Growth and cell wall changes in azuki bean epicotyls I. Changes in wall polysaccharides during intact growth

Measurement of endogenous growth rates and the mechanical propertyof the cell wall in various regions of light-grown azuki beanepicotyls revealed diat the minimum stress-relaxation time (T_o)was the shortest in the upper region (0–30 mm below theapex) of the epicotyl, where vigorous endogenous growth tookplace, and became longer toward the basal region, which wasmature and not growing. In the upper region of the epicotyl, a lower percentage of a-celluloseand a higher percentage of pectic substances than in the lowerregion were found. The percentage of hemicellulose content wasalmost constant over the whole epicotyl. Major components ofnoncellulosic neutral sugars in the cell wall were galactoseand xylose. The percentage of the galactose content to the noncellulosicpolysaccharide was highest in the upper region and lowest inthe basal region of the epicotyl, and a clearly negative correlationbetween the galactose composition and the T_o value was obtained.On the contrary, the percentage of die xylose content was highestin the basal region and lowest in die upper region, and a clearlypositive correlation between die xylose composition and theT_o value was obtained. During die endogenous growth of die intactepicotyl, all die neutral sugars, particularly galactose, increasedin die upper region, whereas in die middle and basal regions,only xylose increased. Similar changes in sugar compositionswere observed during IAA-induced elongation of die segment excisedfrom various regions of die epicotyl. (Received July 27, 1978; )     

Low-molecular-mass polypeptide components of a photosystem II preparation from the thermophilic cyanobacterium Thermosynechococcus vulcanus

Using a recently introduced electrophoresis system [Kashino et al. (2001) Electrophoresis 22: 1004], components of low-molecular-mass polypeptides were analyzed in detail in photosystem II (PSII) complexes isolated from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (formerly, Synechococcus vulcanus). PsbE, the large subunit polypeptide of cytochrome b(559), showed an apparent molecular mass much lower than the expected one. The unusually large mobility could be attributed to the large intrinsic net electronic charge. All other Coomassie-stained polypeptides were identified by N-terminal sequencing. In addition to the well-known cyanobacterial PSII polypeptides, such as PsbE, F, H, I, L, M, U, V and X, the presence of PsbY, PsbZ and Psb27 was also confirmed in the isolated PSII complexes. Furthermore, the whole amino acid sequence was determined for the polypeptide which was known as PsbN. The whole amino acid sequence revealed that this polypeptide was identical to PsbTc which has been found in higher plants and green algae. These results strongly suggest that PsbN is not a member of the PSII complex. It is also shown that cyanobacteria have cytochrome b(559) in the high potential form as in higher plants.     

Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants

Summary In order to study possible functions of the inclusion body matrix protein (IBMP) encoded by gene VI of cauliflower mosaic virus (CaMV), the XbaI fragment containing the gene VI of a Japanese strain of CaMV (CaMV S-Japan) was transferred to tobacco plants by Ti mediated transformation. Eight out of 18 kanamycin resistant plants (40%) expressed detectable levels of IBMP. Those transgenic plants expressing IBMP produced leaves with light green color, and their growth was suppressed as compared with control plants. Symptom-like necrotic spots also appeared on the leaves and stems of the mature transgenic plants. Furthermore, in these transgenic plants, pathogenesis-related proteins 1a, 1b and 1c were highly expressed and the activity of 1,3--glucanase was increased up to eightfold. From these results, we concluded that expression of the IBMP is associated with symptom development.     

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.     

Cyclical changes in the fine structure of bovine endometrial gland cells

Summary Cyclical changes in bovine endometrial gland cells were investigated in six heifers, three at estrus and three at day twelve of the estrous cycle in the luteal phase. The epithelium is generally low at estrus but high in the luteal phase. There are ciliated and non-ciliated cells. The ciliated cells are fewer and lighter and show inconspicuous cyclical changes.The secretory cells show more prominent changes. At estrus, their free border is flat with short microvilli. The conspicuous rough-surfaced endoplasmic reticulum may synthesize protein for later secretion. The Golgi complex seems inactive. The high number of cytosegresomes and dense bodies might express cell regression caused by endocrine changes.In the luteal phase, the cells are lighter with long microvilli. The Golgi apparatus shows vacuoles and immature secretory droplets. Secretory vacuoles with light contents occur in the apical cytoplasm. Some of them appear to discharge their contents into the lumen. This is interpreted as evidence of merocrine secretion. Accumulations of tubular, smooth-surfaced endoplasmic reticulum, masses of glycogen granules, and several fat droplets are present.Some lymphocytes and degranulated granulocytes are seen near the basement membrane, more frequently at estrus.Financial support for this study was received from Anslaget för främjande av medicinsk forskning vid Veterinärhögskolan.The authors express their gratitude to Prof. A. Bane and Dr. J.-E. E. Ringmar, Department of Obstetrics and Gynecology, for their help with the selection and clinical control of the animals and for keeping them in good condition.Post doctoral fellow, No. 43-KO-52 (1968) from the Educational Ministry of Japan.     

Voltammetric detection of inorganic phosphate using ion-channel sensing with self-assembled monolayers of a hydrogen bond-forming receptor

A voltammetric ion-channel sensing for phosphate based on gold electrodes modified with the self-assembled monolayers of a bis-thiourea receptor was developed to detect phosphate. The working principle of this voltammetric sensor conceptually mimics that of ligand gated ion-channel proteins, as to chemically stimulated changes in membrane permeability. The response to analytes is based on the change in electron transfer rate constant of the redox reaction of [Fe(CN)(6)](4-/3-) marker, before and after binding of phosphate to the receptor on the electrode surface; where the electrostatic repulsion between a phosphate-receptor complex and the marker induced the decrease in the rate constant. In a solution of pH 7.0, a high selectivity was observed for phosphate and the sensor was virtually insensitive at all to many of other anions, such as SO(4)(2-), AcO(-), NO(3)(-), and Cl(-). The sensor response was obtained with phosphate concentrations above 5.0 x 10(-4) M using cyclic voltammetry and differential pulse voltammetry.     

Tyrosine phosphorylation-dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6-PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type-specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity.     

Enteral tube feeding alters the oral indigenous microbiota in elderly adults

Enteral tube feeding is widely used to maintain nutrition for elderly adults with eating difficulties, but its long-term use alters the environment of the oral ecosystem. This study characterized the tongue microbiota of tube-fed elderly adults by analyzing the 16S rRNA gene. The terminal restriction fragment length polymorphism (T-RFLP) profiles of 44 tube-fed subjects were compared with those of 54 subjects fed orally (average age, 86.4 ± 6.9 years). Bar-coded pyrosequencing data were also obtained for a subset of the subjects from each group (15 tube-fed subjects and 16 subjects fed orally). The T-RFLP profiles demonstrated that the microbiota of the tube-fed subjects was distinct from that of the subjects fed orally (permutational multivariate analysis of variance [perMANOVA], P < 0.001). The pyrosequencing data revealed that 22 bacterial genera, including Corynebacterium, Peptostreptococcus, and Fusobacterium, were significantly more predominant in tube-fed subjects, whereas the dominant genera in the subjects fed orally, such as Streptococcus and Veillonella, were present in much lower proportions. Opportunistic pathogens rarely detected in the normal oral microbiota, such as Corynebacterium striatum and Streptococcus agalactiae, were often found in high proportions in tube-fed subjects. The oral indigenous microbiota is disrupted by the use of enteral feeding, allowing health-threatening bacteria to thrive.     

Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage

The p53 tumor suppressor is activated in the cellular response to genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or induction of apoptosis; however, a molecular mechanism responsible for these distinct functions remains unclear. Recent studies revealed that phosphorylation of p53 on Ser(46) was associated with induction of p53AIP1 expression, resulting in the commitment of the cell fate into apoptotic cell death. Moreover, upon exposure to genotoxic stress, p53DINP1 was expressed and recruited a kinase(s) to p53 that specifically phosphorylated Ser(46). Here, we show that the pro-apoptotic kinase, protein kinase C delta (PKCdelta), is involved in phosphorylation of p53 on Ser(46). PKCdelta-mediated phosphorylation is required for the interaction of PKCdelta with p53. The results also demonstrate that p53DINP1 associates with PKCdelta upon exposure to genotoxic agents. Consistent with these results, PKCdelta potentiates p53-dependent apoptosis by Ser(46) phosphorylation in response to genotoxic stress. These findings indicate that PKCdelta regulates p53 to induce apoptotic cell death in the cellular response to DNA damage.