biophysical studies of integrin-mediated cell adhesion and reveals insight into one mechanism utilized by cells to perceive environmental changes.     

A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis

The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.     

Rapid degradation of Cdt1 upon UV-induced DNA damage is mediated by SCFSkp2 complex

Cdt1 is a licensing factor for DNA replication, the function of which is tightly controlled to maintain genome integrity. Previous studies have indicated that the cell cycle-dependent degradation of Cdt1 is triggered at S phase to prevent re-replication. In this study, we found that Cdt1 is degraded upon DNA damage induced by either UV treatment or gamma-irradiation (IR). Although the IR-triggered degradation of Cdt1 was caffeine-insensitive, the UV-triggered degradation of Cdt1 was caffeine-sensitive. This indicates that the cells treated with UV utilize the checkpoint pathway, which differs from that triggered by IR. A recent study has suggested that Cdt1 is phosphorylated, ubiquitylated, and degraded at the G(1)/S boundary in the normal cell cycle. Treatment with MG132, a proteasome inhibitor, inhibited the degradation of Cdt1 and resulted in the accumulation of the phosphorylated form of Cdt1 after UV treatment. In the case of UV treatment, phosphorylation of Cdt1 induced the recruitment of Cdt1 to a SCF(Skp2) complex. Moreover, ectopic overexpression of Cdt1 after UV treatment interfered the inhibition of DNA synthesis. These results indicate that Cdt1 is a target molecule of the cell cycle checkpoint in UV-induced DNA damage.     

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker

Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.     

Plant-pollinator interactions in New Caledonia influenced by introduced honey bees

The flora of New Caledonia is characterized by remarkably high species diversity, high endemicity, and an unusual abundance of archaic plant taxa. To investigate community-level pollination mutualism in this endemic ecosystem, we observed flower visitors on 99 plant species in 42 families of various types of vegetation. Among the 95 native plant species, the most dominant pollination system was melittophily (bee-pollinated, 46.3%), followed by phalaenophily (moth-pollinated, 20.0%), ornithophily (bird-pollinated, 11.6%), cantharophily (beetle-pollinated, 8.4%), myophily (fly-pollinated, 3.2%), chiropterophily (bat-pollinated, 3.2%), and anemophily (wind-pollinated, 3.2%). The prevalence of ornithophily by honeyeaters shows an ecological link to pollination mutualism in Australia. The relative dominance of phalaenophily is unique to New Caledonia, and is proposed to be related to the low diversity of the original bee fauna and the absence of long-tongued bees. Although some archaic plants maintain archaic plant-pollinator interactions, e.g., Zygogynum pollinated by micropterigid moths, or Hedycarya pollinated by thrips and staphylinid beetles, the most dominant organism observed on flowers was the introduced honey bee, Apis mellifera. The plant species now visited by honey bees are thought to have originally been pollinated by native solitary short-tongued bees. Our data suggest that the unique systems of pollination mutualism in New Caledonia are now endangered by the establishment of highly invasive honey bees.     

Branched-chain amino acids as a protein- and energy-source in liver cirrhosis

Protein-energy malnutrition (PEM) is a common manifestation in cirrhotic patients with reported incidences as high as 65-90%. PEM affects largely the patients' quality of life and survival. Thus, diagnosis of and intervention for PEM is important in the clinical management of liver cirrhosis. Supplementation with branched-chain amino acids (BCAA) is indicated to improve protein malnutrition. As an intervention for energy malnutrition, frequent meal or late evening snack has been recently recommended. Plasma amino acid analysis characterizes the patients with liver cirrhosis to have decreased BCAA. Such reduction of BCAA is explained by enhanced consumption of BCAA for ammonia detoxication and for energy generation. Supplementation with BCAA raises in vitro the synthesis and secretion of albumin by cultured rat hepatocytes without affecting albumin mRNA expression. BCAA recover the impaired turnover kinetics of albumin both in rat cirrhotic model and in cirrhotic patients. Longer-term supplementation with BCAA raises plasma albumin, benefits quality of life issues, and finally improves survival in liver cirrhosis. Recent interests focused on the timing of administration of BCAA, since daytime BCAA are usually consumed by energy generation for physical exercise of skeletal muscles. Nocturnal BCAA seem to be more favorable as a source of protein synthesis by giving higher nitrogen balance. This minireview focuses on the basic and clinical aspects of BCAA as a pharmaco-nutritional source to control PEM in liver cirrhosis.     

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.     

Three new thermotolerant methylotrophic yeasts, Candida krabiensis sp. nov., Candida sithepensis sp. nov., and Pichia siamensis sp. nov., isolated in Thailand

Five strains of thermotolerant methylotrophic yeasts isolated in Thailand were found to represent three new species in the genera Pichia and Candida, based on phylogenetic analysis of D1/D2 domain of 26S rDNA, in addition to the morphological, physiological, biochemical and chemotaxonomic characterization. Three strains, FS96 and FS101 from flowers and M02 from tree flux, were characterized by ubiquinone Q7, multilateral budding, and the formation of hat-shaped ascospores that are liberated at maturation. These strains showed identical nucleotide sequences in the D1/D2 domain and formed a cluster with Candida thermophila, "Pichia salicis" and Pichia angusta. They differed by 1.9% of nucleotide substitutions from Candida thermophila, the nearest species. They were considered to represent a single new species and are described as Pichia siamensis sp. nov. Two strains, N051 and S023, isolated from soil did not produce ascospores, proliferated by multilateral budding, did not demonstrate urease or DBB color reaction, and lacked sexual stages. These characteristics correspond to the genus Candida. Strains N051 and S023 differed by 2.8% and 1.9% of nucleotide substitutions in the D1/D2 domain from the nearest species, Candida nemodendra and Candida ovalis, respectively, and are considered to represent respective new species. N051 and S023 are described as Candida krabiensis sp. nov. and Candida sithepensis sp. nov., respectively.