Codon-anticodon interaction at the ribosomal P (peptidyl-tRNA) site

A method for binding tRNA to ribosomes, introduced by Watanabe [Watanabe, S. (1972) J. Mol. Biol. 67, 443-457], permits nonenzymatic binding of N-acetyl-Phe-tRNA(Phe) to either the ribosomal aminoacyl-tRNA (A) or peptidyl-tRNA (P) site with almost 100% specificity. We used this method to analyze a possible codon-anticodon interaction at the P site for NH(2)-blocked aminoacyl-tRNA and deacylated tRNA. N-Acetyl-Phe-tRNA(Phe) bound only to the P site of poly(U)-programmed 70S ribosomes, not to poly(A)-programmed ribosomes. The reverse mRNA dependence was found for N-acetyl-Lys-tRNA(Lys). A series of purified deacylated tRNAs was analyzed in the poly(U) and poly(A) system for abilities to block P-site binding of N-acetyl-aminoacyl-tRNA and to direct the N-acetyl-aminoacyl-tRNA to the A site. Only the cognate tRNA was as effective as the bulk tRNA at a concentration of less than 1/20th that of bulk tRNA. tRNAs whose corresponding codons are identical or similar (same base character) in the first two codon positions showed a low but significant effect. The other noncognate tRNAs were unable to direct the NH(2)-blocked aminoacyl-tRNAs to the A site. Chlortetracycline interfered neither with the P-site binding of NH(2)-blocked aminoacyl-tRNA nor with the effects of deacylated tRNAs. Furthermore, the translocation blocker viomycin affected neither the binding to the A site nor that to the P site. These effects of both antibiotics indicate that both kinds of tRNA do not bind transiently in the A site before filling the P site and that codon-anticodon interaction takes place at the P site.     

Lymphatic reprogramming of microvascular endothelial cells by CEA-related cell adhesion molecule-1 via interaction with VEGFR-3 and Prox1

Here, we demonstrate that carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed and co-localized with podoplanin in lymphatic endothelial cells (LECs) of tumor but not of normal tissue. CEACAM1 overexpression in human dermal microvascular endothelial cells (HDMECs) results in a significant increase of podoplanin-positive cells in fluorescence-activated cell sorting analyses, while such effects are not observed in CEACAM1 overexpressing human umbilical vein endothelial cell (HUVECs). This effect of CEACAM1 is ceased when HDMECs are transfected with CEACAM1/y- missing the tyrosine residues in its cytoplasmic domain. CEACAM1 overexpression in HDMECs leads to an up-regulation of vascular endothelial growth factor C, -D (VEGF-C, -D) and their receptor vascular endothelial growth factor receptor 3 (VEGFR-3) at mRNA and protein levels. HDMECs transfected with CEACAM1 but not those with CEACAM1/y- show enhanced expression of the lymphatic markers Prox1, podoplanin, and LYVE-1. Furthermore, Prox1 silencing in HDMECs via small interfering RNA blocks the CEACAM1-induced increase of VEGFR-3 expression. Number and network of endothelial tubes induced by VEGF-C and -D are enhanced in CEACAM1-overexpressing HDMECs. Moreover, VEGF-A treatment of CEACAM1-silenced HDMECs restores their survival but not that with VEGF-C and VEGF-D. These data imply that the interaction of CEACAM1 with Prox1 and VEGFR-3 plays a crucial role in tumor lymphangiogenesis and reprogramming of vascular endothelial cells to LECs. CEACAM1-induced signaling effects appear to be dependent on the presence of tyrosine residues in the CEACAM1 cytoplasmic domain.     

Acute induction of gene expression in brain and liver by insulin-induced hypoglycemia

The robust neuroendocrine counterregulatory responses induced by hypoglycemia protect the brain by restoring plasma glucose, but little is known about molecular responses to hypoglycemia that may also be neuroprotective. To clarify these mechanisms, we examined gene expression in hypothalamus, cortex, and liver 3 h after induction of mild hypoglycemia by a single injection of insulin, using cDNA microarray analysis and quantitative real-time PCR. Real-time PCR corroborated the induction of six genes (angiotensinogen, GLUT-1, inhibitor of kappaB, inhibitor of DNA binding 1 [ID-1], Ubp41, and mitogen-activated protein kinase phosphatase-1 [MKP-1]) by insulin-induced hypoglycemia in the hypothalamus: five of these six genes in cortex and three (GLUT-1, angiotensinogen, and MKP-1) in liver. The induction was due to hypoglycemia and not hyperinsulinemia, since fasting (characterized by low insulin and glucose) also induced these genes. Four of these genes (angiotensinogen, GLUT-1, ID-1, and MKP-1) have been implicated in enhancement of glucose availability, which could plausibly serve a neuroprotective role during acute hypoglycemia but, if persistent, could also cause glucose-sensing mechanisms to overestimate plasma glucose levels, potentially causing hypoglycemia-induced counterregulatory failure. Although using cDNA microarrays with more genes, or microdissection, would presumably reveal further responses to hypoglycemia, these hypoglycemia-induced genes represent useful markers to assess molecular mechanisms mediating cellular responses to hypoglycemia.     

Prediction of eye and skin color in diverse populations using seven SNPs

An essential component in identifying human remains is the documentation of the decedent's visible characteristics, such as eye, hair and skin color. However, if a decedent is decomposed or only skeletal remains are found, this critical, visibly identifying information is lost. It would be beneficial to use genetic information to reveal these visible characteristics. In this study, seven single nucleotide polymorphisms (SNPs), located in and nearby genes known for their important role in pigmentation, were validated on 554 samples, donated from non-related individuals of various populations. Six SNPs were used in predicting the eye color of an individual, and all seven were used to describe the skin coloration. The outcome revealed that these markers can be applied to all populations with very low error rates. However, the call-rate to determine the skin coloration varied between populations, demonstrating its complexity. Overall, these results prove the importance of these seven SNPs for potential forensic tests.     

Endostatin inhibits angiogenesis by stabilization of newly formed endothelial tubes

Endostatin decreased vascular endothelial growth factor (VEGF)-induced formation of endothelial tubes and microvessels sprouting from aortic rings and blocked their network. After cessation of treatment, the survival time of endostatin plus VEGF-treated tubes was approximately doubled in comparison to VEGF alone. Endostatin antibody blocked VEGF-induced endothelial tube formation and disrupted existing tubes. Endostatin immunostaining was localized between endothelium and basement membrane and in inter-endothelial junctions of new, but not of quiescent, blood vessels. In tumors grown in SCID mice, endostatin immunostaining was stronger accompanying blood vessel maturation and was significantly prominent in vessels of tumor marginal zone where angiogenesis is highly active. These data indicate a new antiangiogenic action of endostatin stabilizing and maturating endothelial tubes of newly formed blood vessels. Thus, strategies accelerating vascular stabilization and maturation could be promising in tumor therapy. This revised version was published online in June 2006 with corrections to the Cover Date.     

Einflüsse des Knorpelwachstums auf die Formung des Mittelgesichts

Ohne ZusammenfassungMit 48 AbbildungenHerrn Professor Dr. Dr. G. Korkhaus zum 75. Geburtstag     

Verification of eye and skin color predictors in various populations

Validation of testing methods is an essential feature in all scientific endeavors, but it is particularly important in forensics. Due to the sensitive nature of these investigations and the limited sample size it is crucial to validate all employed procedures. This includes novel forensic phenotypic DNA tests, to learn more of their capabilities and limitations before incorporating them as routine methods. Ideally, validations are performed on large sample sets that mimic real cases. Recently, three phenotypic predictors, two for eye colors and one for skin color have been published (Spichenok et al., 2011; Walsh et al., 2011). These predictors are well-defined by a selection of single nucleotide polymorphisms (SNPs) and unambiguous instructions on how to interpret the genotypes. These standardized approaches have the advantages that they can be applied in diverse laboratories leading to the same outcome and offer the opportunity for validation. For these tests to be used on the characterization of human remains, they should be validated on various populations to perform reliably without prior knowledge of ethnic origin. Here, in this study, these eye and skin color predictors were validated on new sample sets and it could be confirmed that they can be applied in various populations, including African-American, South Asian (dark), East Asian (light), European, and mixed populations. The outputs were either predictive or inconclusive. Predictions were then compared against the actual eye and skin colors of the tested individuals. The error-rates varied; they were low for the predictors that describe the eye and skin color exclusively (non-brown or non-blue and non-white or non-dark, respectively) and higher for the predictor that describes individual eye colors (blue, brown, and intermediate/green), because of uncertainties with the green eye color prediction. Our investigation deepens the insight for these predictors and adds new information.