Phosphorogenic and spontaneous formation of tris(bipyridine)ruthenium in peptide scaffolds

Redox‐active ruthenium complexes have been widely used in various fields; however, the harsh conditions required for their synthesis are not always conducive to their subsequent use in biological applications. In this study, we demonstrate the spontaneous formation of a derivative of tris(bipyridine)ruthenium at 37°C through the coordination of three bipyridyl ligands incorporated into a peptide to a ruthenium ion. Specifically, we synthesized six bipyridyl‐functionalized peptides with randomly chosen sequences. The six peptides bound to ruthenium ions and exhibited similar spectroscopic and electrochemical features to tris(bipyridine)ruthenium, indicating the formation of ruthenium complexes as we anticipated. The photo‐excited triplet state of the ruthenium complex formed in the peptides exhibited an approximately 1.6‐fold longer lifetime than that of tris(bipyridine)ruthenium. We also found that the photo‐excited state of the ruthenium complexes was able to transfer an electron to methyl viologen, indicating that the ruthenium complexes formed in the peptides had the same ability to transfer charge as tris(bipyridine)ruthenium. We believe that this strategy of producing ruthenium complexes in peptides under mild conditions will pave the way for developing new metallopeptides and metalloproteins containing functional metal‐complexes.     

Interactions of in vitro selected fluorogenic peptide aptamers with calmodulin

Objectives

We examined the importance of aptamer usage under the same condition as the selection process by employing the previously selected aptamers for calmodulin (CaM) which includes a non-natural fluorogenic amino acid, 7-nitro-2,1,3-benzoxadiazole.

Results

We added five amino acids at the N-terminus which was employed for the selection and then we tested the affinity and selectivity for CaM binding. Surface plasmon resonance and fluorescence measurements showed that the additional amino acids for one of the aptamers drastically improved binding affinity to CaM, indicating the importance of aptamer use under the same conditions as the selection process. Such drastic improvement in affinity was not observed for the sequence which had been reported previously. Nuclear magnetic resonance data identified that the primary binding site is located in a C-terminal of CaM and the additional residues enhance interactions with CaM.

Conclusions

We found that the addition of the common sequence, which was employed for ribosome display, makes the affinity of a selected peptide as strong as the previously reported peptide.

     

Hybrid incompatibilities in interspecific crosses between tetraploid wheat and its wild diploid relative <Emphasis Type="Italic">Aegilops umbellulata</Emphasis>

Key message

Herbaspirillum rubrisubalbicans decreases growth of rice. Inoculation of rice with H. rubrisubalbicansincreased the ACCO mRNA levels and ethylene production. The H. rubrisubalbicans riceinteractions were further characterized by proteomic approach.

Abstract

Herbaspirillum rubrisubalbicans is a well-known growth-promoting rhizobacteria that can also act as a mild phyto-pathogen. During colonisation of rice, RT-qPCR analyses showed that H. rubrisubalbicans up-regulates the methionine recycling pathway as well as phyto-siderophore synthesis genes. mRNA levels of ACC oxidase and ethylene levels also increased in rice roots but inoculation with H. rubrisubalbicans impaired growth of the rice plant. A proteomic approach was used to identify proteins specifically modulated by H. rubrisubalbicans in rice and amongst the differentially expressed proteins a V-ATPase and a 14-3-3 protein were down-regulated. Several proteins of H. rubrisubalbicans were identified, including the type VI secretion system effector Hcp1, suggesting that protein secretion play a role colonisation in rice. Finally, the alkyl hydroperoxide reductase, a primary scavenger of endogenous hydrogen peroxide was also identified. Monitoring the levels of reactive oxygen species in the epiphytic bacteria by flow cytometry revealed that H. rubrisubalbicans is subjected to oxidative stress, suggesting that the alkyl hydroperoxide reductase is an important regulator of redox homeostasis in plant-bacteria interactions.

     

CH/pi interaction in the conformation of peptides. A database study.

A study was carried out, with use of the Cambridge Structural Database, to examine the role of the CH/pi interaction in the conformation of peptides. A number of short intramolecular CH/pi distances have been shown in the crystal structure of peptides bearing at least an aromatic residue in the sequence. The molecular structure in the crystal was inspected individually to know whether the conformation is merely a consequence of the so-called packing forces, or the CH/pi interaction plays a role. It has been demonstrated that the CH/pi interaction constitutes one of the key factors in controlling the conformation of peptides.     

The Length and Viscosity Dependence of End-to-End Collision Rates in Single-Stranded DNA

Intramolecular dynamics play an essential role in the folding and function of biomolecules and, increasingly, in the operation of many biomimetic technologies. Thus motivated we have employed both experiment and simulation to characterize the end-to-end collision dynamics of unstructured, single-stranded DNAs ranging from 6 to 26 bases. We find that, because of the size and flexibility of the optical reporters employed experimentally, end-to-end collision dynamics exhibit little length dependence at length scales <11 bases. For longer constructs, however, the end-to-end collision rate exhibits a power-law relationship to polymer length with an exponent of −3.49 ± 0.13. This represents a significantly stronger length dependence than observed experimentally for unstructured polypeptides or predicted by polymer scaling arguments. Simulations indicate, however, that the larger exponent stems from electrostatic effects that become important over the rather short length scale of these highly charged polymers. Finally, we have found that the end-to-end collision rate also depends linearly on solvent viscosity, with an experimentally significant, nonzero intercept (the extrapolated rate at zero viscosity) that is independent of chain length—an observation that sheds new light on the origins of the “internal friction” observed in the dynamics of many polymer systems.     

A case with glucagonoma syndrome--endocrine and metabolic studies

The results of clinical endocrine and metabolic studies on a 57-year-old female with surgically and autopsy verified glucagonoma syndrome were presented. All of the clinical manifestations of glucagonoma syndrome so far reported in the literature were noted but there was no evidence indicating the presence of multiple endocrine adenomatosis. The plasma IRG level was always more than 20 times above the normal, and the IRG response to insulin and tolbutamide injection was abnormal and the results of the other endocrinological studies revealed less remarkable features, if any. The surgically removed metastatic tumor of the liver contained an enormous amount of IRG and an appreciable amount of IRI, indicating that the elevated plasma IRG was mainly of tumor origin. These results clearly indicate that in glucagonoma there is some abnormality in glucagon release from the tumor. In addition to these findings, hypocalcemia, cardiac left ventricular hypertrophy and gastrointestinal dysfunction reportedly due to hyperglucagonemia were also seen in this patient.     

Regional Difference in Sex Steroid Action on Formation of Morphological Sex Differences in the Anteroventral Periventricular Nucleus and Principal Nucleus of the Bed Nucleus of the Stria Terminalis

Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-α (ERα), but not estrogen receptor-β (ERβ). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ERα, or ERβ genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ERα genes, but not the ERβ gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ERα, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ERα mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ERα mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ERα during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.     

A case with glucagonoma syndrome--heterogeneity of glucagon and insulin

The heterogeneity of glucagon and insulin in plasma and tissue extracts from a 57-year-old female with glucagonoma syndrome with surgically and autopsy verified islet-cell tumors was studied by Bio-Gel P-10 filtration. The preoperative plasma immunoreactive glucagon (IRG) level was 20.2 ng/ml, and plasma glucagon-like immunoreactivity(GLI) 25.8 ng/ml. The column chromatography of the preoperative plasma revealed three or four IRG components and four GLI components. Among these, peak II, the large glucagon immunoreactivity (LGI) peak, considered a candidate for proglucagon, was prominent, along with peak III. The resected metastatic liver tumor contained an enormous amount of IRG and an appreciable amount of immunoreactive insulin (IRI), indicating that the elevated plasma IRG was mainly of tumor origin. The IRG pattern of the tumor tissue extract revealed a small quantity of IRG in peaks I and II, and a large amount in peak III; control pancreatic tissue extract manifested a similar elution pattern. The IRI elution pattern of the tumor tissue extract revealed two major IRI peaks which migrated close to the elution volume of cytochrome C and insulin, respectively. This is a quite different pattern from the control pancreatic tissue extract in which the RI peak was localized in the elution volume of the insulin. We conclude that the present metastatic liver tumor produced not only enormous amounts of glucagon but heterogeneous peptides which contained immunological insulin determinants within their.