Lignin is a complex phenolic plant polymer that is essential for mechanical support, defense, and water transport in higher plants. The AC-rich motif, Pal-box is an important cis-acting element for gene expression in phenylpropanoid biosynthesis. We isolated a cDNA clone (Ntlim1) encoding a Pal-box binding protein by Southwestern screening. The deduced amino acid sequence of Ntlim1 is highly similar to members of the LIM protein family that contain a zinc finger motif. Moreover, Ntlim1 had a specific DNA-binding ability and transiently activated transcription of a beta-glucuronidase reporter gene driven by the Pal-box sequence. The results of transient expression assays with tobacco cultured cells showed that fusion proteins between GFP and Ntlim1 can enter nuclei. Transgenic tobacco plants with antisense Ntlim1 showed low levels of transcripts from some key phenylpropanoid pathway genes such as phenylalanine ammonia-lyase, hydroxycinnamate CoA ligase and cinnamyl alcohol dehydrogenase. Furthermore, a greater than 20% reduction in lignin content was observed in transgenic tobacco with antisense Ntlim1.

This study was performed to assess the influence of smoking on periodontal disease severity. Data concerning periodontal status and smoking habits were collected from 889 periodontal patients: 340 male and 549 female, 21 to 76 years of age, 47.4% being non smokers and 52.6% smokers. Periodontal parameters, recorded by the same examiner (PMC), were: gingival recession (GR), Pocket depth (PD), Probing attachment level (PAL), and mobility (M). The influence of age, sex and tobacco consumption on these periodontal parameters was statistically evaluated using an analysis of variance (ANOVA) with covariates. A non-linear effect model was also fitted by taking the natural logarithms of the response variables (GR, PD, PAL) closer to biomedical phenomena. Mobility was analyzed by a chi2-test. The effect of smoking on periodontitis showed no association with age or with sex. Smoking, age and sex were shown to be statistically significant for periodontitis, by performing both univariate (t-test for equal means) and multivariate tests. p-values for smoking and periodontitis were: GR (p=0.000), PD (p=0.000), PAL (p=0.000) and M (P=0.015). Smoking one cigarette per day, up to 10, and up to 20, increased PAL by 0.5%, 5% and 10%, respectively. The impact of tobacco is comparable to the impact resulting from the factor of age in this sample, increasing PAL by 0.7% for each year of life. Comparison between smokers of less than 10 cigarettes per day (PAL mean 3.72 mm +/-0.86) and non-smokers (PAL mean 3.84 +/- 0.89) showed no differences in PAL (p=0.216), while comparison for smokers from 11 to 20 cigarettes (PAL mean 4.36 +/- 1.23) and for more than 20 cigarettes (PAL mean 4.50 +/- 1.04) demonstrated significant differences (p=0.000). These findings suggest that: (1) tobacco increases periodontal disease severity; (2) this effect is clinically evident above consumption of a certain quantity of tobacco.

1-Aminocyclopropane-1-carboxylate (ACC) synthase was rapidly induced in mesocarp tissue of Cucurbita maxima after wounding in the cut surface layer in 1 mm thickness (ca. 9 cells) (first layer) in both the enzyme activity and the levels of transcript. This led to a rapid accumulation of ACC and hence ethylene production. In the inside tissue (1-2 mm) (second layer), no significant induction of ACC synthase was observed, which resulted in a low level of ACC, although ethylene was evolved at a much lower rate than the first one. In contrast to ACC synthase, ACC oxidase was induced markedly in both the first and second layers and the development of its activity and the levels of mRNA remained high until later stages. It was considered that wound ethylene was closely associated with the development of ACC oxidase, since 2,5-norbornadiene (NBD), an inhibitor of ethylene action, substantially suppressed it. Phenylalanine ammonia-lyase (PAL) greatly increased in activity after wounding similarly to that of ACC synthase, in which increase in PAL activity occurred predominantly in the first layer. Induction of peroxidase activity after wounding had a close correlation in profile with that of ACC oxidase in that marked increases in the activity were observed in both the first and second layers and were strongly suppressed by NBD application. Four peroxidase isozymes were found by PAGE, among which a fraction was newly detected after wounding.

TolR is a part of the Pal/Tol system which forms a five-member, membrane-spanning, multiprotein complex that is conserved in Gram-negative bacteria. The Pal/Tol system helps to maintain the integrity of the outer membrane and has been proposed to be involved in several other cellular processes including cell division. Obtaining the structure of TolR is of interest not only to help explain the many proposed functions of the Pal/Tol system but also to gain an understanding of the TolR homologues ExbD and MotB and to provide more targets for antibacterial treatments. In addition, the structure may provide insights into how colicins and bacteriophages are able to enter the cell. Here we report the solution structure of the homodimeric periplasmic domain of TolR from Haemophilus influenzae, determined with conventional, NOE-based NMR spectroscopy, supplemented by extensive residual dipolar coupling measurements. A novel method for assembling the dimer from small-angle X-ray scattering data confirms the NMR-derived structure. To facilitate NMR spectral analysis, a TolR construct containing residues 59-130 of the 139-residue protein was created. The periplasmic domain of TolR forms a C 2-symmetric dimer consisting of a strongly curved eight-stranded beta-sheet, generating a large deep groove on one side, while four helices cover the other face of the sheet. The structure of the TolR dimer together with data from the literature suggests how the periplasmic domain of TolR is most likely oriented relative to the cytoplasmic membrane and how it may interact with other components of the Pal/Tol system, particularly TolQ.

To examine the phenotype of the sinusoidal endothelial cells (SECs) surrounding tumor cells and the process of capillarization in hepatocellular carcinoma (HCC), 51 primary HCCs, 4 adrenal metastases, and 3 portal tumor thrombi were immunohistochemically stained with monoclonal antibodies (MAbs) for CD4, CD14 (lipopolysaccharide-binding protein complex receptors), and CD32 (Fc gamma receptor II), which are specifically found on the SECs in normal liver, but not on ordinary vascular endothelial cells (ECs). Immunostaining was also performed for CD36 (thrombospondin receptors), EN4 antigen (Ag) (a pan-vascular endothelial cell Ag), PAL-E Ag (a venous and capillary EC Ag), factor VIII-related Ag (FVIIIRAg), and laminin. MAb 25F9, which identifies macrophages, was simultaneously used with the other MAbs to distinguish macrophages from SECs in HCCs (HCC SECs). CD4, CD14, and/or CD32 were found on HCC SECs only in 12 well-differentiated primary HCCs showing a thin trabecular or pseudoglandular tumor cell arrangement. These 12 tumors were smaller than those without CD4-, CD14-, and/or CD32-positive SECs (P < .05). Among them, 7, 5, and 11 tumors were negative or only partially positive for laminin, PAL-E Ag, and FVIIIRAg, respectively. Staining for laminin and PAL-E Ag showed an inverse relationship to the expression of CD4, CD14, and CD32 on HCC SECs and the tumor differentiation. In conclusion, the phenotypes of the SECs in early and well-differentiated HCC are thought to be similar to those of the SECs in normal liver. With progressing tumor dedifferentiation the HCC SECs lose the phenotypes peculiar to liver SECs and acquire the characteristics of capillary ECs, though both types of phenotypical change occur independently of each other.

OBJECTIVE

To identify adults and children as under- (UR), acceptable (AR), or over-reporters (OR) of energy intake (EI) using energy expenditure measured by doubly labelled water (DLW) (EE(DLW)), and to use this as a reference to determine the sensitivity and specificity of (i) EE measured by heart rate (EE(HR)), and (ii) the Goldberg cut-off technique for classifying subjects into the same categories.

METHODS

Retrospective analysis of a dataset comprising concurrent measurements of EE(DLW), EE(HR), basal metabolic rate (BMR), and EI by weighed record (EI(WR)) on 14 adults and 36 children. EI by diet history (EI(DH)) was also measured in the children only. EI(WR):EE(DLW) provided the reference definition of subjects as UR, AR or OR. Three strategies for classifying mis-reporters based on EE(HR) and Goldberg cut-offs were then explored. Sensitivity and specificity were calculated respectively as the proportion of UR and non-UR correctly identified.

RESULTS

Approximately 80% of all subjects were AR. For EI(WR) and EI(DH) respectively, the sensitivity of EE(HR) was 0.50 and 1.00, and specificity was 0.98 and 1.00. Although designating subjects as having low, medium or high activity levels (EE(HR):BMR(meas)) and calculating cut-offs based on appropriate WHO physical activity level PALs did not change sensitivity, specificity dropped to 0.98 (EI(WR)) and 0.97 (EI(DH)). Cut-offs based on a PAL of 1.55 reduced sensitivity to 0.33 (EI(WR)) and 0.00 (EI(DH)), but specificity remained unchanged. The sensitivity of all cut-offs based on physical activity level (PALs) for EI(WR) was 0.50 (adults) and 0.25 (children).

CONCLUSIONS

If the precision of EE(HR) was improved, it may be useful for identifying mis-reporters of EI.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.

Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae.

We have recently shown that the locus of enterocyte effacement (LEE) of the bovine enterohemorrhagic E. coli RW1374 (O103:H2) resides within a large pathogenicity island (PAI), integrated in the vicinity of the phenylalanine tRNA gene pheV. Here we describe an additional, but LEE-negative genomic island in RW1374 in the vicinity of another phenylalanine tRNA gene, pheU, the sequence of which is identical to pheV. These two genomic islands revealed identity of the left, but a relative variability of their right end sequences. To investigate the mechanism of LEE-PAI distribution in E. coli, we analysed similar junctions in the pheU/pheV loci of additional EPEC and EHEC strains the LEE location of which had not been determined before. By hybridisation of NotI restriction fragments with probes specific for LEE, pheV locus, and pheU locus, the LEE was found linked to either one of these two loci. The results agreed well with recently published phylogenetic data and indicate that in the clones of diarrheagenic E. coli (Dec) Dec 11 and Dec 12, forming the phylogenetic cluster EPEC 2, and in the strains of the most typical serotypes of the Dec 8, belonging to the phylogenetic cluster EHEC 2, the LEE was linked with pheV and not with the pheU locus as previously assumed. Sequence comparison with other pheU- and pheV-located genomic islands from different E. coli pathotypes (uropathogenic E. coli, septicemic E. coli) as well as from Shigella indicated the same structural features at the junctions. These conserved structures suggested a common DNA cassette, serving as common vehicle for horizontal gene transfer of various PAls. In addition, the elements suggest an origin from a common pheU-located ancestor and integration into the chromosome through site-specific recombination. Our results indicate that pheU/pheV-located genomic islands played an important role in the evolution of several PAls in E. coli and related pathogens.

OBJECTIVE

Chronic hyperglycaemia in patients with Type II (non-insulin-dependent) diabetes mellitus often leads to a decline in glucose-responsive insulin secretion from pancreatic beta cells, a phenomenon called glucose toxicity. Upon hyperglycaemia, glycation reaction occurs in the beta cells and induces oxidative stress. To understand the molecular basis of the beta-cell glucose toxicity, we investigated the possible effects of glycation on the expression and enzymatic activity of glucokinase, which plays a crucial part in glucose-responsive insulin secretion.

METHODS

Glycation and reactive oxygen species were induced in HIT-T15 cells by treatment with D-ribose and effects on glucokinase gene transcription, glucokinase protein amount, glucose phosphorylation activity, and DNA-binding activities of putative glucokinase gene transcription factors were evaluated.

RESULTS

When glycation was induced in HIT-T15 cells, the activity of the human glucokinase gene beta-cell-type promoter was suppressed substantially (83% reduction at 60 mmol/l D-ribose). Also, similar reductions in mRNA and protein amounts of glucokinase and in the Vmax of its enzymatic activity were observed. In agreement with the reduction in the promoter activity, the two major transcription factors of the glucokinase gene, the Pal-binding factor and PDX-1, reduced their binding to their target sequences in the glucokinase gene promoter in glycation-induced HIT cells. Because these effects of D-ribose were counteracted by aminoguanidine or N-acetylcysteine, reactive oxygen species, generated by the glycation reaction, appears to be involved in the phenomena.

CONCLUSIONS

The induction of the glycation reaction, which is known to occur in pancreatic beta cells in chronic hyperglycaemia, suppresses the glucokinase gene transcription and its enzymatic activity. Thus, hyperglycaemia-dependent inhibition of glucokinase activity could in part explain beta-cell glucose toxicity.

The objective of this research was to carry out an in vitro and in vivo study of the biological performance of PLLA/beta-TCP composite materials, to estimate the scope of their potential applications in bone surgery. Samples with increasing beta-TCP (0-60% w/w) contents were processed by injection molding. The in vitro study consisted of an evaluation of inflammatory potential by assaying the IL-1alpha secreted by monocytes, and then cell proliferation (counting) and phenotype expression (PAL and I collagen) in human osteogenous cells. The in vivo study was carried out using cylindrical implants of composite materials composed of composite materials containing 0 or 60% beta-TCP and pure beta-TCP, respectively. The implants were inserted in femoral sites in rabbits, using the Kathagen protocol. Each animal received a 60% implant, with either a 0 or a 100% implant in the contralateral femur, so that the materials could be compared with one another. Five animals were examined for each material and implantation period, giving a total of 30 animals. This study showed that adding increasing percentages of beta-TCP to a lactic acid polymer matrix stimulated the proliferation of human osteogenous cells and synthesis of the extracellular bone matrix in a dose-dependent manner. In vivo results indicate that, in comparison with pure PLA, tricalcium phosphate-containing composite materials had faster degradation kinetics, caused less inflammatory reaction, and promoted contact osteogenesis. The composite material containing 60% beta-TCP demonstrated a similar performance to pure tricalcium phosphate bone grafts in terms of osteogenesis, and is apparently compatible with the production of intra-osseous implants for situations representing high levels of mechanical strain.

The biosynthesis of alpha-amidated peptides from their glycine-extended precursors is catalyzed by the sequential action of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The two enzymes are part of a bifunctional, integral membrane protein precursor, peptidylglycine alpha-amidating monooxygenase (PAM). The major forms of PAM mRNA in the adult rat atrium differ by the presence or absence of optional exon A, a 315-nucleotide segment separating the PHM and PAL domains. Using antipeptide antibodies specific to the PHM, exon A, PAL, and cytoplasmic domains of rat PAM, carbonate-washed atrial membranes were found to contain proteins corresponding to rPAM-1 and rPAM-2. Digestion of atrial membranes with a variety of endoproteinases released PHM and PAL catalytic activities. Dose-response curves indicated that both catalytic activities were extremely resistant to inactivation by trypsin. Endoproteolytic digestion of atrial membranes with trypsin, chymotrypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 35-kDa PHM fragment. Digestion with trypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 42-kDa PAL fragment. In contrast to the stability exhibited by the PHM and PAL domains, the cytoplasmic domain of PAM was destroyed by most of the enzymes; only digestion with endoproteinase Lys-C generated a stable fragment. Digestion with endoproteinase Arg-C removed the carboxyl-terminal tail from PAM but failed to release the PHM or PAL domains from the membranes. The PHM fragments generated by some of the endoproteinases showed a tendency to adhere to the membranes. Thus the bifunctional PAM protein consists of independent catalytic domains separated from each other and from the putative transmembrane domain by flexible regions accessible to attack by a wide variety of endoproteinases.

Erwinia rhapontici is able to convert sucrose into isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) and trehalulose (1-O-alpha-D-glucopyranosyl-D-fructose) by the activity of a sucrose isomerase. These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen. This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption. Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated. Here we describe for the first time the cloning and characterization of the palatinose (pal) genes from Erwinia rhapontici. To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned. The pal gene products of Erwinia rhapontici were shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms. The palE, palF, palG, palH, palK, palQ, and palZ genes were oriented divergently with respect to the palR and palI genes, and sequence analysis suggested that the first set of genes constitutes an operon. Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of the palI gene and the palEFGHKQZ genes is oppositely regulated at the transcriptional level. Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose. Palatinose activation is inhibited by sucrose. Functional expression of palI and palQ in Escherichia coli revealed sucrose isomerase and palatinase activity, respectively.

OBJECTIVE

To provide an objective analysis of surgical performance of robotic-assisted laparoscopic hysterectomy (RALH) with lymphadenectomy for endometrial cancer during the learning phase of the procedure and to assess opportunities for improvement.

METHODS

From July 2006 to March 2008, 100 patients with endometrial cancer underwent RALH with lymphadenectomy using the da Vinci Robotic Surgical System. Data were analyzed for operative time (OT), estimated blood loss (EBL), length of stay (LOS), intra-operative complications, surgical-pathologic factors, and post-operative complications using an intent-to-treat analysis. A comparison of the data on a quartile (Q) basis was performed for the 100 RALH cases and separately for the 65 cases that had a complete pelvic-and-aortic lymphadenectomy (PAL).

RESULTS

Age and body mass index (BMI) did not change significantly during the study. More grade 3 tumors were treated in the last 50 cases (22% vs. 10%, p<0.05). Stage III tumors were identified in 18.7% cases in Q2-4 and none in Q1 (p<0.05). The number of patients undergoing complete PAL and the number of aortic lymph nodes (LN) removed per case increased each quarter. There were 4 (4%) conversions to laparotomy. Delayed vaginal cuff healing decreased from 16% in Q1 to 0% in Q3-4. No case required blood transfusion. Comparing first 10 cases to the last 10 cases, the total LN counts increased from 15 to 21 nodes, the aortic LN counts increased from 4.7 to 8.0, and the OT decreased from 203 to 160 min. Intra-surgeon analysis revealed an improvement in the total LN yields from first 50 to second 50 cases for each surgeon.

CONCLUSIONS

Operative times decreased and aortic dissections improved with increasing LN counts during the first 100 cases of RALH. Furthermore, patient safety and improvement in surgical performance was demonstrated.

The aim of the present study was to analyze the effect of LPS on the localization and differentiation of splenic B lymphocytes. Therefore, we used a double immunoperoxidase technique which enabled us to detect both the IgM+ IgD- marginal zone lymphocytes and the IgM+ IgD+ follicular lymphocytes in the same tissue section. Next to a dramatic disappearance of the predominantly IgM+ IgD- lymphocytes in the marginal zone shortly after an intravenous injection of LPS, an increased number of these cells could be found in the splenic follicles. The present results strongly suggest that the IgM+ IgD- cells in the splenic follicles represent immigrating marginal zone lymphocytes, and not differentiating follicular B cells, because no IgM+ IgD- cells could be observed in the follicles of draining lymph nodes shortly after a subcutaneous injection of a similar amount of LPS. These observations support the suggestion that LPS induces a migration of marginal zone lymphocytes into the follicles. The present results also showed the formation of IgD plasmablasts in the inner PALS and around the terminal arterioles of the spleen after LPS administration. The induction of IgD plasmablasts appeared to be a specific effect of LPS which may be related to its toxic properties.

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1-560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.

In this study, tetrodotoxin (TTX) inactivation was employed to evaluate the involvement of the rat's orbitofrontal cortex (OFC) in hippocampus-dependent spatial memory using Morris water maze (MWM) and place avoidance learning (PAL) tasks. In Experiment 1, rats trained in MWM task with two blocks of four trials per day for 3 consecutive days received bilateral injections of either TTX or saline into the OFC 60 min before each daily training session. The acquisition of spatial memory was evaluated 24h after the last training day and it was shown an impairment by the TTX. In Experiment 2, bilateral intra-OFC injections of TTX or saline were made immediately after two blocks of four trials. Testing 24h later, it was revealed that TTX also impairs spatial memory consolidation. In Experiments 3 and 4, rats were trained in a single 30-min session to avoid a 60 degrees segment of the stable circular (80-cm diameter) arena, entering which was punished by a mild shock (PAL task) and retention was tested 24h later in a 30-min extinction session. Bilateral injections of TTX or saline were made into the OFC 60 min before training or immediately after training. Again, TTX impaired the place avoidance retention when it was injected into the OFC either before (acquisition phase) or after (consolidation phase) training. These findings indicate that functional integrity of the OFC is necessary for both the acquisition and the consolidation of hippocampus-dependent spatial memory in rats.

According to the report of the World Health Organization (1985), total energy expenditure (TEE) in human subjects can be calculated as BMR x physical activity level (PAL). However, other reports have pointed out limitations in the suggested procedure related to the % body fat of the subjects. The purpose of the present study was to evaluate the World Health Organization (1985) procedure in thirty-four healthy women with BMI 18-39 kg/m2. BMR and TEE were measured using indirect calorimetry (BMRmeas) and the doubly-labelled water method (TEEref) respectively. When assessed using the doubly-labelled water and skinfold-thickness methods, the women had 34 (SD 8) and 33 (SD 6) % body fat respectively. On the basis of guidelines provided by the World Health Organization (1985), 1.64 was selected to represent the average PAL of the women. Furthermore, PAL was also assessed by means of an accelerometer (PALacc), heart-rate recordings (PAL(HR)) and a questionnaire (PALq). These estimates were: PALacc 1.71 (SD 0.17), PAL(HR) 1.76 (SD 0.24), PALq 1.86 (SD 0.27). These values were lower than TEEref/BMRref, which was 1.98 (SD 0.21). BMR assessed using equations recommended by the World Health Organization (1985) (BMRpredicted) overestimated BMR by 594 (SD 431) kJ/24 h. However, when TEE was calculated as BMRpredicted x PALacc, BMRpredicted x PAL(HR) and BMRpredicted x PALq respectively, average results were in agreement with TEEref. Furthermore, TEE values based on BMRpredicted and PALacc, PAL(HR), PALq as well as on PAL = 1.64, minus TEEref, were significantly correlated with body fatness. When the same PAL value (1.64) was used for all subjects, this correlation was particularly strong. Thus, the World Health Organization (1985) procedure may give TEE results that are biased with respect to the body fatness of subjects.

In order to study the precise localization pattern of anti-TNP antibody-forming cells (AFCs) during the early primary immune response against TNP conjugated TD (thymus-dependent) and TI-2 (thymus-independent type-2) antigens, rats received an intravenous injection with either TNP-keyhole limpet haemocyanin (KLH) or with TNP-Ficoll. Anti-TNP AFCs developed in the spleen already at 2 days after injection of the antigens as demonstrated with our immunoenzyme technique for the detection of specific AFCs. In order to obtain information on the relationship between the non-lymphoid cells in the marginal zone (MZ) and the localization of AFCs, simultaneous staining for marginal metallophils and MZ macrophages (MZM) was performed using the monoclonal antibody ED3. AFCs were not found in the marginal zone (MZ), but the bulk of the cells in the white pulp were found in the outer part of the periarteriolar lymphocyte sheaths (PALS) close to the border between PALS and MZ. The precise localization of the anti-TNP AFCs in the outer part of the PALS resembled the localization of marginal metallophils but the latter cells were mainly present in the outer part of the follicles. So, the present results did not indicate a close relationship between marginal zone macrophages or marginal metallophils and anti-TNP AFCs, neither in the immune response to TD antigens nor in that to T1-2 antigens.

Increasing the precision of measurements of total energy expenditure in population-based epidemiological studies is important for accurately quantifying the relationship between this exposure and disease. Current questionnaire-based methods cannot accurately quantify total energy expenditure, although they may provide an estimate of the frequency of vigorous activities. Heart rate monitoring with individual calibration has been advocated as a method for assessing energy expenditure in field studies and has been compared with the 'gold standard' techniques of doubly-labelled water and indirect calorimetry. However the method has previously only been used on small and selected populations. This study was, therefore, established to test the feasibility of using heart rate monitoring in a population-based study of adults. A total of 167 individuals aged 30-40 years were randomly selected and underwent 4 d heart-rate monitoring. Only three individuals could not complete the protocol. The mean physical activity level (PAL) measured over 4 d was 1.89 (SD 0.40) in men and 1.76 (sd 0.31) in women. There was no difference between mean PAL on weekend days compared with weekdays (mean paired difference 0.0008, 95% CI -0.06 +0.06). The estimate of mean PAL was not correlated with BMI, percentage body fat or the waist:hip ratio. It was, however, correlated with cardio-respiratory fitness as measured by VO2(max) per kg (Spearman rank correlation coefficient 0.50 in men and 0.42 in women). The pattern of energy expenditure was assessed by calculating the percentage of daytime hours in which PAL was greater than five times basal energy expenditure. This measure was strongly correlated with the mean PAL in both men (Spearman correlation coefficient 0.77) and women (0.71). We conclude that heart-rate monitoring is a feasible method for assessing the pattern and total level of energy expenditure in medium-sized epidemiological studies. It may also prove useful as the reference technique for calibrating questionnaires to estimate energy expenditure in larger scale studies.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.

We investigated the distribution of SP+ nerve fibers in the spleen of adult male Fischer 344 rats. SP+ nerve fibers entered the spleen with the splenic artery in the hilar region, arborized along the venous sinuses, and extended from these larger plexuses into trabeculae and the surrounding red pulp. In the white pulp, SP+ nerve fibers were found in the marginal zone, and in the outer regions of the PALS among T lymphocytes. No SP+ nerve fibers were observed in association with the splenic capsule, the central arteries of the white pulp, or the follicles. SP levels in rat spleen were 5.7 +/- 0.4 ng/g wet wt. On the basis of the present findings of SP presence in nerve fibers in the spleen, and published evidence for SP receptors on lymphocytes and macrophages, we suggest that SP derived from nerve fibers in the spleen can act as a neurotransmitter with cells of the immune system as targets. These SP nerve fibers may be an important neural link between the nervous system and the immune system and may participate in modulation of immune reactivity and inflammatory responses.

In Japan, EBV positive rate in immunocompetent patients with nodal lymphomas is less than 10% in B-cell and 20-50% in T cell lymphoma. Among extranodal lymphomas, EBV positive rate is higher in pyothorax-associated lymphoma (PAL), nasal NK/T-cell lymphoma, and adrenal lymphoma. PAL is non-Hodgkin's lymphoma that develops from chronic pyothorax resulted from artificial pneumothorax for the treatment of lung tuberculosis or tuberculous pleuritis. This disease was originally described by Dr. Aozasa as a distinctive clinicopathologic entity in 1987, and now listed as the disease entity in the WHO classification of Tumours, Pathology & Genetics, Tumours of the Lung, Pleura, Thymus and Heart (2004).

The effects of cadmium (Cd), a well-known environmental pollutant with high toxicity to plants, were tested on root growth, cell viability, phenylalanine ammonia-lyase (PAL) soluble plus cell wall-bound peroxidase (POD) activities, hydrogen peroxide (H(2)O(2)) levels, and the content and monomeric composition of lignin in soybean (Glycine max) roots. Three-day-old seedlings were cultivated in half-strength Hoagland's solution (pH 6.0), with or without 25-100 μM CdCl(2) in a growth chamber (25°C, 12/12-h light/dark photoperiod, irradiance of 280 μmolm(-2)s(-1)) for 24h. In general, root length and the fresh and dry weights decreased followed by loss of cell viability after Cd treatment. PAL activity, soluble and cell wall-bound POD activities, and H(2)O(2) and lignin contents increased significantly after Cd exposure. The lignin monomeric composition of Cd-exposed roots revealed a significant increase of p-hydroxyphenyl (H) and syringyl (S) units. These results suggest that the effects caused by Cd may be due to excessive production of monolignols forming lignin, which solidifies the cell wall and restricts root growth.