Association between underweight and taste sensitivity in middle- to old-aged nursing home residents in Sri Lanka: a cross-sectional study.

Low taste sensitivity may be one factor related to undernutrition, which is a major problem in developing countries. The purpose of this cross-sectional study was to examine the association between underweight, one indicator of undernutrition, and taste sensitivity in middle- to old-aged Sri Lankan nursing home residents. Participants were 946 residents with BMI of <25·0 from 25 nursing homes. Data were obtained on height, weight, taste sensitivity, subjective taste ability, sex, age, ethnicity, number of years in nursing homes, activities of daily living (ADL), frequency of exercise, bowel movements, smoking status, drinking status, current number of chronic diseases, number and kinds of medications used, self-reporting questionnaire 20 (SRQ20), subjective smell ability, number of teeth present, Eichner index and flow rate of saliva. Low sensitivity to bitter taste, being male, old age, low ADL, smoking experience, drinking experience, fewer medications used and no use of medication for hypertension and diabetes were each associated with underweight (P < 0·05). In a multilevel Poisson regression model adjusted for sex, age, ADL, smoking status, drinking status, number of medications used, use of medication for hypertension and diabetes and flow rate of saliva, subjects with low sensitivity (>0·003% quinine hydrochloride dihydrate) to bitter taste had a significant 1·70 times higher prevalence ratio (95% confident interval 1·04-2·80) for underweight compared with those with high sensitivity (0·0001% quinine hydrochloride dihydrate). These results suggest that low taste sensitivity to bitter taste may be one factor related to underweight.

The squamous cell carcinoma antigens as relevant biomarkers of atopic dermatitis.

BACKGROUND: Although it is thought that both Th1- and Th2-type inflammations are involved in the pathogenesis of atopic dermatitis (AD), it is controversial which immune response is more involved in regulating the clinical severity of AD. We recently found that the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 are novel biomarkers of bronchial asthma, downstream of IL-4 and IL-13. OBJECTIVE: We examined whether SCCA1 and SCCA2 could also serve as biomarkers of AD, reflecting its Th2-type immune responses, and whether the expression level of SCCA was correlated with clinical severity of AD. METHOD: We compared the expression of SCCA1 and SCCA2 at the mRNA and protein levels in both involved and uninvolved skin of AD patients and in normal control skin. We next analysed induction of SCCA by IL-4 or IL-13 in keratinocytes. Finally, we compared the serum level of SCCA with laboratory parameters reflecting Th2-type inflammation and clinical severity in AD patients. RESULTS: SCCA1 and SCCA2 were highly expressed in involved skin of AD patients, compared with their uninvolved skin, at both mRNA and protein levels. SCCA protein was dominantly expressed in suprabasal keratinocytes in the epidermis of AD patients. Either IL-4 or IL-13, but not IFN-gamma or TNF, induced production of SCCA in keratinocytes. These result suggest that SCCA is induced in AD skin, probably due to direct actions of IL-4 and/or IL-13 on keratinocytes. Serum levels of SCCA were well correlated with eosinophil numbers and serum lactate dehydrogenase levels, and weakly with serum IgE levels, in AD patients. Furthermore, serum levels of SCCA were strongly correlated with clinical severity. CONCLUSIONS: Th2-type inflammation dominantly regulates the clinical severity of AD, and SCCA is a relevant biomarker of AD, reflecting both Th2-type inflammation and clinical severity.

Biting suppresses stress-induced expression of corticotropin-releasing factor (CRF) in the rat hypothalamus.

Corticotropin-releasing factor (CRF) expressed in the hypothalamus plays an important role in mediating behavioral responses to stressors. Restraining the body of an animal has been shown to activate and induce an enhanced expression of CRF in paraventricular neurons of the rat hypothalamus. Since aggressive biting behavior is known to suppress stress-induced noradrenaline secretion in the central nervous system and the formation of gastric ulcers, we investigated the effect of biting on restraint-induced CRF expression in the rat hypothalamus. The number of CRF-expressing neurons in the paraventricular nucleus increased significantly after short time restraint (30 or 60 min) followed by a 180-minute post-restraint period. Biting of a wooden stick during the restraint stress significantly suppressed the restraint-induced enhancement of CRF expression in the paraventricular nucleus. These observations suggest a possible anti-stress effect of biting and an important role of para-functional masticatory activity in coping with stressful events.

Identification of AFLP markers closely linked to the rhm gene for resistance to Southern Corn Leaf Blight in maize by using bulked segregant analysis.

Southern Corn Leaf Blight (SCLB) is an important disease in warm-temperate and tropical corn-producing areas throughout the world. We applied a combination of the amplified fragment length polymorphism (AFLP) technique and bulked segregant analysis (BSA) to a large F2 population in order to identify molecular markers linked to the rhm gene for resistance to SCLB. One co-dominant AFLP marker, p7m36, was mapped to a position 1.0 cM from rhm, and we converted this marker to an STS (sequence-tagged site) marker. Combined with the previously identified agrP144, this new marker may be useful for map-based cloning of the rhm gene and marker-assisted selection for rhm.

Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF.

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in alpha2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.

Regulation of gene expression by herbal medicines--a new paradigm of gene therapy for multifocal abnormalities of genes.

Japanese herbal medicine has long been considered as only supplementary therapy to Western medicine. However, we discovered that an herbal mixture, Saiko-keishi-to-ka shakuyaku (SK, TJ-960), showed regulatory function of gene expression such as increased expression of seizure-related gene PTZ-17, proto-oncogene c-fos and heat shock protein HSP 72. These results provide a scientific basis for an important ancient concept and usage of herbal mixtures as a "therapy against diseases which will be suffered in the future". Our results also give an adequate provide break-throughs for therapy and even prevention of intractable epilepsy, Alzheimer's disease, developmental disorders during pregnancy and the postnatal period, and also probably for prevention of metastasis or relapse of various cancers.

Molecular mechanism of regulation of pentylenetetrazol-induced calcium entry by 3'-untranslated region of a seizure-related cDNA, PTZ-17, in Xenopus oocytes.

To determine the molecular mechanism of regulation of pentylenetetrazol (PTZ)-induced calcium entry by the seizure-related gene, PTZ-17, the role of the 3'-untranslated region (3'UTR) and also interaction between 3'UTR and intracellular factors were investigated. PTZ-induced calcium inward current in Xenopus oocytes injected with PTZ-17 RNA varied in magnitude among strains of mice: RNA derived from the DBA/2 mouse, which has a high susceptibility to convulsions, showed the largest current and that from the BALB/c mouse with a low susceptibility to convulsions showed no PTZ response. The sequence of 3'UTR showed alterations among mouse strains: 3'UTR of BALB/c showed a sequence alteration from T to G and that of DBA/2 showed a GTG insertion compared with that of B6. The 3'UTR also regulated the translation of chloramphenicol acetyltransferase (CAT) RNA depending on its sequence. A particular region within the 3'UTR demonstrated interaction with 60- and 47-kDa proteins. Sequence alterations in this region corresponded to disappearance or increase in PTZ-induced calcium entry. These findings suggest that a particular region within 3'UTR of the seizure-related gene, PTZ-17, is involved in PTZ-induced calcium entry via interaction between mRNA and specific RNA-binding proteins.

Nervous diseases and Kampo (Japanese herbal) medicine: a new paradigm of therapy against intractable nervous diseases.

Doctors who learned exclusively western medicine probably understand a priori Kampo (Japanese herbal) medicine merely as a kind of folk medicine which is not so effective and only a supplementary therapy to western medicine. We have been performing experiments on the mechanism of epileptogenesis mainly at the cellular level for a long time. During the research process, we unexpectedly encountered Kampo (Japanese herbal) medicine, and also performed research on the mechanism of action of a herbal mixture prescription, saiko-keishi-to-ka-shakuyaku (SK, TJ-960). Recently we discovered that SK acts to induce the best functional state of neurons and consequently intractable nervous symptoms disappear. SK has protective effects against neuron damage, normalizing effects on developmental defects of El mouse neurons, complete preventive effects on stress-induced increased c-fos and HSP 72 expression, complete suppression effects on the Reilly syndrome, complete normalizing effects on expression of the seizure-related gene, PTZ-17, and also, surprisingly, complete suppression effects on amyloid beta protein-induced neuron death. Such wide ranging effects which are preferable to functional maintenance and development of neurons can not be obtained by pure chemical drugs. These findings suggest that we should effectively use such ancient herbal prescriptions which show excellent preventive effects against neuron damage, enforcing action on natural healing forces and even regulatory action against adverse expression of genes, at least to prevent intractable nervous diseases, such as epilepsy, Alzheimer's disease and developmental defects of neurons during pregnancy and after birth. We should also create a future medicine, the 'third medicine', which is situated in a higher dimension than that of contemporary oriental and western medicines. For this purpose, it is necessary to perform research on the mechanism of action of Kampo (Japanese herbal) medicine.

Preparation of soluble recombinant T cell receptor alpha chain by using a calmodulin fusion expression system.

We have isolated a full length T cell receptor alpha chain (TCR alpha) cDNA derived from a bee venom phospholipase A2-specific mouse suppressor T cell hybridoma. A bacterial fusion expression system was constructed using rat calmodulin as a fusion partner for production of soluble TCR alpha. In this system, calmodulin-TCR alpha fusion protein was expressed at a high level in the soluble fraction of bacterial cell lysate, and could be purified by binding of calmodulin portion of the protein to phenyl-Sepharose. Using this system, fusion proteins containing a TCR alpha peptide corresponding to the complete extracellular region, V alpha-J alpha region or C alpha extracellular region were isolated. TCR alpha peptides were then released from the fusion proteins by digestion with thrombin which recognizes a linker sequence between calmodulin portion and TCR alpha segment. Polyclonal antibodies against constant region of TCR alpha chain (C alpha) were obtained by immunization of rabbits with the recombinant C alpha peptide. ELISA for TCR protein was established by using the polyclonal antibodies and the monoclonal antibody specific for C alpha region.

Cloning and characterization of pentylenetetrazol-related cDNA, PTZ-17.

cDNAs related to pentylenetetrazol-induced bursting activity in neurons were screened by a differential hybridization method using normal and pentylenetetrazol-treated primary cultured neurons from the cerebral cortex of mice. Twenty clones of candidate cDNA with expression increased or decreased by treatment with pentylenetetrazol were obtained. One of them, PTZ-17, was sequenced. Injection of PTZ-17 derived RNA into Xenopus oocytes showed a large calcium inward current with extracellular application of pentylenetetrazol.

A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5'- and 3'-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev.

We previously constructed a multiribozyme expression vector by combining cis- and trans-acting ribozymes and we showed that several ribozymes, each directed against a different target in the HIV genome and acting independently in a 'shotgun' manner, markedly increased the efficiency of cleavage of HIV RNA in vitro [Ohkawa et al., Proc. Natl Acad. Sci. USA 90, 11302 (1993)]. However, the cis-acting ribozymes that had trimmed the 5' and 3' ends of each trans-acting ribozyme were designed merely to await for degradation by RNases when they were used in vivo. Since several trans-activator proteins are essential for viral replication of HIV-1, we wondered whether a decoy function could be coupled with the cleavage activity of ribozymes. We therefore introduced the TAR or the RRE sequence into the stem II region of each cis-acting ribozyme. When the activity of each resulting cis-acting ribozyme that had been endowed with the decoy function was examined in vitro, it was found to retain almost full trimming activity. Moreover, cis-acting ribozymes with either the TAR or the RRE sequence were shown to be able to trap Tat or Rev protein successfully. It is, therefore, possible to endow the stem II region with a specific protein-binding function without the loss of ribozyme function. Thus, cis-acting ribozymes, endowed with the decoy function, can first trim the 5' and 3' ends of each trans-acting ribozyme and are then still available for trapping trans-activator proteins possibly prior to their degradation by RNases when they are to be used in vivo. Furthermore, it is also expected that the reduction in production of HIV RNA that is achieved by sequestering the trans-activator proteins might provide the trans-acting ribozymes, targeted to HIV RNA, with a better chance of eliminating the remaining HIV RNA.

A hammerhead ribozyme inhibits the proliferation of an RNA coliphage SP in Escherichia coli.

Ribozymes are potentially powerful tools for the suppression of intracellular gene expression. However, the few reports that exist of their activities in bacteria have described mixed success. Chuat and Galibert (Chuat, J.-C., and Galibert, F. (1989) Biochem. Biophys. Res. Commun. 162, 1025-1029) failed to detect any trans-activities of hammerhead ribozymes in Escherichia coli, while Sioud and Drlica (Sioud, M., and Drlica, K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7303-7307) reported complete inhibition of expression of the gene for a nonbacterial protein, HIV-1 integrase, by trans-acting hammerhead ribozymes in E. coli. It is of interest to determine whether ribozymes can really be used in natural bacterial systems (Altman, S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10898-10900). We now report that a ribozyme designed to cleave the A2 gene of RNA coliphage SP, when transcribed from a plasmid in E. coli caused failure of the proliferation of progeny phage. Inactive ribozymes with altered catalytic sequences did not affect phage growth. These results indicate that it is mainly the catalytic activity of the ribozyme and not its function as an antisense molecule that is responsible for suppressing the proliferation of the RNA phage. Moreover, an analysis based on numbers of plaque-forming units and the function of the A2 protein indicated that antisense RNA may successfully compete with ribosomes in targeting mRNA while ribozymes in this study may not compete with ribosomes in naturally occurring bacterial transcription/translation-coupled systems.

Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus.

The kinetic behavior of ribozymes derived from two types of multiple-ribozyme expression vector were examined. In some cases, multiple ribozymes were expressed as a single RNA molecule and all the ribozymes were simply connected in tandem (connected type). In other cases, multiple ribozymes were flanked by cis-acting ribozymes at both their 5' and 3' ends so that, upon transcription, multiple ribozymes were trimmed at both their 5' and 3' ends, with resultant liberation of multiple independent ribozymes (shotgun type). When levels of ribozyme expression were examined for the shotgun-type vector, the level of the ribozyme transcript was found to be proportional to the number of units (n) connected in tandem. Accordingly, the activities of the shotgun-type ribozymes, in terms of the cleavage of HIV-1 RNA in vitro, were also found to be proportional to the number of units connected in tandem (n). By contrast, the activities of the connected-type ribozymes reached plateau values at around n = 3. These results indicate that, when the shotgun-type expression system is used, it is theoretically possible to generate various independent ribozymes, each specific for a different target site, without sacrificing the activity of any individual ribozyme.

Multiple site-specific cleavage of HIV RNA by transcribed ribozymes from shotgun-type trimming plasmid.

We have constructed a multiple-ribozyme expression system (designated shotgun type expression system). This system made it possible to express multiple ribozymes without reducing of the activities of each ribozyme. By using this system, multiple ribozymes specific for five different target sites in HIV-1 RNA were transcribed; all of them functioned independently of the others, and each cleaved the RNA at the respective predetermined target site only. Transient HIV-1 infection assay revealed that this kind of construct can disarm AIDS virus up to 96%, at least in a cultured cell. This proves that our system is effective in vivo as well.

Construction of a tRNA-embedded-ribozyme trimming plasmid.

We have combined Cotten and Birnstiel's tRNA-embedded ribozymes and our 5'- and 3'-trimming system. Although the activity of the tRNA-embedded ribozyme was ca. 30% lower than those of naked ribozymes, since the stability of the former in bovine serum was higher than those of naked ribozymes, the tRNA-embedded ribozymes appear useful especially when the 5'- and 3'-trimming units are concatenated in tandem.

Activities of HIV-RNA targeted ribozymes transcribed from a 'shot-gun' type ribozyme-trimming plasmid.

We have constructed a "shot-gun" type ribozyme-trimming system. By concatenating several units, each consisting of a trans-acting ribozyme (targeted to HIV-RNA) and cis-acting ribozymes (trimming 5'- and 3'-ends of the trans-acting ribozyme), several kinds of trans-acting ribozymes can be liberated upon transcription and self-cleavage. Since each liberated HIV-RNA-targeted ribozymes can work independently, they can simultaneously cleave HIV-RNA at several different sites. Ribozymes were targeted at relatively conserved GUC-containing sites at LTR, gag and tat regions.

Inhibitory effect of peony root extract on pentylenetetrazol-induced EEG power spectrum changes and extracellular calcium concentration changes in rat cerebral cortex.

To elucidate the mechanism of anticonvulsant action of peony root and to determine the relative contributions of pure component substances, the water, water/acetone and methanol extracts of peony roots, paeoniflorin, albiflorin and pentagalloylglucose were studied in rats using the EEG power spectrum changes induced by pentylenetetrazol administration and the extracellular calcium and potassium concentration changes related to seizure activity. The water extract of peony roots, albiflorin and pentagalloylglucose given orally completely inhibited the EEG power spectrum changes as well as the extracellular calcium and potassium concentration changes related to seizure activity. The water/acetone and methanol extracts and paeoniflorin were relatively less potent. These findings suggested that the anticonvulsant action of peony roots is due primarily to albiflorin and the gallotannin fraction. Albiflorin and pentagalloylglucose appear to manifest their anticonvulsant action due to inhibition of the seizure-related decrease of extracellular calcium and consequent intracellular calcium increase.

Highly conserved coding sequences in polychlorinated biphenyl (PCB)-degraders of Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715.

Sixteen strains that utilize biphenyl or polychlorinated biphenyl (PCB) as the sole source of carbon and energy have previously been isolated. Nucleotide sequence of the bphABCXD operon (11 kb) of Pseudomonas pseudoalcaligenes KF707 has now been determined. When bphD region is compared with the previously reported bphD sequence of another PCB-degrader, Pseudomonas putida KF715, homologies at the level of nucleotides as well as amino acids are as high as 96%. Moreover, both bphABCXD operon (KF707) and bphABCD operon (KF715) share the identical transcriptional initiation site. Since these aromatic hydrocarbon-degraders are coded on chromosomes, whereas other majorities of hydrocarbon-degraders are coded on plasmids, there must be a mechanism governing the chromosomal gene-shuffling.