Dynamics of the Phanerochaete carnosa transcriptome during growth on aspen and spruce.

BACKGROUND: The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. RESULTS: Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. CONCLUSIONS: The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.

Increased systemic levels of inflammatory mediators following one-stage full-mouth scaling and root planing.

BACKGROUND AND OBJECTIVE: Full-mouth scaling and root planing (FM-SRP) acts as a potent inflammatory stimulus immediately after treatment; however, systemic inflammation typically improves in the long term. The contribution of FM-SRP to systemic biological and acute-phase responses is largely unknown. The purpose of this prospective intervention study was to assess the systemic and local biological responses after FM-SRP. MATERIAL AND METHODS: Thirty-one patients with generalized moderate-to-severe chronic periodontitis received 1-stage FM-SRP. Measurement of clinical parameters and body temperature as well as collection of subgingival plaque, peripheral blood and gingival crevicular fluid was performed before and after treatment 2 or 3 times. Quantification of periodontopathic bacteria in the sulcus and measurement of corresponding serum IgG titers were performed. Systemic and local inflammatory markers such as endotoxin, high-sensitive C-reactive protein (hs-CRP) and 6 inflammatory cytokines were assessed using high-sensitivity assays. RESULTS: Compared to baseline values, FM-SRP resulted in a substantial improvement in clinical parameters (P < .05), lower bacterial counts (P < .01) and a significant decrease of IgG titers against Porphyromonas gingivalis (P < .001) 6 weeks after treatment. Comparing baseline parameters to those at 1 day post-treatment, there was a statistically significant elevation in body temperature (P = .007). In addition, a 5-fold increase in hs-CRP (P < .001), a remarkable increase in interferon-γ (P < .001) and a slight increase in interleukin (IL)-12p70 (P = .001) were detected in serum samples. In the gingival crevicular fluid, marked increases in hs-CRP (P < .001), IL-5 (P = .001), IL-6, IL-12p70 and tumor necrosis factor-α (P < .001 for the latter 3 markers) were noted 1 day after treatment. Endotoxin levels were below measurable limits for most time points. CONCLUSION: FM-SRP resulted in clinical and microbiological improvement 6 weeks post-treatment, but produced a moderate systemic acute-phase response including elevated inflammatory mediators 1 day post-treatment.

Increased serum PCSK9, a potential biomarker to screen for periodontitis, and decreased total bilirubin associated with probing depth in a Japanese community survey.

BACKGROUND AND OBJECTIVES: Previous reports suggest that several serum biomarkers play roles in the pathogenesis, inflammatory response, and oxidative stress in periodontitis caused by bacterial infections, linking chronic periodontitis to atherosclerotic vascular disease (ASVD). The aim of this preliminary study was to investigate, in a Japanese cross-sectional community survey, potential serum biomarkers of periodontitis that are associated with ASVD and chronic periodontitis. MATERIAL AND METHODS: The study cohort included a total of 108 male subjects who underwent annual health examinations. Serum biomarkers (high-sensitivity C-reactive protein [hs-CRP], proprotein convertase subtilisin/kexin type 9 [PCSK9], interleukin-6, tumor necrosis factor-α, soluble CD14, myeloperoxidase, matrix metalloproteinase-3, adiponectin, total bilirubin [TBIL], and serum lipids) were analyzed to determine their association (if any) with periodontal parameters. Aortic stiffness was evaluated using the brachial-ankle aortic pulse wave velocity (PWV) index and the cardio-ankle vascular index (CAVI). RESULTS: The concentrations of PCSK9 and hs-CRP were increased (P = .001 and .042, respectively), and the concentration of TBIL was decreased (P = .046), in subjects with periodontal disease (determined as a probing depth of ≥4 mm in at least one site) compared with periodontally healthy subjects. The ratio of low-density lipoprotein cholesterol (LDL-C) to high-density lipoprotein cholesterol and the concentrations of triglycerides, remnant-like particles-cholesterol, and oxidized LDL were elevated in subjects with periodontal disease compared with periodontally healthy subjects (P = .038, .007, .002, and .049, respectively). Multivariate regression analyses indicated that the number of sites with a pocket depth of ≥4 mm was associated with the concentration of PCSK9 and inversely associated with the concentration of TBIL independently (standardized ß = .243, P = .040; standardized ß = -.443, P = .0002; respectively). Analysis of receiver operating characteristic curves of PCSK9 indicated moderate accuracy for predicting the presence of disease sites (probing depth ≥ 4 mm) (area under the curve = 0.740). No significance in the values of PWV and CAVI was observed between subjects with periodontal disease and periodontally healthy subjects. CONCLUSION: In Japanese male subjects, the concentrations of serum PCSK9 and TBIL were correlated with periodontal parameters. Moreover, PCSK9 could be a candidate biomarker for diagnosing chronic periodontitis, and may also have potential to evaluate the risk for periodontitis to cause ASVD. Longitudinal studies of larger populations are necessary to confirm the exact association of periodontitis with increased serum PCSK9 and decreased TBIL.

Effects of paraoxonase 1 on the cytodifferentiation and mineralization of periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Single nucleotide polymorphisms (SNPs) of paraoxonase 1 (PON1) are known to be associated with the pathogenesis of osteoporosis and periodontitis. However, the effects of PON1 on the osteoblastic differentiation of periodontal ligament (PDL) cells are unclear. In this study, we examined the effects of PON1 on the osteoblastic differentiation of PDL cells, and analysed the role of PON1 SNPs on the pathogenesis of aggressive periodontitis (AgP) in the Japanese population. MATERIAL AND METHODS: Human PDL (HPDL) cells were exposed to the PON1 plasmid and PON1 inhibitor, 2-hydroxyquinoline, and cultured in mineralization medium. Expression of calcification-related genes and calcified nodule formation were assessed by real-time PCR, an alkaline phosphatase (ALPase) activity assay and Alizarin red staining. Sanger sequencing was performed to evaluate whether PON1 SNPs are associated with the pathogenesis of AgP in Japanese people. RESULTS: During osteoblastic differentiation of HPDL cells, expression of PON1 mRNA increased in a time-dependent manner. PON1 stimulated an increase in expression of mRNA for calcification-related genes, as well as ALPase activity. In contrast, 2-hydroxyquinoline clearly inhibited the expression of calcification-related genes, ALPase activity and calcified nodule formation in HPDL cells. Moreover, there was a statistically significant difference in the minor allele frequency of PON1 SNP rs854560 between the Japanese control database and patients with AgP in the Japanese population (P = .0190). CONCLUSION: PON1 induced cytodifferentiation and mineralization of HPDL cells, and PON1 SNP rs854560 may be associated with the pathogenesis of AgP in the Japanese population.

Sphingomyelin Phosphodiesterase 3 Enhances Cytodifferentiation of Periodontal Ligament Cells.

Sphingomyelin phosphodiesterase 3 ( Smpd3), which encodes neutral sphingomyelinase 2 (nSMase2), is a key molecule for skeletal development as well as for the cytodifferentiation of odontoblasts and alveolar bone. However, the effects of nSMase2 on the cytodifferentiation of periodontal ligament (PDL) cells are still unclear. In this study, the authors analyzed the effects of Smpd3 on the cytodifferentiation of human PDL (HPDL) cells. The authors found that Smpd3 increases the mRNA expression of calcification-related genes, such as alkaline phosphatase (ALPase), type I collagen, osteopontin, Osterix (Osx), and runt-related transcription factor (Runx)-2 in HPDL cells. In contrast, GW4869, an inhibitor of nSMase2, clearly decreased the mRNA expression of ALPase, type I collagen, and osteocalcin in HPDL cells, suggesting that Smpd3 enhances HPDL cytodifferentiation. Next, the authors used exome sequencing to evaluate the genetic variants of Smpd3 in a Japanese population with aggressive periodontitis (AgP). Among 44 unrelated subjects, the authors identified a single nucleotide polymorphism (SNP), rs145616324, in Smpd3 as a putative genetic variant for AgP among Japanese people. Moreover, Smpd3 harboring this SNP did not increase the sphingomyelinase activity or mRNA expression of ALPase, type I collagen, osteopontin, Osx, or Runx2, suggesting that this SNP inhibits Smpd3 such that it has no effect on the cytodifferentiation of HPDL cells. These data suggest that Smpd3 plays a crucial role in maintaining the homeostasis of PDL tissue.

Visit-to-visit blood pressure variability, average BP level and carotid arterial stiffness in the elderly: a prospective study.

In a cross-sectional study, visit-to-visit blood pressure (BP) variability was shown to be associated with artery remodelling. Here, we investigated the impact of visit-to-visit BP variability and average BP on the carotid artery remodelling progression in high-risk elderly according to different classes of antihypertension medication use/non-use. BP measurements and carotid ultrasound were performed in the common carotid artery in 164 subjects (mean age 79.7 years at baseline, 74.7% females) with one or more cardiovascular risk factors. Based on 12 visits (1 × /month for 1 year), we calculated visit-to-visit BP variability expressed as the standard deviation (s.d.), coefficient of variation (CV), maximum BP, minimum BP and delta (maximum-minimum) BP. We measured mean intima-media thickness (IMT) as well as stiffness parameter ß were measured at baseline and at the mean 4.2-year follow-up. In a multiple regression analysis, the maximum, minimum, s.d. and average of systolic BP (SBP) were significantly associated with a change in ß-values between the baseline and follow-up after adjustment for age, smoking, lower high-density lipoprotein level, baseline ß-value and follow-up period. There were no significant associations between the visit-to-visit BP variability measures and the change in mean IMT. Significant associations of maximum, minimum, s.d. and average SBP were found with increased ß-values in the subjects without calcium channel blocker (CCB) use and in the subjects using renin-angiotensin system inhibitors (RASIs). Thus, exaggerated visit-to-visit SBP variability and a high average SBP level were significant predictors of progression in carotid arterial stiffness in high-risk elderly without CCBs use and in those using a RASI.

Japanese nurse practitioner practice and outcomes in a nursing home.

AIM: By describing the practice of a Japanese nurse practitioner, this descriptive case study discusses role development and outcomes before and after the intervention. BACKGROUND: One of the first Japanese nurse practitioners intervened at a nursing home during the government-designated trial period for nurse practitioner practice. CONCLUSION: Because of the nurse practitioner's meticulous observation and timely care provision to the residents in collaboration with the physician and the other staff in the facility, comparative data showed improvement in daily health status management of every resident and decreased deterioration of residents' health conditions requiring ambulance transfer and hospitalization.

Effects of the proteasome inhibitor, bortezomib, on cytodifferentiation and mineralization of periodontal ligament cells.

BACKGROUND AND OBJECTIVE: The proteasome inhibitor, bortezomib, is known to induce osteoblastic differentiation in a number of cell lines, such as mesenchymal stem cells and osteoblastic precursor cells. As periodontal ligament (PDL) cells are multipotent, we examined whether bortezomib may induce the differentiation of PDL cells into hard-tissue-forming cells. MATERIAL AND METHODS: A mouse PDL clone cell line, MPDL22 cells, was cultured in mineralization medium in the presence or absence of bortezomib. Expression of calcification-related genes and calcified-nodule formation were evaluated by real-time PCR and Alizarin Red staining, respectively. RESULTS: Bortezomib increased the expression of calcification-related mRNAs, such as tissue nonspecific alkaline phosphatase isoenzyme (ALPase), bone sialoprotein (Bsp), runt-related transcription factor 2 (Runx2) and osteopontin, and calcified-nodule formation in MPDL22 cells. These effects were induced, in part, by increasing the cytosolic accumulation and nuclear translocation of ß-catenin, leading to an increase in expression of bone morphogenetic protein (Bmp)-2, -4 and -6 mRNAs. In addition, bortezomib enhanced BMP-2-induced expression of Bsp and osteopontin mRNAs and increased calcified-nodule formation in MPDL22 cells. CONCLUSION: Bortezomib induced cytodifferentiation and mineralization of PDL cells by enhancing the accumulation of ß-catenin within the cytosol and the nucleus and increasing the expression of Bmp-2, -4 and -6 mRNAs. Moreover, bortezomib enhanced the BMP-2-induced cytodifferentiation and mineralization of PDL cells, suggesting that bortezomib may be efficacious for use in periodontal regeneration therapy.

The first nurse practitioner graduate programme in Japan.

AIM: This paper describes the establishment of the first Japanese nurse practitioner graduate programme and legislative activities to institutionalize nurse practitioners in Japan. BACKGROUND: To address the super-ageing population, Oita University of Nursing and Health Sciences initiated the first academic graduate level nurse practitioner programme in Japan, based upon the global standard defined by the International Council of Nurses. CONCLUSION: In 2010, Oita University of Nursing and Health Sciences graduated the first nurse practitioner. We believe that nurse practitioners will be highly valued in Japan for thoughtful nursing care to the fragile elders living in rural and urban Japan.

Natural killer T cells mediate alveolar bone resorption and a systemic inflammatory response in response to oral infection of mice with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: T and B cells are known to be involved in the disease process of periodontitis. However, the role of natural killer T cells in the pathogenesis of periodontitis has not been clarified. MATERIALS AND METHODS: To examine the role of these cells, C57BL/6J (wild-type), CD1d(-/-) and α-galactosylceramide (αGC)-stimulated wild-type mice were orally infected with Porphyromonas gingivalis strain W83. RESULTS: Apart from CD1d(-/-) mice, the level of alveolar bone resorption was elevated by the infection and was further accelerated in αGC-stimulated mice. The infection induced elevated levels of serum amyloid A and P. gingivalis-specific IgG in the sera, although the degree of elevation was much smaller in the CD1d(-/-) mice. Infection-induced RANKL elevation was only observed in αGC-stimulated mice. Although the cytokines produced by splenocytes were mainly T-helper 1 type in wild-type mice, those in αGC-stimulated mice were predominantly T-helper 2 type. In the liver, the infection demonstrated no effect on the gene expression for interferon-γ, interleukin-4 and RANKL except αGC-stimulated mice in which the infection upregulated the gene expressions. CONCLUSION: This study is the first to show that natural killer T cells upregulated systemic and local inflammatory responses induced by oral infection with P. gingivalis, thereby contributing to the progression of alveolar bone resorption.

Oral infection with Porphyromonas gingivalis and systemic cytokine profile in C57BL/6.KOR-ApoE shl mice.

BACKGROUND AND OBJECTIVE: Periodontal infection affects atherosclerotic diseases, such as coronary heart diseases. Mouse models have revealed that oral infection with Porphyromonas gingivalis induces changes in inflammatory- and lipid metabolism-related gene expression, regardless of the development of atherosclerotic lesions. However, the serum protein expression profile in the oral infection model has not been investigated. The present study aimed to analyse the effect of oral infection with P. gingivalis on the expression levels of multiple cytokines in the serum in apolipoprotein E-deficient mice by using a cytokine antibody array. MATERIAL AND METHODS: C57BL/6.KOR-Apoe(shl) mice were orally infected with P. gingivalis five times at 3 day intervals and were then killed. Splenocytes were isolated and analysed for proliferative activity and immunoglobulin G (IgG) production in response to in vitro restimulation with P. gingivalis. The expression levels of various cytokines in the sera were analysed using a mouse antibody array glass chip. RESULTS: Splenocytes from P. gingivalis-infected mice demonstrated significantly greater proliferation and IgG production in response to P. gingivalis compared with those from sham-infected mice. Antibody array analysis revealed the selective upregulation of matrix metalloproteinase 3, intercellular adhesion molecule 1, insulin-like growth factor binding protein 2 and chemokine (C-X-C motif) ligand 7 and the downregulation of interleukin-17, tumor necrosis factor-α and L-selectin. CONCLUSION: These data demonstrate that oral infection with P. gingivalis induces alterations in systemic cytokine production. These cytokines could play roles in the development not only of periodontitis but also of atherosclerosis.

Mirror visual feedback alleviates deafferentation pain, depending on qualitative aspects of the pain: a preliminary report.

OBJECTIVES: Following lesions in somatosensory pathways, deafferentation pain often occurs. Patients report that the pain is qualitatively complex, and its treatment can be difficult. Mirror visual feedback (MVF) treatment can improve deafferentation pain. We sought to classify the qualities of the pain in order to examine whether the potential analgesic effect of MVF depends on these qualities. METHODS: Twenty-two patients with phantom limb pain, or pain related to spinal cord or nerve injury, performed a single MVF procedure. Before and after the MVF procedure, we evaluated phantom limb awareness, movement representation of the phantom or affected/paralysed limb, pain intensity on an 11-point numerical rating scale (0-10) and the qualities of the pain [skin surface-mediated (superficial pain) vs deep tissue-mediated (deep pain)] using lists of pain descriptors for each of the two categories. RESULTS: Fifteen of the patients perceived the willed visuomotor imagery of the phantom or affected/paralysed limb after the MVF procedure. In most of the patients, a reduction in pain intensity and a decrease in the reporting of deep-pain descriptors were linked to the emergence of willed visuomotor imagery. CONCLUSIONS: In this pilot study, we roughly classified the pain descriptor items into two types for evaluating the qualities of deafferentation pain. We found that visually induced motor imagery by MVF was more effective for reducing deep pain than superficial pain. This suggests that the analgesic effect of MVF treatment does depend on the qualities of the pain. Further research will be required to confirm that this effect is a specific consequence of MVF.

Prism adaptation to optical deviation alleviates pathologic pain.

BACKGROUND: The human visual and somatosensory systems are interdependent. Using a visual subjective body-midline (SM) judgment task, we previously confirmed that pathologic pain and deafferentation can modify visuospatial perception, indicating that altered somatosensory experience can modify visual perception. Conversely, in the present study we investigated whether a change in visual experience can modify perception of pathologic pain. METHODS: We used prism adaptation (PA) to modify subjects' visual experience. Five patients with complex regional pain syndrome (CRPS) adapted to wedge prisms, producing a 20-degree visual displacement toward the unaffected side. Further, we used several types of prisms in a longitudinal single-case study. Wearing prismatic goggles, the subjects performed a target-pointing task once a day for 2 weeks. We evaluated pain intensity and visual SM judgment to measure the adaptive aftereffects at three time points: before PA (pre-test), immediately after the first PA exposure (IA-test), and after a 14-day sequence of PA exposure (post-test). RESULTS: PA toward the unaffected side alleviated pathologic pain and other CRPS pathologic features, when measured at post-test. None of the IA-test results showed an analgesic effect. In the longitudinal study, sham PA and 5-degree PA did not produce any effects, and PA toward the affected side actually exacerbated the subjective pain. CONCLUSIONS: Our findings suggest that vision can influence pathologic pain, and preliminarily suggest that prism adaptation has a direction-specific and reproducible effect on not only pathologic pain but also other CRPS pathologic features. Thus, prism adaptation may be a viable cognitive treatment for CRPS.

Pathologic pain distorts visuospatial perception.

Spatial perception is achieved by integrating multisensory information. Using visual subjective body midline (vSM) judgments in patients with unilateral limb pain (complex regional pain syndrome [CRPS]), we found that their vSM deviated toward the affected side; however, deafferentation of the affected limb caused a transient pain decrease and a transient shift of the vSM deviation toward the unaffected side. Our results indicate that the persistent pain state in CRPS distorts visuospatial perception.

Functional characteristics of NaS2, a placenta-specific Na+-coupled transporter for sulfate and oxyanions of the micronutrients selenium and chromium.

NaS2 is a Na+-coupled transporter for sulfate that belongs to the SLC13 gene family. This transporter was originally cloned from high endothelial venule endothelial cells, but nothing is known about the functional characteristics of this transporter except that it transports sulfate in a Na+-coupled manner. Northern blot analysis indicates that NaS2 is expressed most robustly in placenta. In the present study, we cloned NaS2 from rat placenta and characterized its transport function in detail using the Xenopus laevis oocyte expression system. Rat NaS2 consists of 629 amino acids and is highly similar to human NaS2. In situ hybridization studies with mouse placental sections show that NaS2 transcripts are expressed primarily in trophoblasts of the labyrinth zone. The expression of the transporter is confirmed in primary cultures of trophoblasts isolated from human placenta. When expressed in X. laevis oocytes, rat NaS2 mediates Na+-coupled transport of sulfate. The transport of sulfate is inhibited by oxyanions of selenium, chromium, arsenic, molybdenum, and phosphorous, suggesting that the transporter may mediate the transport of these oxyanions in addition to sulfate. The Kt for sulfate is 153+/-30 microM and the Na+:sulfate stoichiometry is 3:1. The transport process is electrogenic as evidenced from the inhibition of the uptake process by K+-induced depolarization. We conclude that NaS2 is a placenta-specific Na+-coupled, electrogenic, transporter for sulfate expressed in trophoblasts and that it is also responsible for the transport of oxyanions of the micronutrients selenium and chromium.

Biological functions of SLC5A8, a candidate tumour suppressor.

SLC5A8 is a candidate tumour suppressor gene that is silenced in colon cancer, gastric cancer and possibly other cancers in humans. This gene codes for a transporter belonging to the Na(+)/glucose co-transporter gene family (SLC5). The cancer-associated silencing of the gene involves hypermethylation of CpG islands present in exon 1 of the gene. SLC5A8 is expressed in colon, ileum, kidney and thyroid gland. The protein coded by the gene mediates the Na(+)-coupled and electrogenic transport of a variety of monocarboxylates, including short-chain fatty acids, lactate and nicotinate. It may also transport iodide. The normal physiological function of this transporter in the intestinal tract and kidney is likely to facilitate the active absorption of short-chain fatty acids, lactate and nicotinate. One of the short-chain fatty acids that serves as a substrate for SLC5A8 is butyrate. This fatty acid is an inhibitor of histone deacetylases and is known to induce apoptosis in a variety of tumours including colonic tumour. Since butyrate is produced in the colonic lumen at high concentrations by bacterial fermentation of dietary fibre, we speculate that the ability of SLC5A8 to mediate the entry of this short-chain fatty acid into colonic epithelial cells underlies the potential tumour suppressor function of this transporter.

Hyaluronan synthase in trabecular meshwork cells.

BACKGROUND/AIMS: Hyaluronan is present in the trabecular meshwork where it is involved in the pathophysiology of aqueous outflow environment. In this study, the expression and regulation of hyaluronan synthase (HAS), which is the enzyme synthesising hyaluronan, in trabecular meshwork cells were investigated. METHODS: Cultured bovine trabecular meshwork cells (BTMCs) were used. HAS expression in BTMCs was examined by RT-PCR. The effects of transforming growth factor beta (TGF-beta) and platelet derived growth factor BB (PDGF-BB) on HAS expression in BTMCs were examined by quantitative RT-PCR. The HAS2 expression by TGF-beta and PDGF-BB at the protein level was also confirmed immunohistochemically. The production of hyaluronan from BTMCs was detected by high performance liquid chromatography (HPLC). RESULTS: Three HAS isoforms were expressed in BTMCs at the mRNA level. Among HAS isoforms, only the expression of HAS2 mRNA was increased by the administration of TGF-beta or PDGF-BB. HAS2 upregulation by these growth factors was also confirmed at the protein level. Further, hyaluronan production from BTMCs was stimulated by TGF-beta or PDGF-BB. CONCLUSION: Expression of HAS in trabecular meshwork may maintain the hyaluronan content in the aqueous outflow pathway. Its production is regulated by TGF-beta and PDGF-BB. The regulation of the expression of HAS in trabecular meshwork might be useful for modulating the aqueous outflow environment.

Estimation of joint fluid volume in the knee joint of rabbits by measuring the endogenous calcium concentration.

OBJECTIVES: The purpose of this study was to develop a new, simple, and more efficient method for estimating joint fluid volume in the knee of experimental animals by measuring endogenous calcium concentration, which is maintained rigidly at a constant level in the body fluid. METHODS: Calcium concentrations in the plasma and joint cavity lavage were colorimetrically measured in normal and papain-induced arthritic rabbits. The joint fluid volume was estimated by dividing the total calcium concentration in the joint cavity lavage (microg/joint) by calcium concentration in the plasma (microg/ml). To confirm the relevancy of this method, radioactivity in the plasma and joint cavity lavage was determined after intravenously injecting 3H2O. The correlation between the joint fluid volumes obtained from the two different methods was examined to evaluate the validity of the method involving measurement of the calcium concentration. Results The joint fluid volumes of normal rabbits were estimated at 401 micro/joint and that in the arthritic rabbits at 680 microl/joint by measuring calcium concentration, respectively. These values were not significantly different from those estimated by radioactivity in the plasma and joint cavity lavage (normal: 425 microl/joint, arthritis: 761 microl/joint). A statistically significant correlation was observed between the values obtained from the two methods (r = 0.985). This method is considered useful for the evaluation of therapeutic medicine for arthritis or for hydrarthrosis research using experimental animal models.

Enzymatic hydrolysis of poly(lactide)s: effects of molecular weight, L-lactide content, and enantiomeric and diastereoisomeric polymer blending.

Nonblended poly(L-lactide) (PLLA) films having different molecular weights and nonblended poly(lactide) (PLA) films, enantiomeric blend films from PLLA and poly(D-lactide) (PDLA), and diastereoisomeric blend films of poly(DL-lactide) (PDLLA) with either PLLA or PDLA, having different L-lactide (LLA) contents (X(LLA)s) in the range of 0-0.99, were amorphous made by melt-quenching. The effects of molecular weight, X(LLA), and average L- and D-lactyl unit sequence length (l(L) and l(D), respectively) on the enzymatic hydrolysis of the films were investigated in the presence of proteinase K. The enzymatic hydrolysis rate (R(EH)) of PLLA estimated by gravimetry increased monotonically with the inverse of number-average molecular weight (M(n)). The extrapolation of R(EH) of PLLA to M(n)(-1) = 0, where no exo-chain-scission takes place, gave a positive R(EH) value (1.75 microg/(mm(2).h)), meaning that the enzymatic hydrolysis of PLLA proceeds via both endo- and exo-chain-scission. The R(EH) of the nonblended films as well as the enantiomeric and diastereoisomeric blend films decreased monotonically with the decease in X(LLA) and finally became zero for X(LLA) below 0.3. The R(EH) values of the blend films of PLLA and PDLLA with PDLA (l(D) = infinity) were lower than expected, while the R(EH) values of the blend films of PLLA with PDLLA (l(D) = 4) agreed completely with the expected values. These results reveal that the nonblended PLA films are enzymatically hydrolyzable when X(LLA) and l(L) are higher than 0.3 and 3, respectively, and l(D) is lower than 10 and that the presence of long D-lactyl unit sequences (l(D) > 4) as in PDLA hinders the enzymatic hydrolysis of long L-lactyl unit sequences even when long D- and L-lactyl unit sequences are present in the different molecules.