GGC and StuI polymorphism on the androgen receptor gene in endometrial cancer patients

Androgens have an anti-proliferative effect on endometrial cells. Human androgen receptor (AR) gene contains two polymorphic short tandem repeats of GGC and CAG, and a single-nucleotide polymorphism on exon 1 that is recognized by the restriction enzyme, StuI. Prior studies have shown that the lengths of the CAG repeat are inversely and linearly related to AR activity and associated with endometrial cancer. However, little is known about the GGC repeat and the StuI polymorphism of the AR gene. Thus, we investigated whether these AR polymorphisms are risk factors for endometrial cancer. To test this hypothesis, the genetic distributions of these polymorphisms were investigated in blood samples from endometrial cancer patients and healthy controls. The allelic and genotyping profiles were analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and direct DNA sequencing, and analyzed statistically. The GGC repeat was significantly longer in endometrial cancer patients as compared to normal healthy controls. In general, an increased risk of endometrial cancer was found with increasing GGC repeat. The relative risk for the 17 GGC repeat was greater than 4, as compared to controls. However, the StuI polymorphism was not significantly different between patients and controls. The findings suggest that increased numbers of GGC repeat on the AR gene may be a risk factor for endometrial cancer.     

Dose-rate effect on transgenerational mutation frequencies in spermatogonial stem cells of the medaka fish

The estimation of transgenerational genetic risk of radiation exposure to non-human species is crucial for the protection of ecosystems. Here we determined the frequency of specific-locus mutations at the five pigmentation loci in medaka spermatogonial stem cells after gamma irradiation at 0.03 cGy/min and 95 cGy/min. At each total dose, the mutation frequency was significantly lower in the 0.03-cGy/min group than in the 95-cGy/min group, suggesting a dose-rate effect. The ratio of the induced mutation frequency at 0.03 cGy/min to that at 95 cGy/min was approximately 0.42 from 0 to 1.9 Gy and approximately 0.33 from 1.9 to 4.75 cGy. In the mouse, this ratio is estimated to be 0.33 (Russell and Kelly, Proc. Natl. Acad. Sci. USA 79, 542-544, 1982). It is thus possible that the magnitude of the dose-rate effect on transgenerational mutation frequencies is comparable between mouse and medaka spermatogonia, suggesting similar dose-rate effects among vertebrates.     

LuxS-based signaling affects Streptococcus mutans biofilm formation

Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.     

Expression of the adrenomedullin gene in adipose tissue

Adrenomedullin (AM) is a potent vasodilating peptide originally isolated from human pheochromocytoma cells. This report concerns the expression and secretion of AM from adipose tissue. Northern blot analysis demonstrated marked expression of AM mRNA in mouse adipose tissue. Expression levels in adipose tissues were 2.5-3.2 times higher than in the kidney. AM mRNA level in mature adipocytes was 7.3 times higher than in the stroma-vascular fraction of adipose tissue. In mature adipocyte culture, time-dependent increase of AM peptide concentration in the culture medium was detected. AM expression was also detected in human subcutaneous adipose tissue. Adipose AM expression significantly increased in obesity mouse model, high-fat diet fed mice and ob/ob mice. These results suggest that adipose tissue, especially mature adipocytes, is major source of AM in the body, and that adipocyte-derived AM plays a pathophysiological role in obesity.     

Quantitative analysis of helix-coil transition of block copolypeptide, Glu12-Ala12, by combined use of CD and NMR spectroscopy

To investigate helix-coil transition mechanisms, conformations of Glu12-Ala12, EA, in aqueous solution have been studied in detail over the pH range from 2 to 8 and the temperature range from 20 to 60 degrees C using CD and NMR spectroscopy. The 750-MHz NMR spectra displayed excellent dispersion of the backbone amide proton signals, and permitted essentially complete sequence-specific resonance assignments. These assignments, together with short- and medium-range nuclear Overhauser effect (NOE) constraints and coupling constants, enable us to analyze conformational characteristics of all the residues in the EA peptide individually. A combined use of CD and NMR techniques reveals that the EA peptide assumes a stable alpha-helix from Glu12 to Ala19 in 0.1 M NaCl solution at 20 degrees C above pH 7. The alpha-helix is getting longer as decreasing pH. Below pH 4, the peptide assumes the longest alpha-helix from Glu3 to Ala23. The important observation of the present study is that the helix-coil transition occurs stepwise, residue by residue, from both the N- and C-termini of the alpha-helix. No conformational equilibrium between the helical and random-coil states is detected for the residues in the central region of the alpha-helix. Quantitative analysis of temperature-induced helix-to-coil transitions at various pHs provides a pH-independent residual enthalpy change delta H(r) = 0.95 kcal res(-1). Similar values have been reported for a 50-residue alanine-rich peptide (1.2 kcal res(-1)), poly-L-glutamate (1.1 kcal res(-1)), poly-L-lysine (1.1 kcal res(-1)), and poly-L-alanine (0.86 kcal res(-1)). Those investigations, along with our present result, suggest that delta H(r) is mainly determined by the transformation of the backbone associated with the disruption of the intramolecular hydrogen bond. These results should increase our understanding of the helix-coil transition.     

Dual effects of adenovirus-mediated thrombopoietin gene transfer on hepatic oval cell proliferation and platelet counts

Thrombopoietin (TPO) is the growth factor for megakaryocytes and platelets, however, it also acts as a potent regulator of stem cell proliferation. To examine the significance of TPO expression in proliferation of hepatic oval cells, the effect of adenovirus-mediated TPO gene transfer into livers of the Solt-Farber model, which mimics the condition where liver regeneration is impaired, was examined. Hepatic TPO mRNA peaked its expression at 2 days after gene transduction and then gradually decreased. The peripheral platelet number began to increase at 4 days (P<0.05) and reached its plateau at 9 days (P<0.01). Oval cells expressed c-Mpl, a receptor for TPO as well as immature hematopoietic and hepatocytic surface markers such as CD34 and AFP. The proliferating cell nuclear antigen-positive oval cells in rats into which adenovirus-TPO gene was transferred at 7 and 9 days were significantly greater than those in adenovirus-LacZ gene transferred (P<0.05, each), and the total numbers of oval cells in the adenovirus-TPO gene transferred at 9 and 13 days were also significantly greater than those in adenovirus-LacZ gene transferred (P<0.05, each). Expression of SCF protein was increased at 4, 7, and 9 days by TPO gene administration and that of c-Kit was increased at 4 and 7 days. These data suggest that adenovirus-mediated TPO gene transfer stimulated oval cell proliferation in liver as well as increasing peripheral platelet counts, emphasizing the significance of the TPO/c-Mpl system in proliferation of hepatic oval cells.     

Addition of a peptide fragment on an alpha-helical depsipeptide induces alpha/3(10)-conjugated helix: synthesis, crystal structure, and CD spectra of Boc-Leu-Leu-Ala-(Leu-Leu-Lac)3-Leu-Leu-OEt

The depsipeptide Boc(1)-Leu(2)-Leu(3)-Ala(4)-Leu(5)-Leu(6)-Lac(7)-Leu(8)-Leu(9)-Lac(10)-Leu(11)-Leu(12)-Lac(13)-Leu(14)-Leu(15)-OEt(16) (1) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized from the peptide Boc-Leu-Leu-Ala-OEt (2) and a depsipeptide, Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (3). Single crystals of 1 were successfully obtained and the structure has been solved by direct methods (such as Sir2002 and Shake-and-Bake). Interestingly, 1 adopts an alpha/3(10)-conjugated helix containing a kink at the junction of peptide and depsipeptide segments, Leu3-Lac7. This is significantly different from the conformation of 3, which has a straight alpha-helical structure with standard phi and psi angles. Microcrystalline CD spectra were also studied to compare structural properties of 1 and 3. The differences between alpha/3(10)- and alpha-helices appear in these CD spectra.     

A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.     

Identification of ASK and clock-associated proteins as molecular partners of LKP2 (LOV kelch protein 2) in Arabidopsis

The ADO/FKF/LKP/ZTL family of proteins of Arabidopsis thaliana Heynh. have a LOV domain, an F-box motif, and a kelch repeat region. LKP2 is a member of this family and functions either within or very close to the circadian oscillator in Arabidopsis. Promoter-GUS fusion studies revealed that the LKP2 gene was highly active in rosette leaves. In CaMV 35S:LKP2-GFP plants, GFP-associated fluorescence was detected in nuclei, suggesting that LKP2 is a nuclear protein. Yeast two-hybrid analysis demonstrated that LKP2 interacted with some Arabidopsis Skp1-like proteins (ASK), as do other ADO/FKF/LKP/ZTL family proteins, suggesting that LKP2 can form an SCF (Skp1-Cullin-F-box protein) complex that functions as a ubiquitin E3 ligase. LKP2 interacted not only with itself but also with other members of the family, LKP1 and FKF1. The two-hybrid analysis also demonstrated that LKP2 interacted with TOC1, a clock component, but not with CCA1 or LHY, negative regulators of TOC1 gene expression. The LOV domain of LKP2 was shown to be necessary and sufficient for the interaction with TOC1. An interaction between LKP2 and APRR5, a paralogue of TOC1, was also observed, but LKP2 did not interact with APRR3, APRR7, or APRR9, other paralogues of TOC1.