Mid-term results of alumina ceramic unlinked total elbow arthroplasty with cement fixation for patients with rheumatoid arthritis.

Aims: The aim of this study was to report the mid-term clinical outcome of cemented unlinked J-alumina ceramic elbow (JACE) arthroplasties when used in patients with rheumatoid arthritis (RA). Patients and Methods: We retrospectively reviewed 87 elbows, in 75 patients with RA, which was replaced using a cemented JACE total elbow arthroplasty (TEA) between August 2003 and December 2012, with a follow-up of 96%. There were 72 women and three men, with a mean age of 62 years (35 to 79). The mean follow-up was nine years (2 to 14). The clinical condition of each elbow before and after surgery was assessed using the Mayo Elbow Performance Index (MEPI, 0 to 100 points). Radiographic loosening was defined as a progressive radiolucent line of >1 mm that was completely circumferential around the prosthesis. Results: The mean MEPI scores significantly improved from 40 (10 to 75) points preoperatively to 95 (30 to 100) points at final follow-up (p < 0.0001). Complications were noted in ten elbows (ten patients; 11%). Two had an intraoperative humeral fracture which was treated by fixation and united. One had a postoperative fracture of the olecranon which united with conservative treatment and one had a radial neuropathy which resolved. Further surgery was required for one with a dislocation, three with an ulnar neuropathy and one with a postoperative humeral fracture. Revision with removal of the components was performed in one elbow due to deep infection. There was no radiographic evidence of loosening around the components. With any revision surgery or revision with implant removal as the endpoint, the rates of survival up to 14 years were 93% (95% confidence interval (CI), 83.9 to 96.6) and 99% (95% CI 91.9 to 99.8), respectively, as determined by Kaplan-Meier analysis. Conclusion: With the appropriate indications, the mid-term clinical performance of the cemented JACE TEA is reliable and comparable to other established TEAs in the management of the elbow in patients with RA. Cite this article: Bone Joint J 2018;100-B:1066-73.

Factors associated with the practice of nursing staff sharing information about patients' nutritional status with their colleagues in hospitals.

BACKGROUND/OBJECTIVES: Nursing staff have an important role in patients' nutritional care. The aim of this study was to demonstrate how the practice of sharing a patient's nutritional status with colleagues was affected by the nursing staff's attitude, knowledge and their priority to provide nutritional care. SUBJECTS/METHODS: The participants were 492 nursing staff. We obtained participants' demographic data, the practice of sharing patients' nutritional information and information about participants' knowledge, attitude and priority of providing nutritional care by the questionnaire. We performed partial correlation analyses and linear regression analyses to describe the relationship between the total scores of the practice of sharing patients' nutritional information based on their knowledge, attitude and priority to provide nutritional care. RESULTS: Among the 492 participants, 396 nursing staff (80.5%) completed the questionnaire and were included in analyses. Mean±s.d. of total score of the 396 participants was 8.4±3.1. Nursing staff shared information when they had a high nutritional knowledge (r=0.36, P<0.01) and attitude (r=0.13, P<0.05); however, their correlation coefficients were low. In the linear regression analyses, job categories (ß=-0.28, P<0.01), knowledge (ß=0.33, P<0.01) and attitude (ß=0.10, P<0.05) were independently associated with the practice of sharing information. Nursing staff's priority to provide nutritional care practice was not significantly associated with the practice of sharing information. CONCLUSIONS: Knowledge and attitude were independently associated with the practice of sharing patients' nutrition information with colleagues, regardless of their priority to provide nutritional care. An effective approach should be taken to improve the practice of providing nutritional care practice.

Hypothalamic brain-derived neurotrophic factor regulates glucagon secretion mediated by pancreatic efferent nerves.

Understanding the molecular mechanism of the regulation of glucagon secretion is critical for treating the dysfunction of α cells observed in diabetes. Glucagon-like peptide (GLP)-1 analogues reduce plasma glucagon and are assumed to contribute to their action to lower blood glucose. It has previously been demonstrated that the central administration of brain-derived neurotrophic factor (BDNF) improves glucose metabolism by a mechanism independent of feeding behaviour in obese subjects. Using male rats, we examined whether BDNF influences glucagon secretion from α cells via the the central nervous system. We investigate whether: (i) the central infusion of BDNF stimulates glucagon and/or insulin secretion via the pancreatic efferent nerve from the hypothalamus; (ii) the intraportal infusion of GLP-1 regulates glucose metabolism via the central and peripheral nervous system; and (iii) BDNF receptor and/or BDNF-positive fibres are localised near α cells of islets. The portal glucagon level decreased with the central administration of BDNF (n = 6, in each; P < 0.05); in contrast, there was no significant change in portal insulin, peripheral glucagon and insulin levels with the same treatment. This reduction of glucagon secretion was abolished by pancreatic efferent denervation (n = 6, in each; P < 0.05). In an immunohistochemical study, pancreatic α cells were stained specifically with BDNF and tyrosine-related kinase B, a specific receptor for BDNF, and α cells were also co-localised with BDNF. Moreover, intraportal administration of GLP-1 decreased glucagon secretion, as well as blood glucose, whereas it increased the BDNF content in the pancreas; these effects were inhibited with the central infusion of BDNF antibody (n = 6, in each; P < 0.05). BDNF and GLP-1 affect glucose metabolism and modulate glucagon secretion from pancreatic α cells via the central and peripheral nervous systems.

Long-term infusion of brain-derived neurotrophic factor reduces food intake and body weight via a corticotrophin-releasing hormone pathway in the paraventricular nucleus of the hypothalamus.

Brain-derived neurotrophic factor (BDNF) has been implicated in learning, depression and energy metabolism. However, the neuronal mechanisms underlying the effects of BDNF on energy metabolism remain unclear. The present study aimed to elucidate the neuronal pathways by which BDNF controls feeding behaviour and energy balance. Using an osmotic mini-pump, BDNF or control artificial cerebrospinal fluid was infused i.c.v. at the lateral ventricle or into the paraventricular nucleus of the hypothalamus (PVN) for 12 days. Intracerebroventricular BDNF up-regulated mRNA expression of corticotrophin-releasing hormone (CRH) and urocortin in the PVN. TrkB, the receptor for BDNF, was expressed in the PVN neurones, including those containing CRH. Both i.c.v. and intra-PVN-administered BDNF decreased food intake and body weight. These effects of BDNF on food intake and body weight were counteracted by the co-administration of alpha-helical-CRH, an antagonist for the CRH and urocortin receptors CRH-R1/R2, and partly attenuated by a selective antagonist for CRH-R2 but not CRH-R1. Intracerebroventricular BDNF also decreased the subcutaneous and visceral fat mass, adipocyte size and serum triglyceride levels, which were all attenuated by alpha-helical-CRH. Furthermore, BDNF decreased the respiratory quotient and raised rectal temperature, which were counteracted by alpha-helical-CRH. These results indicate that the CRH-urocortin-CRH-R2 pathway in the PVN and connected areas mediates the long-term effects of BDNF to depress feeding and promote lipolysis.

Young adult-specific hyperphagia in diabetic Goto-kakizaki rats is associated with leptin resistance and elevation of neuropeptide Y mRNA in the arcuate nucleus.

The present study aimed to examine whether hyperphagia, which is frequently observed in type 1 diabetic patients and model animals, also occurs in type 2 diabetic Goto-Kakizaki (GK) rats and, if so, to explore underlying abnormalities in the hypothalamus. GK rats at postnatal weeks 6-12, compared to control Wistar rats, exhibited hyperphagia, hyperglycaemia, hyperleptinemia and increased visceral fat accumulation, whereas body weight was unaltered. The ability of leptin to suppress feeding was reduced in GK rats compared to Wistar rats of these ages. In GK rats, leptin-induced phosphorylation of signal transducer and activator of transcription 3 was significantly reduced in the cells of the hypothalamic arcuate nucleus (ARC), but not of the ventromedial hypothalamus, whereas the mRNA level of functional leptin receptor was unaltered. By real-time polymerase chain reaction and in situ hybridisation, mRNA levels of neuropeptide Y, but not pro-opiomelanocortin and galanin-like peptide, were significantly increased in the ARC of GK rats at 11 weeks, but not 26 weeks. Following i.c.v. injection of a NPY Y1 antagonist, 1229U91, the amount of food intake in GK rats was indistinguishable from that in Wistar rats, thus eliminating the hyperphagia of GK rats. These results demonstrate that young adult GK rats display hyperphagia in association with leptin resistance and increased NPY mRNA level in the ARC.

Preliminary study of autologous blood predonation in pediatric open-heart surgery impact of advance infusion of recombinant human erythropoietin.

We applied an autologous blood predonation protocol using recombinant human erythropoietin in pediatric open-heart surgery. The study included 69 children weighing 8 kg or more. Twice before operation, 8 ml/kg of blood was taken. At each donation, 100 (group 1; n = 20), 200 (group 2; n = 11), or 300 (group 3; n = 13) units/kg of erythropoietin was given subcutaneously. In group 4 (n = 25), 300 units/kg of erythropoietin was given 1 week prior to the first donation, followed by 300 units/kg given at each donation. No harmful events occurred during the donation period. During the donation period, the patients' hematocrit decreased but the hematocrit 1 day after the operation remained at 32.1 +/- 0.6% and was 33.3 +/- 0.6% 2 weeks later. The decrease in hematocrit was minimal in group 4 (39.0 +/- 0.6% before donation to 37.5 +/- 0.5% before operation) compared with that of the other three groups. Among those who completed the protocol, 58 patients (93.5%) were discharged without homologous blood transfusion. We consider our protocol of autologous blood predonation using erythropoietin to be safe and effective for avoiding homologous blood transfusion in pediatric patients. The early infusion of erythropoietin prior to the first donation minimizes the decrease in hematocrit level before operation.

Effect of eicosapentaenoic acid ethyl ester on hypothyroid function.

Thyroid hormones affect reactions in almost all pathways of lipid metabolism. It has been reported that plasma free fatty acid (FFA) concentration in hypothyroidism is generally within the normal range. In this study, however, we show that plasma FFA concentration in some hypothyroid patients is higher than the normal range. Symptoms of thyroid dysfunction in these individuals were less severe than those of patients with lower plasma FFA concentrations. From these findings we hypothesized that the change in FFA concentration must correlate with thyroid function. Using an animal model, we then examined the effect of highly purified eicosapentaenoic acid ethyl ester (EPA-E), a n-3 polyunsaturated fatty acid derived from fish oil, on thyroid function in 1-methyl-2-imidazolethiol (MMI)-induced hypothyroid rats. Oral administration of EPA-E inhibited reduction of thyroid hormone levels and the change of thyroid follicles in MMI-induced hypothyroid rats. These findings suggest that FFA may affect thyroid functions and EPA-E may prevent MMI-induced hypothyroidism.

Purification, characterization, and cDNA cloning of lipoate-activating enzyme from bovine liver.

In mammals, lipoate-activating enzyme (LAE) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. The lipoyl moiety is then transferred to the specific lysine residue of lipoate-dependent enzymes by the action of lipoyltransferase. We purified LAE from bovine liver mitochondria to apparent homogeneity. LAE activated lipoate with GTP at a 1000-fold higher rate than with ATP. The reaction absolutely required lipoate, GTP, and Mg(2+) ion, and the reaction product was lipoyl-GMP. LAE activated both (R)- and (S)-lipoate to the respective lipoyl-GMP, although a preference for (R)-lipoate was observed. Similarly, lipoyltransferase equally transferred both the (R)- and (S)-lipoyl moieties from the respectively activated lipoates to apoH-protein. Interestingly, however, only H-protein carrying (R)-lipoate was active in the glycine cleavage reaction. cDNA clones encoding a precursor LAE with a mitochondrial presequence were isolated. The predicted amino acid sequence of LAE is identical with that of xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III, but an amino acid substitution due to a single nucleotide polymorphism was found. These results indicate that the medium-chain acyl-CoA synthetase in mitochondria has a novel function, the activation of lipoate with GTP.

Autologous blood donation with recombinant human erythropoietin for abdominal aortic aneurysm surgery.

We evaluated the efficacy of autologous blood donation using recombinant human erythropoietin for elective abdominal aortic aneurysm (AAA) surgery regarding postoperative recovery. Twenty-five AAA patients who completed surgery without receiving a homologous blood transfusion were divided into two groups, consisting of a control group (n = 12) who did not bank any autologous blood, and a donation group (n = 13) who did bank more than 800 ml of autologous blood with the use of erythropoietin. The hematocrit (Ht) level of the control group decreased from 41.1% +/- 1.2% before the operation to 36.2% +/- 0.9% just afterwards (P > 0.01). In the donation group, however, the Ht did not change significantly during either the donation period or the perioperative period. The postoperative period before oral food intake and natural defecation were both significantly shorter in the donation group than in the control group. The first day of mobilization was also earlier in the donation group. In conclusion, autologous blood donation using erythropoietin for AAA surgery is therefore considered to promote the early recovery of patients.

Increased urinary phosphate excretion in pseudohypoparathyroidism type II with long-term treatment with phosphodiesterase inhibitor.

A 58-year-old woman was diagnosed to have pseudohypoparathyroidism (PHP) type II because of the absence of an increase of urinary phosphate secretion, despite a marked increase in urinary cAMP excretion on the Ellsworth-Howard test. We treated the patient with a cyclic-nucleotide phosphodiesterase inhibitor, theophylline, resulting in increased urinary phosphate and cAMP excretions. Dibutyl cAMP administration induced the increase in the urinary phosphate excretion. In this case, the unresponsiveness of the urinary phosphate secretion to cAMP was recovered by a high dose of cAMP or long-term administration of a phosphodiesterase inhibitor. These data imply that cAMP responsiveness to renal tubular phosphate reabsorption should be more strictly elucidated in the patient with PHP type II.

Histamine monoclonal antibody for brain immunocytochemistry.

Among five monoclonal antibodies (AHA-1 to 5 mAbs) prepared against glutaraldehyde (GA)-conjugated histamine (HA) in our previous study, only mAb AHA-2 was found to detect HA specifically in rat brain neurons by an immunocytochemistry method (ICC) using GA as a tissue fixative. All the other mAbs, except for AHA-5, reacted with HA in the enterochromaffin-like cells (ECL cells) of rat stomach [Fujiwara et al. (1997) Histochem. Cell Biol. 107, 39-45]. Enzyme-linked immunosorbent assay (ELISA) binding and inhibition tests demonstrated that AHA-2 is specific for HA, with almost no detectable cross-reaction with any other established or putative amino acid neurotransmitters, LH-RH, TRH, or peptides with N-terminal histidines. ELISA assays also suggested that the AHA-2 mAb recognizes a HA epitope structure different from the one recognized by the AHA-1 mAb. The immunostaining patterns with AHA-2 mAb, as seen in the five subgroups of the tuberomammillary nuclei in the rat posterior hypothalamus, were very similar to those described by Inagaki et al. [(1988) Brain Res. 439, 402-405; (1990) Exp. Brain Res. 80, 374-380] and Panula et al. [(1984) Proc. Natl. Acad. Sci. USA 81, 2572-2576; (1988) J. Histochem. Cytochem. 36, 259-269] using polyclonal anti-HA serum. However, it was also noted that moderate numbers of immunoreactive nerve fibers projected into the median eminence. The present HA ICC method using AHA-2 mAb allows highly sensitive HA detection in brain, and thus might permit detailed studies of HA localization hitherto impossible using previously available anti-HA polyclonal antibodies produced against carbodiimide-conjugated HA.

Involvement of growth-related protein in lipopolysaccharide-induced rabbit arthritis: cooperation between growth-related protein and IL-8, and interrelated regulation among TNFalpha, IL-1, IL-1 receptor antagonist, IL-8, and growth-related protein.

We investigated the functional role of a CXC chemokine, growth-related protein (GRO), in the recruitment of neutrophils in lipopolysaccharide (LPS)-induced rabbit arthritis. The amounts of GRO in the synovial fluids (SF) reached the first peak (major) at 2 hours and the second peak (minor) at 9 hours after injection of LPS into the knee joints. Administration of anti-GRO mouse monoclonal antibody inhibited 54% of the peak leukocyte accumulation at 9 hours (neutrophils greater than 95%), which was similar to the inhibition by anti-IL-8 IgG (48%). Co-administration of these inhibitors increased the inhibition up to 70% at 9 hours and also inhibited 65% of the initial phase of leukocyte infiltration at 2 hours (neutrophils greater than 99%), which was not affected by a single administration of each inhibitor. The amounts of GRO in SF at 2 hours were not altered by either anti-TNFalpha mAb or anti-IL-8 IgG, but reduced by rabbit recombinant IL-1 receptor antagonist (rrlL-1Ra) by 39%. The inhibition by rrlL-1 Ra was augmented further to 59% with coadministered anti-TNFalpha mAb. In contrast, the amounts of GRO at 9 hours were reduced by rrlL-1Ra by 67%. There was no additional reduction in the amounts of GRO at 9 hours by either combination of rrlL-1Ra with anti-TNFalpha mAb or anti-IL-8 IgG. Administration of anti-GRO mAb did not alter TNFalpha or IL-8 contents in SF at their peak (2 hours), but reduced the amounts of IL-1beta at 6 hours and IL-1Ra at 9 hours by 42% and 49%, respectively. These results provide evidence for the following: (a) GRO as well as IL-8 are important mediators involved in the recruitment of neutrophils both in the early and the late phase of LPS-induced arthritis, (b) IL-1 produced in the early phase stimulates GRO production, (c) GRO plays a role in the later induction of IL-1beta and IL-1Ra, and (d) induction of GRO is not regulated by IL-8.

Immunocytochemical localization of prolactin-releasing peptide in the rat brain.

A hypothalamic peptide that stimulates PRL release has recently been found as a ligand of an orphan receptor and named PRL-releasing peptide (PrRP). PrRP and its receptor were mainly detected in the hypothalamus and pituitary gland, respectively. Its characteristics suggested PrRP to be a novel hypophysiotropic peptide that stimulates the anterior pituitary PRL cell; however, this remained to be confirmed morphologically. We therefore performed an immunocytochemical study to locate PrRP in the rat brain using the region-specific monoclonal antibodies, P2L-1C and P2L-1T, which recognize the C-terminal and the internal sequence of PrRP, respectively. Our results clearly show that dense immunoreactive nerve fiber networks are present in the paraventricular hypothalamic nucleus, supraoptic nucleus, paratenial thalamic nucleus, basolateral amygdaloid nucleus, and bed nucleus of the stria terminalis. A small number of PrRP nerve fibers was also observed in the neural lobe of the hypophysis. However, no immunopositive fiber was observed in the external region of the median eminence, which is known to be the release site of the classical hypophysiotropic hormones. Also, the distribution of PrRP was not changed during the estrous cycle. We therefore concluded that PrRP probably differs from classical hypothalamic releasing hormones. We found the immunoreactive cell bodies to be mainly in the caudal portion of the dorsomedial hypothalamic nucleus and solitary nucleus. A double immunocytochemical procedure revealed that some PrRP-positive neurons showed synaptic contact with oxytocin-positive cell bodies in the paraventricular hypothalamic nucleus, which suggests that PrRP regulates the function of oxytocin neurons. This is the first report to demonstrate the localization of the novel hypothalamic peptide, PrRP, and we therefore suggest that it takes part in a variety of brain functions. However, it is not yet known how PrRP is transported to the pituitary gland, which is the site that contains the greatest concentration of receptors to this new peptide. Therefore, additional work will be required to resolve this discrepancy between ligand and receptor site location.

Enzyme immunoassay for a characteristic protein in the animal crude drug lumbricus.

A new method was developed to measure the content of a Lumbricus component in a traditional Chinese medicine (TCM). An antiserum specific to Lumbricus was elicited in a rabbit following immunization with a suspension of Lumbricus fragments. A characteristic antigen protein, 70 kDa, was found in Lumbricus and was purified almost to singleness using a column chromatography series of gel filtration and DEAE-Sepharose. A selected antibody enzyme immunoassay (SAEIA) was developed using the antiserum and the purified 70 kDa protein as a solid-phase antigen. The SAEIA was specific to Lumbricus species, and showed no cross-reaction with any crude drugs other than Lumbricus. This SAEIA detected 70 kDa protein in the amount of 10 ng/ml with excellent reproducibility (coefficient of variation=3.0%) and an EC50 of 0.24 microg/ml. Using this assay, Lumbricus levels were easily determined in a Lumbricus-based TCM Kazecoll, but not in the control Kazecoll (Kakkonto) prepared without Lumbricus. The SAEIA for 70 kDa protein was simple, accurate, reproducible and may provide a general analytical method for the quality control of Lumbricus-based TCMs.

[Lower mid-line skin incision and minimal sternotomy in pediatric open heart surgery--an alternative cosmetic approach].

Full median sternotomy has been the standard approach for open heart surgery. However, it leaves an unsightly surgical scar. We therefore designed a lower mid-line skin incision and minimal sternotomy approach as a cosmetic alternative. Since February 1993, 78 pediatric patients with congenital heart disease have undergone this open heart surgery procedure. Their ages ranged from 1 month to 13 years (median: 2 years and 7 months) and body weights ranged from 2.4 to 43 kg (median: 11.5 kg). Thirty-one patients had atrial septal defect, 34 perimembranous ventricular septal defect, 9 subpulmonary ventricular septal defect, and 4 other cardiac anomalies. All cannulations for cardiopulmonary bypass could be performed through this approach. No patients required conversion to standard median sternotomy and no peri- or postoperative complications related to this approach, such as injury to the great artery and vein, air embolism, or sternal deherence, were noted. This approach is technically easy and an excellent cosmetic approach in pediatric open heart surgery.

Synthesis and characterization of selenolipoylated H-protein of the glycine cleavage system.

H-protein of the glycine cleavage system has a lipoic acid prosthetic group. Selenolipoic acid is a lipoic acid analog in which both sulfur atoms are replaced by selenium atoms. Two isoforms of bovine lipoyltransferase that are responsible for the attachment of lipoic acid to H-protein had an affinity for selenolipoyl-AMP and transferred the selenolipoyl moiety to bovine apoH-protein comparable to lipoyl-AMP. Selenolipoylated H-protein was overexpressed in Escherichia coli and purified. Selenolipoylated H-protein was 26% as effective as lipoylated H-protein in the glycine decarboxylation reaction, in which reduction of the diselenide bond of selenolipoylated H-protein is catalyzed by P-protein. The diselenide form of selenolipoylated H-protein was a poor substrate for L-protein, and the rate of reduction was 0.5% of that of lipoylated H-protein. The rate of the overall glycine cleavage reaction with selenolipoylated H-protein was <1% of that with lipoylated H-protein. These results are consistent with the difference in the redox potential between the diselenide and disulfide bonds. In contrast, selenolipoylated H-protein showed three times as high glycine-14CO2 exchange activity as lipoylated H-protein, presumably because the rate of reoxidation of reduced selenolipoylated H-protein is much higher than that of lipoylated H-protein.

Application of a licorice root specific protein to a general method for the assay of licorice root components in traditional Chinese medicines.

A new fuzzy immunoassay method generally applied to ten Glycyrrhizae Radix (GR) preparations of four different botanical origins was studied. Four kinds of antisera were elicited in rabbits immunized with GRs of different botanical origins. The presence of the characteristic GR protein (GRP) was shown using Western blot analyses and selected antibody enzyme immunoassay (SAEIA) methods. A GRP was isolated from one of the GR specimens which was selected using SAEIA methods. The isolated GRP was heated to reduce its binding activity to an anti-GR serum. A new fuzzy SAEIA method generally applicable for assay of the extract of the ten GR specimens was developed using heat-treated GRP as the solid-phase antigen. The fuzzy SAEIA method was successfully applied for the detection and quantitative analysis of the GR component contained in traditional Chinese medicines.

A general enzyme immunoassay for the licorice root component contained in traditional Chinese medicines.

The development and application of a new enzyme immunoassay for general assay of the Glycyrrhizae radix (GR) component in Chinese traditional medicines is described. Three commercial GR-based medicines, Tohoku kanzo (GRTK), Seihoku Kanzo (GRSEK) and Sinkyo kanzo (GRSK) were used as GR specimens. Anti-GRSK serum was elicited from rabbits immunized with GRSK fragments. The presence of common proteins as specific antigens of GR was first established by Western blot analysis of extracts of GRSK, GRSEK or GRTK using anti-GRSK. The specific antigens were applied to develop an ELISA for the assay of GR extract. Anti-GRSK was put in competition with a sample or standard GR extract and immobilized GRSK components in microtiter plate wells. The proportion of antibody binding to the solid-phase GRSK component was detected using an enzyme-labeled second antibody. The ELISA method was specific to GRSK extract and showed low sensitivity for the assay of GRTK extract. The technique of selected antibody enzyme immunoassay (SAEIA) was applied to develop a sensitive general assay method. Solid-phase GRTK extract, rather than immobilized GRSK extract, was used in the SAEIA. The SAEIA possessed the same quantitative working range of between 1 and 100 micrograms/ml for the assay of each extract of GRTK, GRSK and GRSEK. The SAEIA was successful in the detection and quantitative measurement of GR component contents in Chinese traditional medicines.

Development and application of a sandwich enzyme immunoassay for Glycyrrhizae Radix protein (GRP) using monoclonal antibodies.

We have developed mouse monoclonal antibodies (anti-GRP mAb-1-5, all IgG1 sub-isotype mAbs) against Glycyrrhizae Radix protein (GRP), which was recently determined to be a marker protein of Glycyrrhizae Radix (GR). Among these, anti-GRP mAb-1 and 2 were found to recognize different epitopes on the GRP molecule, as demonstrated by ELISA analysis, and were used for the development of a sandwich enzyme immunoassay (SEIA) for GRP in traditional Chinese medicines (TCMs). The SEIA was based on the principle of binding an analyte to anti-GRP mAb2 coated on polystyrene microtiter wells, followed by immunoreaction with biotinylated anti-GRP mAb1 and horseradish peroxidase-streptavidin. The SEIA was specific to GRP in GRs, and showed no cross-reaction with any Leguminosae crude drugs other than GRs. This SEIA detected GRP with excellent reproducibility (coefficient of variation=5.9%), an EC50 of 11.5 ng/well and a detection limit of 0.1 ng/well. The present SEIA was about 10-times more sensitive in detecting GRP than the selected antibody enzyme immunoassay (SAEIA) for GRP previously developed using an antiserum to GR itself. Also, the SEIA has such a low assay background that it allowed us to detect a low concentration of GRP in Kyuki-tyoketsu-in-daiichi-kagen (KTIDK), a TCM consisting of only 2.7% GR. The GRP SEIA was simple, accurate, reproducible and may provide a general analytical method for the quality control of GR-based TCMs.

Effects of magnetic flux density and direction of the magnetic field on growth and CO2 exchange rate of potato plantlets in vitro.

Potato plantlets (Solanum tuberosum L. cv. Benimaru) were cultured in vitro for 20 days on a 10 ml MS agar medium under a magnetic flux density of 0 (control: Cont), 2, 4 or 6 mT (T=Wb m-2) in both upward and downward directions of magnetic field. Air temperature in the head space of the test tubes and photosynthetic flux density on the culture shelf were 25 +/- 1 degrees C and 38 micromoles m-2 s-1 respectively. The results showed that a magnetic flux density of around 4 mT had beneficial effects, regardless of the direction of magnetic field, on the growth promotion and enhancement of CO2 uptake of potato plantlets in vitro The direction of magnetic field at the magnetic flux densities tested had no effects on the growth and CO2 exchange rate.