Flavonoid composition of fruit tissues of citrus species

An HPLC analysis was performed on the concentrations of flavonoids in 42 species and cultivars of the Citrus genus and those of two Fortunella and one Poncirus species according to the classification system established by Tanaka. The composition of 8 flavanones and 9 flavone/ols for these species was determined in the albedo, flavedo, segment epidermis and juice vesicle tissues, and those in the fruit and peel tissues were calculated from the composition data of the tissues. A principal component analysis showed that such neohesperidosyl flavonoids as neoeriocitrin, naringin, neohesperidin, and rhoifolin had large factor loading values in the first principal component for each tissue. The flavonoid composition of citrus fruits was approximately the same within each section of Tanaka's system, except for the species in the Aurantium section and those with a peculiar flavonoid composition such as Bergamot (C. bergamia), Marsh grapefruit (C. paradisi), Sour orange (C. aurantium), and Shunkokan (C. shunkokan). The Aurantium section included both naringin-rich and hesperidin-rich species.     

Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine beta-lactoglobulin

A disulfide bond between cysteine 66 and cysteine 160 of equine beta-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces alpha-helices as observed in the cold denatured state of the wild-type protein.     

Identification of Pip4k2β as a mechanical stimulus responsive gene and its expression during musculoskeletal tissue healing

To investigate the mechano-transduction system of cells, we identified genes responsive to a cyclic mechanical stimulus. MC3T3.E1 cells were cultured on a computer-controlled vacuum-pump-operated device designed to provide a cyclic mechanical stimulus. A maximum elongation of 15% of membrane at 10 cycles/min (3 s extension followed by 3 s relax per cycle) was repeated for 48 h. By means of a differential display, the gene expression pattern of cells exposed to the stimulus was compared with that of unexposed cells. As a result, a gene fragment that was exclusively expressed in mechanically stressed cells was identified. By using expressed sequence tag walking together with the oligo-capping method, this gene was identified as phosphatidylinositol 4-phosphate 5-kinase type II β (initially known as Pip5k2β but now reclassified as Pip4k2β). The specific up-regulation of Pip4k2β upon mechanical stimulus was also confirmed by using another apparatus, viz. a computer-controlled linearized-stepping motor system. To examine the involvement of the cyclic mechanical stimulus in the regulation of Pip4k2β expression in musculoskeletal tissue, we created an Achilles tendon transection model in rabbits. The temporal expression of Pip4k2β was assessed by means of a quantitative reverse-transcribed polymerase chain reaction. In the gastrocnemius muscle, expression of Pip4k2β rapidly decreased 1 week after transection but was restored to normal levels at 4 weeks. In the Achilles tendon, however, expression remained decreased until 4 weeks after transection. We suggest that the expression of Pip4k2β can be used as a marker for cells receiving a suitable mechanical stimulus. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.     

Generation of DNA-free Escherichia coli cells by 2-aminopurine requires mismatch repair and nonmethylated DNA

Undirected mismatch repair initiated by the incorporation of the base analog 2-aminopurine kills DNA-methylation-deficient Escherichia coli dam cells by DNA double-strand breakage. Subsequently, the chromosomal DNA is totally degraded, resulting in DNA-free cells.     

Substrate recognition ability differs among various prokaryotic tRNase Zs

There exists a significant difference in pre-tRNA preference among prokaryotic tRNase Zs. This is an enigma, because pre-tRNAs should form the common L-shaped structure and tRNase Zs should form the common structure based on the alphabeta/betaalpha-fold. To address this issue, we examined six different eubacterial and archaeal tRNase Zs including two newly isolated tRNase Zs for cleavage of 18 different pre-tRNA substrates. Two Thermotoga maritima, one Thermus thermophilus, one Bacillus subtilis, one Thermoplasma acidophilum, and one Pyrobaculum aerophilum enzymes were tested. To our surprise, the newly isolated proteins T. maritima and T. thermophilus showed the weak tRNase Z activity, even though their primary amino acid sequences are, on the whole, quite different from those of the typical tRNase Zs. We confirmed that substrate recognition ability is quite different among those tRNase Zs. In addition, we found that the optimal conditions as a whole differ significantly among the enzymes. From these results, we provided several clues to solve the enigma by showing the potential importance of the 74th-76th nucleotide sequence of pre-tRNA, the flexible arm length of tRNase Z, the divalent metal ion species, and the histidine corresponding His222 in T. maritima tRNase Z.     

Functionally relevant coupled dynamic profile of bacteriorhodopsin and lipids in purple membranes

The dynamics of bacteriorhodopsin (bR) and the lipid headgroups in oriented purple membranes (PMs) was determined at various temperatures and relative humidity (rh) using solid-state NMR spectroscopy. The 31P NMR spectra of the alpha- and gamma-phosphate groups in methyl phosphatidylglycerophosphate (PGP-Me), which is the major phospholipid in the PM, changed sensitively with hydration levels. Between 253 and 233 K, the signals from a fully hydrated sample became broadened similarly to those of a dry sample at 293 K. The 15N cross polarization (CP) NMR spectral intensities from [15N]Gly bR incorporated into fully hydrated PMs were suppressed in 15N CP NMR spectra at 293 K compared with those of dry membranes but gradually recovered at low temperatures or at lower hydration (75%) levels. The suppression of the NMR signals, which is due to interference with proton decoupling frequency (approximately 45 kHz), coupled with short spin-spin relaxation times (T2) indicates that the loops of bR, in particular, have motional components around this frequency. The motion of the transmembrane alpha-helices in bR was largely affected by the freezing of excess water at low temperatures. While between 253 and 233 K, where a dynamic phase transition-like change was observed in the 31P NMR spectra for the phosphate lipid headgroups, the molecular motion of the loops and the C- and N-termini slowed, suggesting lipid-loop interactions, although protein-protein interactions between stacks cannot be excluded. The results of T2 measurements of dry samples, which do not have proton pumping activity, were similar to those for fully hydrated samples below 213 K where the M-intermediates can be trapped. These results suggest that motions in the 10s micros correlation regime may be functionally important for the photocycle of bR, and protein-lipid interactions are motionally coupled in this dynamic regime.     

Regulation of mitochondrial morphology and cell survival by Mitogenin I and mitochondrial single-stranded DNA binding protein

We found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I in this paper, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology. We demonstrated that overexpression of Mitogenin I or mtSSB increased elongated or fragmented mitochondria in mouse C2C12 myoblast cells, respectively. On the other hand, the silencing of Mitogenin I or mtSSB by RNA interference led to an increase in fragmented or elongated mitochondria in the cells, respectively, suggesting that Mitogenin I and mtSSB are involved in the processes of mitochondrial fusion and fission, respectively. In addition, we showed that the silencing of Mitogenin I resulted in an increase in the number of trypan blue-positive cells and the silencing of mtSSB resulted in an enhancement of the sensitivity of the cells to apoptotic stimulation by etoposide. The present results demonstrated that these proteins play a role in cell survival.     

Cytochemical localization of adenylate cyclase activity in the theca folliculi of the mouse graafian follicle

Summary The adenylate cyclase activity in the theca folliculi of the mouse Graafian follicle was investigated using the electron microscopic cytochemistry. Deposits of reaction product are recognized on the plasma membrane of the fibroblast, theca cell and transitional cell from the fibroblast-like cell to the theca cell (partially or incompletely differentiated theca cell) after incubation with adenylyl-imidodiphosphate (AMP-PNP) as an effective substrate for adenylate cyclase. This fact indicates that these cells have the receptor on the plasma membrane, and the adenylate cyclase-cyclic AMP system is important for the steroid secretion or the collagen fiber production. It is difficult to clarify by this method the relationship between the adenylate cyclase activity and the functional differentiation of the theca cell.Supported by a grant from the Japanese Educational ministry     

Glandulocalyx upatoiensis, a fossil flower of Ericales (Actinidiaceae/Clethraceae) from the Late Cretaceous (Santonian) of Georgia, USA

Background and Aims

Ericales are a major group of extant asterid angiosperms that are well represented in the Late Cretaceous fossil record, mainly by flowers, fruits and seeds. Exceptionally well preserved fossil flowers, here described as Glandulocalyx upatoiensis gen. & sp. nov., from the Santonian of Georgia, USA, yield new detailed evidence of floral structure in one of these early members of Ericales and provide a secure basis for comparison with extant taxa.

Methods

The floral structure of several fossil specimens was studied by scanning electron microscopy (SEM), light microscopy of microtome thin sections and synchrotron-radiation X-ray tomographic microscopy (SRXTM). For direct comparisons with flowers of extant Ericales, selected floral features of Actinidiaceae and Clethraceae were studied with SEM.

Key Results

Flowers of G. upatoiensis have five sepals with quincuncial aestivation, five free petals with quincuncial aestivation, 20–28 stamens arranged in a single series, extrorse anther orientation in the bud, ventral anther attachment and a tricarpellate, syncarpous ovary with three free styles and numerous small ovules on axile, protruding-diffuse and pendant placentae. The calyx is characterized by a conspicuous indumentum of large, densely arranged, multicellular and possibly glandular trichomes.

Conclusions

Comparison with extant taxa provides clear evidence for a relationship with core Ericales comprised of the extant families Actinidiaceae, Roridulaceae, Sarraceniaceae, Clethraceae, Cyrillaceae and Ericaceae. Within this group, the most marked similarities are with extant Actinidiaceae and, to a lesser degree, with Clethraceae. More detailed analyses of the relationships of Glandulocalyx and other Ericales from the Late Cretaceous will require an improved understanding of the morphological features that diagnose particular extant groups defined on the basis of molecular data.     

Chronic receptor-mediated activation of Gi/o proteins alters basal t-tubular and sarcolemmal L-type Ca²⁺ channel activity through phosphatases in heart failure

L-type Ca(2+) channels (LTCCs) play an essential role in the excitation-contraction coupling of ventricular myocytes. We previously found that t-tubular (TT) LTCC current density was halved by the activation of protein phosphatase (PP)1 and/or PP2A, whereas surface sarcolemmal (SS) LTCC current density was increased by the inhibition of PP1 and/or PP2A activity in failing ventricular myocytes of mice chronically treated with isoproterenol (ISO mice). In the present study, we examined the possible involvement of inhibitory heterotrimeric G proteins (G(i/o)) in these abnormalities by chronically administrating pertussis toxin (PTX) to ISO mice (ISO + PTX mice). Compared with ISO mice, ISO + PTX mice exhibited significantly higher fractional shortening of the left ventricle. The expression level of Gα(i2) proteins was not altered by the treatment of mice with ISO and/or PTX. ISO + PTX myocytes had normal TT and SS LTCC current densities because they had higher and lower availability and/or open probability of TT and SS LTCCs than ISO myocytes, respectively. A selective PKA inhibitor, H-89, did not affect LTCC current densities in ISO + PTX myocytes. A selective PP2A inhibitor, fostriecin, did not affect SS or TT current density in control or ISO + PTX myocytes but significantly increased TT but not SS LTCC current density in ISO myocytes. These results indicate that chronic receptor-mediated activation of G(i/o) in vivo decreases basal TT LTCC activity by activating PP2A and increases basal SS LTCC activity by inhibiting PP1 without modulating PKA in heart failure.