The functional maturation of M cells is dramatically reduced in the Peyer's patches of aged mice.

The transcytosis of antigens across the follicle-associated epithelium (FAE) of Peyer's patches by microfold cells (M cells) is important for the induction of efficient immune responses to mucosal antigens. The mucosal immune response is compromised by ageing, but effects on M cells were unknown. We show that M-cell density in the FAE of aged mice was dramatically reduced. As a consequence, aged Peyer's patches were significantly deficient in their ability to transcytose particulate lumenal antigen across the FAE. Ageing specifically impaired the expression of Spi-B and the downstream functional maturation of M cells. Ageing also dramatically impaired C-C motif chemokine ligand 20 expression by the FAE. As a consequence, fewer B cells were attracted towards the FAE, potentially reducing their ability to promote M-cell maturation. Our study demonstrates that ageing dramatically impedes the functional maturation of M cells, revealing an important ageing-related defect in the mucosal immune system's ability to sample lumenal antigens.

Glass transition and molecular mobility in polymer thin films.

Extensive studies on polymer thin films to date have revealed their interesting but unusual properties such as film thickness dependence of glass transition temperature Tg and thermal expansivity. Recent studies have shown that the lower Tg is not always related to the higher mobility in polymer thin films, which contradicts our current understanding of the glass transition process. In this work, we report the results of inelastic neutron-scattering measurements on polystyrene thin films using two spectrometers with different energy resolutions as well as ellipsometry measurements. The results are interpreted in terms of cooperatively rearranging region and motional slowing down due to the surface effect that explain plausibly the anomalous relationship between the glass transition temperature Tg and the molecular mobility in thin films.

Bovine anterior pituitary progenitor cell line expresses interleukin (IL)-18 and IL-18 receptor.

In the anterior pituitary gland, inflammatory mediators regulate cell function through an immuno-endocrine pathway. Recent studies have shown that undifferentiated stem cells act as immunomodulators. These studies prompted us to establish a progenitor cell line from the bovine anterior pituitary gland and to detail its function. First, we localised interleukin (IL)-18 by immunohistochemistry to the marginal cell layer of Rathke's pouch that is assumed to embody a stem/progenitor cell compartment of the postnatal pituitary gland. A cloned anterior pituitary-derived cell line from the bovine anterior pituitary gland was established from single cell clone by the limiting dilution method and was designated as bovine anterior pituitary-derived cell line (BAPC)-1. BAPC-1 cells constantly expressed mRNAs for IL-18 and IL-18 receptor, and grew steadily and rapidly in the medium containing epidermal growth factor and basic fibroblast growth factor. The cell line also expressed the mRNAs for the stem/progenitor cell- related factors such as Nanog, Oct-4, Ptch1, Nestin, Notch1, Hes1, Lrp and Fzd4, and the mRNAs for embryonic pituitary-related factors, such as Lhx3, PitX1 and Pit-1. The nuclei of BAPC-1 were immunostained positively for Pit-1, Hes1 and beta-catenin antibodies. Furthermore, BAPC-1 cells expressed mRNAs for cytokine such as IL-1alpha, IL-6, IL-7, IL-12 and IL-15. Stimulation of BAPC-1 cells with IL-18 increased expression of mRNAs for IL-1alpha, IL-6, IL-1beta and IL-8. At day 6 in culture, BAPC-1 cells also express growth hormone mRNA. These results strongly suggest that BAPC-1 is a stem/progenitor cell line and modulates the immuno-endocrine function of the anterior pituitary cells through its cytokine production.

Revisiting silicate substituted hydroxyapatite by solid-state NMR.

Silicon-substituted hydroxyapatite (Si-HAp) has shown promising properties such as high-bone remodeling around implants. So far, the techniques used for the structural characterization of the Si-HAp have given indirect evidence of the presence of silicon inside the structure (by X-ray and neutron diffraction). In this paper, we focus on Si-HAp derivatives obtained by a precipitation method (widely described in the literature). We demonstrate here by solid-state NMR spectroscopy that only a fraction of the silicon atoms are incorporated into the HAp lattice in the form of Q(0) (SiO(4) (4-)) species, for 4.6 wt% Si-HAp. A large amount of silicate units are located outside the HAp structure and correspond to silica-gel units. All results were established through (29)Si MAS, (1)H -->(29)Si CP MAS and T(1)rho((1)H) edited (1)H -->(29)Si CP MAS experiments. This last pulse scheme acted as a powerful editing sequence, leading to unambiguous spectroscopic conclusions, concerning the location of the SiO(4) (4-) moieties.

Dynamic anisotropy and heterogeneity of polystyrene thin films as studied by inelastic neutron scattering.

We studied the dynamic anisotropy and heterogeniety of polystyrene thin films in glassy state with inelastic neutorn scattering. Adjusting the scattering vector to the directions parallel and perpendicular to the film surface, we observed the elastic scattering intensities as a function of temperature. It was found for the 200 A film that the elastic intesity decreased with increasing temperature more rapidly in the perpendicular direction than in the pararell direction, showing the higher mobility in the perpendicular direction. However, such dynamical anisotropy was not observed in the 1000 A film. The decrease in the mobility was observed with the film thickness in both the directions. These results were explained in terms of an interface hard layer. We also evelauated the dynamical heterogeniety from the non-Gaussian parameter A0, which increased with decreasing the film thickness, showing the increase in the dynamical heterogeneity. Assuming a simple bi-layer model consisting of the interface hard layer and the bulk-like layer, we analyzed the thickness dependence of the non-Gaussian parameter A0 and the mean square displacement (u2) to find that the hard layer has a thickness of approximately 130 A and a mean square displacement of approximately 0.018 A2 at 230 K.

Prevention of diet-induced obesity by dietary isomerized hop extract containing isohumulones, in rodents.

OBJECTIVE: Isomerized hop extract (IHE), which consists mainly of isohumulones and is required in the beer brewing process, was investigated for its effects on diet-induced obesity in two strains of mice. DESIGN: C57BL/6N and KK-A(y) mice were fed a standard or high-fat diet containing IHE and their body and tissue weights were measured at various time points. Oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were carried out in high-fat diet-fed C57BL/6N mice. The effects of IHE on intestinal lipid absorption were examined in Wistar rats using a plasma triacylglycerol assay after oral administration of a lipid emulsion. Fecal lipid levels were also measured in these animals after they were fed a high-fat diet containing IHE for 15 days. The effects of IHE on pancreatic lipase activity and the expression of genes involved in hepatic lipid metabolism were also examined using an in vitro assay and quantitative RT-PCR, respectively. RESULTS: Supplementation of high-fat-containing chow with IHE reduced body weight gain and improved glucose tolerance in our experimental mice. A reduction in body weight gain was also observed in C57BL/6N mice fed a standard diet containing IHE. Wistar rats fed a high-fat diet containing IHE showed reduced plasma triacylglycerol levels and an increase in their fecal lipid excretion. Similarly, their pancreatic lipase activity was inhibited and their elevation in plasma triacylglycerol levels seen after the oral administration of lipid emulsion was significantly suppressed. IHE-fed mice showed an increased expression in their lipid oxidation genes and a decreased expression in genes involved in triacylglycerol biosynthesis. CONCLUSION: The inhibition of intestinal dietary fat absorption may be the mechanism by which IHE induces its weight-lowering effects in high-fat diet-fed mice. The modulatory effect of IHE on lipid metabolism may also, at least partly, be responsible for its beneficial effects on body weight gain. These results suggest that IHE may be helpful in humans in preventing diet-induced obesity and perhaps even metabolic syndrome, the latter of which is known to be associated with obesity.

Dynamics of deuterated polystyrene-protonated butadiene diblock copolymer micelles by neutron spin echo.

We report on neutron spin-echo (NSE) measurements on deuterated styrene-protonated butadiene diblock copolymer micelles in deuterated n-decane to investigate the dynamics of butadiene blocks in the corona. Before the NSE measurements, we performed small-angle neutron-scattering (SANS) measurements on the micelles to evaluate the structure to give a basis for the discussion of the dynamics. In the SANS study, we have estimated the form factor P(Q) in terms of a hard-core-shell model from the direct evaluation without curve-fitting procedure while a more flexible core-shell model with the structure factor S(Q) gives a better fit to the observed data. The observed normalized intermediate scattering function I(Q,t)/I(Q,0) by NSE does not show the collective motions corresponding to the so-called breathing mode but rather single chain motion (Zimm modes) for both the 2 and 20 wt % micelle solutions. The Zimm decay rate Gamma(z) in the micelle solution is slow compared with that in the homopolymer solution. This slowing down is assigned to the effective high concentration in the corona. The differences in Gamma(z) between concentrated solutions and the 20% micellar solution are attributed to end-tethering effect of the corona chains on the core surface. The possible reasons why the breathing mode was not observed in the present micelle system are discussed on the basis of chain density in the corona.

Neutron spin-echo studies on dynamic and static fluctuations in two types of poly(vinyl alcohol) gels.

We report neutron spin-echo measurements on two types of poly(vinyl alcohol) (PVA) gels. The first is PVA gel in a mixture of dimethyl sulfoxide (DMSO) and water with volume ratio 60/40 , and the second is PVA gel in an aqueous borax solution. The observed normalized intermediate scattering functions I (Q,t) /I (Q,0) are very different between them. The former I (Q,t) /I (Q,0) shows a nondecaying component in addition to a fast decay, but the latter does not have the nondecaying one. This clearly indicates that the fluctuations in the former PVA gel consist of static and dynamic fluctuations whereas the latter PVA gel does include only the dynamic fluctuations. The dynamic fluctuations of the former and latter gels have been analyzed in terms of a restricted motion in the network and Zimm motion, respectively, and the origins of these motions will be discussed.

Contraction and reexpansion of polymer thin films.

We report x-ray reflectivity measurements on polystyrene thin films supported on silicon wafer. In annealing experiments, we found fast and slow contraction processes in the thin films above the glass transition temperature. The former is the normal relaxation (annealing) process observed in bulk, and the latter is unexpected and enhanced in thin films below approximately 20 nm. In addition, we found unexpected extremely slow reexpansion processes in the glassy state. These unexpected very slow processes are discussed in terms of lateral contraction and expansion processes driven by entropic changes at the interfaces and the difference of the expansivities between polystyrene and silicon wafer.

Apparent nonergodic behavior of supercooled liquids above the glass transition temperature.

A speckle pattern can be observed in the polarized component of light scattered from glass forming liquids far above their glass transition temperature. This speckle pattern fluctuates with characteristic time that corresponds to the relaxation time of the additional ultraslow component in the correlation function and is about seven orders of magnitude longer than the relaxation time of the alpha-process. This slow process is out of the experimental time window when the alpha-process is measured by means of the photon correlation spectroscopy and results in an apparent nonergodicity which can be seen as a baseline offset in the ensemble-averaged correlation function. In contrast, the time-averaged field correlation functions which have been measured in practically all light scattering studies always decay to zero. The slow process contributes a q-dependent excess intensity to the polarized component of scattered light. The values of the nonergodicity parameters obtained from the static and dynamic light scattering experiments are equal. Both the slow component and the excess intensity result from denser regions of fractal character which develop in glass-forming liquids on approaching the glass transition.

Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system.

The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels revealed titers which correlated highly with respective hemagglutinin inhibition (HI) antibody titers which ranged from <1:8 to 1:256 (correlation coefficient among them was 0.850). ELISA OD levels and HI titers increased at 5 and 7 days post-inoculation, respectively, in a horse inoculated intranasally with LP/93. Respective antibody levels were observed to change in an essentially parallel manner during a period of 1 month. Similarly, ELISA OD levels correlated with HI titers in horses during a period of 6 weeks following intramuscular inoculation with inactivated single-strain vaccines containing LP/93, A/equine/Kentucky/1/81 (H3N8) or A/equine/Rome/5/91 (H3N8). A similar pattern was also observed in eight horses throughout a 10-week period following inoculation with a commercially available inactivated trivalent vaccine containing A/equine/Newmarket/1/77(H7N7), A/equine/Kentucky/81 and LP/93. From these results, it is suggested that this ELISA system could be used for disease diagnosis and surveillance of HI antibody titers among vaccinated horses.

Further evidence of spinodal decomposition during the induction period of polymer crystallization: time-resolved small-angle x-ray scattering prior to crystallization of poly(ethylene naphthalate)

Aiming to clarify spinodal decomposition of polymers in the induction period of crystallization, time-resolved small-angle x-ray scattering measurements have been made in situ for poly(ethylene naphthalate) while it was crystallized from the glass, or in the case of the so-called glass crystallization. It is confirmed for this polymer that in the very beginning of the induction period a scattering peak appears at around 0.03 A (-1) in scattering vector q, which corresponds to a characteristic wavelength of 200 A in density fluctuations, and grows with time. Time evolution of this scattering peak is described by the kinetics of the spinodal decomposition as previously reported for the glass crystallization of poly(ethylene terephthalate).

Acylated delphinidin 3-rutinoside-5-glucosides in the flowers of Petunia reitzii.

Two acylated anthocyanins were isolated from selected individuals of Petunia reitzii, and identified to be delphinidin 3-O-[6-O-(4-O-(4-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-tr ans-p-coumaroyl)-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside]- 5-O-[beta-D-glucopyranoside] and delphinidin 3-O-[6-O-(4-O-(4-O-(beta-D-glucopyranosyl)-trans-p-coumaroyl)-alph a-L-rhamnopyranosyl)-beta-D-glucopyranoside]-5-O-[beta-D-glucopyranoside ]. Nine known anthocyanins were also identified.

Ultra-small-angle neutron scattering studies on phase separation of poly(vinyl alcohol) gels

Time-resolved light scattering measurements during the gelation process of a poly(vinyl alcohol) (PVA) solution in a mixture of dimethyl sulfoxide and water (H2O) have shown that a spinodal decomposition (SD) type phase separation takes place in the early stage of gelation. In this case the kinetics of SD is valid only before macroscopic gelation occurs because the growth rate is slowed down by gelation. Such SD type phase separation makes the solution opaque as it proceeds, and hence the structural change can no longer be followed by light scattering. To investigate the structure of the opaque PVA gel as well, we have employed an ultra-small-angle neutron scattering technique using a Bonse-Hart camera. These observations reveal that even after the macroscopic gelation the structure due to the microphase separation on a spatial scale of several &mgr;m continues to grow against the elasticity. This may be because at first the gel structure is too soft to suppress the growth of the microphase separation, but within 24 h after the quenching the growth terminates. On the basis of the results, we will discuss a possible mechanism of the microphase separation after gelation.

A case of cervical cancer with aggressive tumor growth: possible autocrine growth stimulation by G-CSF and Il-6.

BACKGROUND: Patients with tumors that produce hematopoietic growth factors, such as G-CSF, sometimes exhibit leukemoid reactions. Squamous cell carcinomas of the uterine cervix producing such factors are rarely reported. CASE: A 56-year-old woman had severe leukocytosis (>50 x 10(3)/microl) and fever without evidence of infection after radical hysterectomy for treatment of her cervical cancer. The parametrial recurrent mass appeared after the operation and exhibited extremely aggressive growth. Serum levels of G-CSF and IL-6 were extremely high, and immunohistochemical analysis and RT-PCR assays revealed that G-CSF and IL-6 as well as their receptors were produced in tumor cells. CONCLUSIONS: This is a case of cervical cancer which exhibited an aggressive clinical course, possibly due to autocrine stimulation of cell growth by G-CSF and IL-6.

Demethylating reagent 5-azacytidine inhibits telomerase activity in human prostate cancer cells through transcriptional repression of hTERT.

Telomerase activation is thought to be a critical step in cellular immortality and oncogenesis. Several reagents including differentiation-inducing and antineoplastic agents are known to inhibit telomerase activity, although the molecular mechanisms through which they inhibit telomerase activity remain unclear. Demethylating reagents have recently been used as potential antineoplastic drugs for some types of cancers including those of the prostate. In the present study, we examined the effect of the demethylating reagent 5-azacytidine (5-aza-CR) on telomerase activity using cells of two prostate cancer cell lines, DU-145 and TSU-PR1. 5-aza-CR treatment significantly reduced telomerase activity in TSU-PR1 cells, but not in DU-145 cells, although growth inhibition was observed to a similar extent in both cell lines. Reverse transcription-PCR analyses revealed that inhibition of telomerase activity was accompanied by down-regulation of telomerase catalytic subunit (hTERT) mRNA expression. Transient expression assays showed that 5-aza-CR repressed the transcriptional activity of the hTERT promoter and that the E-box within the core promoter was responsible for this down-regulation. Western blot analyses revealed that 5-aza-CR reactivated p16 expression and repressed c-Myc expression in TSU-PR1 cells but not in DU-145 cells. Overexpression of p16 in TSU-PR1 cells led to significant repression of c-Myc transcription. These findings suggest that 5-aza-CR inhibits telomerase activity via transcriptional repression of hTERT, in which p16 and c-Myc may play a key role.

Identification and characterization of negative regulatory elements of the human telomerase catalytic subunit (hTERT) gene promoter: possible role of MZF-2 in transcriptional repression of hTERT.

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of human telomerase and is a critical determinant of the enzymatic activity of telomerase. Expression of hTERT is known to be regulated mainly at the transcriptional level. In the present study, using transient expression assays, we identified a 400 bp silencer of the hTERT promoter between -776 and -378 upstream of the proximal core promoter. The inhibitory effects of this silencer were enhanced with cellular differentiation. A computer-assisted homology search identified multiple binding motifs for myeloid-specific zinc finger protein 2 (MZF-2) within this region. Mutation introduced in these sites resulted in significant activation of hTERT transcription. Gel shift assays demonstrated that MZF-2 proteins specifically bound to these sites. Overexpression of MZF-2 in cells led to down-regulation of hTERT transcription as well as telomerase activity. These findings suggest that the 400 bp region upstream of the hTERT core promoter that we identified functions as a negative regulatory region and that MZF-2 may be an effector of negative regulation of hTERT.

Computation of motion direction by quail retinal ganglion cells that have a nonconcentric receptive field.

One type of retinal ganglion cells prefers object motion in a particular direction. Neuronal mechanisms for the computation of motion direction are still unknown. We quantitatively mapped excitatory and inhibitory regions of receptive fields for directionally selective retinal ganglion cells in the Japanese quail, and found that the inhibitory regions are displaced about 1-3 deg toward the side where the null sweep starts, relative to the excitatory regions. Directional selectivity thus results from delayed transient suppression exerted by the nonconcentrically arranged inhibitory regions, and not by local directional inhibition as hypothesized by Barlow and Levick (1965).

Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription.

Telomerase activation is a critical step in cellular immortality and oncogenesis. The activity of telomerase is known to be correlated with cell proliferation, but its regulation by cell cycle regulators is not well understood. In the present study, we examined the effects of p53 on telomerase activity. Wild-type p53 was introduced into SiHa cells via a recombinant adenoviral vector, Ad5CMV-p53, and change in telomerase activity was examined by quantitative telomerase assay. Telomerase activity in the Ad5CMV-p53-infected cells was significantly repressed 36 h after infection following down-regulation of human telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] mRNA expression, whereas no change in telomerase activity was observed in the cells infected with control vector AdSCMV-beta-gal. Interestingly, repression of telomerase activity was an early event that preceded cell growth inhibition or apoptosis induced by p53 overexpression, suggesting that p53 directly regulates telomerase activity. Transient expression assays using hTERT-promoter reporter constructs revealed that overexpression of p53 significantly repressed promoter activity of hTERT. 5'-Truncation of the promoter sequences revealed that the proximal core promoter region containing multiple binding sites for transcription factor Spl was responsible for p53-mediated transcriptional repression. Mutations in these binding sites for Spl led to failure of p53 to repress transcription. These findings suggest that p53 repressed telomerase activity through down-regulation of hTERT transcription and that interaction of p53 with Sp1 or other transcription factors may be involved in this regulation.