GFR alpha3, a component of the artemin receptor, is required for migration and survival of the superior cervical ganglion.

GFR alpha3 is a component of the receptor for the neurotrophic factor artemin. The role of GFR alpha3 in nervous system development was examined by generating mice in which the Gfr alpha3 gene was disrupted. The Gfr alpha3-/- mice exhibited severe defects in the superior cervical ganglion (SCG), whereas other ganglia appeared normal. SCG precursor cells in the mutant embryos failed to migrate to the correct position, and they subsequently failed to innervate the target organs. In wild-type embryos, Gfr alpha3 was expressed in migrating SCG precursors, and artemin was expressed in and near the SCG. After birth, SCG neurons in the mutant mice underwent progressive cell death. These observations suggest that GFR alpha3-mediated signaling is required both for the rostral migration of SCG precursors and for the survival of mature SCG neurons.

A novel class of enkephalinase inhibitors containing a C-terminal sulfo group.

A new series of sulfonic acids were synthesized and tested for their enkephalinase inhibitory activity. Among them, the most potent was N-(2-benzyl-3-mercaptopropionyl)metanilic acid 10i with an IC50 value of 0.27 nM. Several other analogues (10a,b,j,n,o,gg,hh) showed the inhibitory activity comparable to or greater than thiorphan (IC50 = 2.6 nM), a C-terminal carboxyl-containing inhibitor of enkephalinase. Thus compounds containing a C-terminal sulfo group, instead of the C-terminal carboxyl group, were found to show a remarkably high level of inhibition of enkephalinase. The analgesic activity of 10b, (S)-10b, and (R)-10b was also evaluated by the phenylbenzoquinone writhing test.

An anomalous side reaction of the Lys303 mutant aromatic L-amino acid decarboxylase unravels the role of the residue in catalysis.

Aromatic L-amino acid decarboxylase (AADC) in which the pyridoxal 5'-phosphate (PLP)-binding residue Lys303 was replaced by an alanine residue is virtually inactive as a catalyst. On reaction of the normal substrate L-dopa with this mutant AADC, the absorption at around 330 nm gradually increased with concomitant decrease of the absorption of the free PLP molecule at 390 nm. Analysis of the 330-nm absorbing species on HPLC and spectrophotometry showed that it is a 1:1 adduct of PLP and dopamine, probably the Pictet-Spengler type adduct formed from the PLP-dopamine Schiff base. The product dopamine was not found outside of the enzyme, showing that the PLP-dopamine Schiff base undergoes adduct formation rather than yields product dopamine. The Pictet-Spengler adduct of PLP with L-dopa was not detected, whereas the corresponding adduct was formed when the carboxylate group of L-dopa was esterified with a methyl group to block the decarboxylation reaction. This suggests that the PLP-L-dopa Schiff base may undergo the Pictet-Spengler reaction, but its rate is much smaller than that of decarboxylation. These results indicate that Lys303 is not essential for the decarboxylation step, but has an important role in the product release, and possibly the transaldimination process.

Functionally important residues of aromatic L-amino acid decarboxylase probed by sequence alignment and site-directed mutagenesis.

To identify functional residues of rat liver L-aromatic amino acid decarboxylase (AADC), we aligned the sequences of 13 group II amino acid decarboxylases and performed mutational analysis on the residues that were invariant or conservatively substituted. Replacements of His192, Asp252, Asp271, Ser296, Lys303, Tyr332, and Arg355 with alanine residues decreased the AADC activity (kcat/K(m)) by more than 10(4)-fold. Conservative replacements of [Asp252-->Glu], [Lys303-->Arg], and [Tyr332-->Phe] also resulted in decreases in activity by more than 10(4)-fold, indicating that both the chemical properties and the shape of these residues are essential for catalysis. The presence of a Schiff base between the amino group of Lys303 and the coenzyme pyridoxal 5'-phosphate is important for catalysis, probably at the transaldimination step. The enzyme activity was essentially unaffected by conservative mutation of [Arg355-->Lys], showing that the presence of a basic group at position 355 is necessary and sufficient for the catalysis. Replacement of [Thr246-->Ala], [His269-->Ala], and [Trp363-->Leu] yielded mutant enzymes that were 1-8% as active as the wild-type enzyme; these residues are not essential for the catalysis but are considered to contribute to the activity through conformational or other effects. The roles of the catalytically important residues of the group II amino acid decarboxylases probed in this study were discussed in the light of their relationship with the residues of other pyridoxal enzymes.

Total elbow arthroplasty with use of a nonconstrained humeral component inserted without cement in patients who have rheumatoid arthritis.

BACKGROUND: Most total elbow prostheses that are currently in use require cement for fixation of each component. We developed a new (type-5) prosthesis that does not need cement for fixation. METHODS: The humeral component is made of cobalt-chromium alloy, and its stem is porous-coated with a plasma spray of titanium alloy. There are two options for the ulnar component: an all-polyethylene type and a metal-backed type with a porous-coated stem. Forty-three elbows in thirty-seven patients who had rheumatoid arthritis were treated with total elbow replacement arthroplasty with use of the type-5 prosthesis. The humeral component was implanted without cement in all elbows, whereas the ulnar component was implanted without cement in eleven elbows and was fixed with cement in the remaining thirty-two. The elbows were followed for an average of three years and ten months (range, two years and six months to five years and six months). RESULTS: The clinical results were assessed according to a modification of the Mayo Clinic Performance Index. At the time of the latest follow-up, the overall result was excellent for six elbows, good for thirty-one, and fair for six. All elbows had been rated as poor before the operation. There was almost complete relief of pain in twenty-nine elbows and mild or occasional pain in the remaining fourteen. Flexion increased markedly, from an average of 104 degrees preoperatively to an average of 133 degrees postoperatively; this difference was highly significant (p < 0.001, Student t test). In contrast, extension (flexion contracture) worsened slightly, from an average of 38 degrees preoperatively to an average of 42 degrees postoperatively; this difference was significant (p < 0.05). There was one postoperative dislocation of the elbow, and ectopic bone formed in another, with recurrence of ankylosis. Both elbows had a reoperation, and a good result eventually was obtained. There were no instances of postoperative infection or neuropathy of the ulnar nerve. Radiographically, there were no radiolucent lines at the bone-metal interface of any of the humeral or ulnar stems that had been implanted without cement, suggesting solid fixation by osseointegration. CONCLUSIONS: The results of total elbow arthroplasty with use of this prosthesis appear promising. There was a high rate of relief of pain as well as of restoration of adequate function in patients in whom the elbow was severely affected by rheumatoid arthritis.

Effects of selected metal oxides on the dechlorination and destruction of PCDD and PCDF.

OCDD and OCDF spiked silica/graphite based model fly ash containing various copper compounds and metal oxides were thermally treated under oxygen deficient conditions. All copper compounds tested showed a considerable dechlorination/hydrogenation reaction at 260 degrees C. After 30 min at 340 degrees C, less than 1% of the spiked OCDD and OCDF was recovered as T4CDD/F to OCDD/F. Other compounds tested demonstrated a lower rate of dechlorination compared to the copper compounds. However, all other metal oxides showed a small dechlorination effect at 260 degrees C, which was considerably increased at 340 degrees C. The model fly.ash containing the different copper compounds or metal oxides showed comparable PCDD and PCDF isomer patterns after thermal treatment. However, small differences were observed among the different tested compounds. The PCDD and PCDF isomer patterns on the model fly ashes were similar to patterns found during dechlorination experiments on fly ashes from waste incineration processes. Model fly ash containing Ca(OH)2 exhibited the highest destruction potential, but a low dechlorination potential. In contrast, model fly ash containing any of the remaining compounds tested, was found to predominantly dechlorinate the spiked OCDD and OCDF.

Neutrophil CD64 expression in the diagnosis of local musculoskeletal infection and the impact of antibiotics.

We examined the usefulness of neutrophil CD64 expression in detecting local musculoskeletal infection and the impact of antibiotics on its expression. Of 141 patients suspected of musculoskeletal infection, 46 were confirmed by microbiological culture to be infected and 95 had infection excluded. The median CD64 count of patients with localised infection was 2230 molecules per cell (interquartile range (IQR) 918 to 4592) and that of the patients without infection was 937 molecules per cell (IQR 648 to 1309) (p < 0.001). The level of CD64 correlated with the CRP level in patients with infection, but not in those without infection (r = 0.59, p < 0.01). Receiver operator characteristic curve analysis revealed that CD64 was a good predictor of local infection. When the patients were subdivided into two groups based on the administration of antibiotics at the time of CD64 sampling, the sensitivity for detecting infection was better in those who had not received antibiotics. These results suggest that measurement of CD64 expression is a useful marker for local musculoskeletal infection.

Cementless or hybrid total elbow arthroplasty with titanium-alloy implants. A study of interim clinical results and specific complications.

Twenty-six patients (32 elbows) with rheumatoid arthritis had a total elbow arthroplasty with insertion of a cementless prosthesis. The humeral component was made of titanium alloy and it was fixed without cement in all elbows. The patients were followed for an average of 3 years 1 month (range, 2 years 2 months to 4 years 4 months). A good result was seen in 25 elbows, a fair result in 2, and a poor result in 5. The reason for the poor results was a breakage of the humeral component at the junctional portion of its stem. In all five of these elbows a marked resorption of bone mass within the condylar portion of the humeral component was observed on the lateral radiograph. The five elbows with a poor result had a revision operation, and in each of these black staining of the soft tissues within the joint was seen. This tissue metallosis due to wear debris of the titanium alloy was responsible for the osteolysis within the condylar portion. It became clear from this study that even in a non-weight-bearing joint, such as the elbow, titanium alloy may wear and result in tissue metallosis when used as a bearing surface of the implant. However, it was also found that in the majority of the elbows an establishment of the biologic fixation of the porous-coated stem could be achieved by use of this alloy.

Meniscal suture. An experimental study in the dog.

A longitudinal tear was made and then sutured in the peripheral quarter of the medial meniscus of 43 dogs. The sutured menisci were examined using histologic and microangiographic techniques at regular intervals during a 12-week period. Biomechanical tests were also performed. The healing process was promoted not only by the synovial blood supply but also by the peripheral blood supply. Even at the end of 12 weeks, the healing tissue was mainly composed of fibrous tissue, but chondrocytelike cells were also found. The tensile forces were calculated and compared with the opposite leg. The maximum tensile strength reached 80% of that of the opposite side at the end of the 12-week period.

Pharmacokinetics of pentazocine and its occupancy of opioid receptors in rat brain.

In order to assess quantitatively the pharmacodynamic process of pentazocine (PTZ), time courses of its plasma concentration and of the occupation of specific opioid receptors in the brain were investigated after intravenous (i.v.) administration of PTZ to rats. The plasma concentration of PTZ was determined by HPLC and the pharmacokinetic parameters were analyzed using nonlinear least-squares analysis. Measurement of ex vivo receptor occupation was made by comparing the specific [3H]naloxone (opioid receptor antagonist) binding in vitro to the crude P2-synaptosomal fractions between vehicle-treated rats (control) and PTZ-treated rats. Following the i.v. administration of PTZ, the occupancy of specific opioid receptors decreased rapidly until 10 min, depending on the two pharmacological doses (2.5 and 10 mg/kg). The results strongly suggest the fast binding kinetics of PTZ in terms of its association with and dissociation from specific opioid receptor sites in the brain in addition to its fast rate of disappearance from the brain compartment. Furthermore, we demonstrated that the time profile of receptor occupancy correlated well (r = 0.8650) with that of the unbound concentration in plasma until 120 min after the i.v. administration of PTZ to rats.

Pharmacokinetic/pharmacodynamic relationship of eptazocine, a narcotic-antagonist analgesic, in rats.

The relationship between the pharmacokinetics and the pharmacodynamics of eptazocine, a narcotic-antagonist analgesic, was investigated in rats. The analgesic effect of eptazocine (2.5, 5 and 10 mg/kg) following intravenous (i.v) administration was evaluated by both the Randall-Selitto method and the D'Amour-Smith method. The analgesic effects were determined before and at designed intervals for a period of 120 min after eptazocine administration, and are expressed as area under the effect-time curve (AUC(E)). The plasma concentration of eptazocine was determined by fluorescence HPLC and was analyzed with a two compartment open model using the nonlinear least-squares method. Eptazocine produced a dose-dependent analgesic effect. It was demonstrated that eptazocine has a linear relationship between AUC(E) and the area under the plasma concentration-time curve (AUC) following i.v. administration for three different doses ranging from 2.5 to 10 mg/kg.

Fibroblast attachment to Arg-Gly-Asp peptide-immobilized poly(gamma-methyl L-glutamate).

The attachment of MRC-5 human fibroblasts was investigated on poly(gamma-methyl L-glutamate) (PMLG), and upon cell adhesion peptides Arg-Gly-Asp-Ser (RGDS)- and Gly-Arg-Gly-Asp-Ser (GRGDS)-immobilized PMLG (RGDS-PMLG and GRGDS-PMLG). The peptides were immobilized by their N-terminal amine to activated PMLG surfaces. Prior to peptide immobilization, the aminolysis of PMLG surfaces was performed with hydrazine hydrate (HA), ethylenediamine (EDA), and hexamethylenediamine (HMDA) and was followed by the activation with hexamethylene diisocyanate. Surface characterization of these films was carried out by means of a Fourier transform IR (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) attachment. The amount of immobilized RGDS could be controlled by the reaction time of the aminolysis. The effects of HA, EDA, and HMDA as a spacer on the cell attachment were also investigated, and it was suggested that a longer spacer promoted the cell attachment via specific receptor-ligand interaction.

Amino acid sequences of trypsin inhibitors from oriental pickling melon (Cucumis melo L. var. Conomon Makino) seeds.

Three inhibitors (CMCTI-I, II, and III) were isolated from oriental pickling melon (Cucumis melo L. var. Conomon Makino) seeds by acetone precipitation, gel filtration, and reversed phase chromatography. The amino acid sequences of these inhibitors were: [table; see text] The reactive sites (P1 and P1' sites) of these inhibitors are presumed to be the Lys-Ile indicated by an arrow, comparing them with other squash family inhibitors. All three inhibitors can inhibit lysyl endopeptidase and trypsin at the enzyme-inhibitor ratio of 1:1.

Accommodation causes with-the-rule astigmatism in emmetropes.

PURPOSE: To measure the changes in astigmatism when bilateral emmetropes accommodate. METHODS: Bilateral emmetropes accommodative responses were measured with an improved photorefractometer PR-1100, which measured binocular refraction in all meridians simultaneously as a fixation target was shown in natural space. RESULTS: The accommodative responses in the vertical meridian are greater than those in the horizontal meridian. In the horizontal meridian, the accommodative responses are less than the accommodative stimulus. CONCLUSIONS: When accommodating the majority of bilateral emmetropes show with-the-rule astigmatism and lag of accommodation in the horizontal meridian.

Transepithelial transport of organic anions across the choroid plexus: possible involvement of organic anion transporter and multidrug resistance-associated protein.

Transport characteristics of 17beta-estradiol 17beta-D-glucuronide (E217betaG), a dual substrate of the transporters for cellular uptake (organic anion-transporting polypeptide 1 or oatp1) and cellular excretion (multidrug resistance-associated protein 1or MRP1), in the rat choroid plexus were studied in vivo and in vitro. The uptake of E217betaG into isolated choroid plexus was mediated by an energy-dependent system with a Km of 3.4 microM. Together with the previous finding that oatp1 is localized on the apical membrane of choroid plexus, these results suggest that oatp1 is responsible for the uptake of this ligand. After intracerebroventricular administration, elimination of E217betaG from cerebrospinal fluid was probenecid sensitive and much more rapid than that of inulin; less than 2% of the administered E217betaG and 40 to 50% of inulin remained in the cerebrospinal fluid 20 min after intracerebroventricular administration. In addition, the amount of E217betaG associated with choroid plexus at 20 min was negligible, suggesting the presence of an efficient excretion system on the basolateral membrane of choroid plexus. Expression of MRP1 was detected in choroid plexus. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analyses indicated that the expression level of MRP1 in choroid plexus is about four or five times higher than that in the lung, one of the tissues exhibiting high expression of MRP1. Together with the in vivo vectorial transport of E217betaG, these results can be accounted for by assuming that there is basolateral localization of MRP1 in choroid plexus. Combined, oatp1 and MRP1 may synergistically mediate the efficient transcellular transport of E217betaG across choroid plexus.

The binocular accommodative response in uncorrected ametropia.

PURPOSE: To measure the changes in astigmatism and in the degree of anisometropia when ametropes respond to accommodative stimulation. METHODS: The accommodative responses of ametropes were binocularly measured with an improved photorefractometer (PR-1100), which objectively measured binocular refraction in all meridians simultaneously, when a visual fixation target was shown in natural space at 5 and 0.5 m. RESULTS: (1) Changes of astigmatism and anisometropia occur during binocular accommodation. (2) Changes of astigmatism mainly result from lag of accommodation in the horizontal meridian. (3) Changes of anisometropia result from the effort to focus. (4) The interaction of both eyes is suggested as the cause of the changes of astigmatism and anisometropia with accommodation. (5) In the majority of strong anisometropes (nonoverlapping group in this paper), the magnitude of anisometropia decreases (approaching isometropia) with accommodation. CONCLUSION: Changes in astigmatism and anisometropia can occur during accommodation, and these interactions of both eyes with accommodation may contribute to the development and maintenance of binocular function.

The retinoic acid-inactivating enzyme CYP26 is essential for establishing an uneven distribution of retinoic acid along the anterio-posterior axis within the mouse embryo.

Retinoic acid (RA), a derivative of vitamin A, plays a pivotal role in vertebrate development. The level of RA may be determined by the balance between its synthesis and degradation. We have examined the role of CYP26, a P450 enzyme that may degrade RA, by generating mutant mice that lack CYP26. CYP26(-/-) mice exhibited anomalies, including caudal agenesis, similar to those induced by administration of excess RA. The concentration of endogenous RA, as revealed by marker gene activity, was markedly increased in the tailbud of the mutant animals, in which CYP26 is normally expressed. Expression of T (Brachyury) and Wnt3a in the tailbud was down-regulated in CYP26(-/-) mice, which may underlie the caudal truncation. The lack of CYP26 also resulted in homeotic transformation of vertebrae as well as in misspecification of the rostral hindbrain associated with anterior expansion of RA-positive domains. These results suggest that local degradation of RA by CYP26 is required for establishing an uneven distribution of RA along the anterio-posterior axis, which is essential for patterning the hindbrain, vertebrae, and tailbud.

Two-step regulation of left-right asymmetric expression of Pitx2: initiation by nodal signaling and maintenance by Nkx2.

Pitx2 is left--right (L--R) asymmetrically expressed initially in the lateral plate and later in primordial visceral organs. The transcriptional regulatory mechanisms that underlie L--R asymmetric expression of Pitx2 were investigated. Mouse Pitx2 has a left side-specific enhancer (ASE) that mediates both the initiation and maintenance of L--R asymmetric expression. This element contains three binding sites for the transcription factor FAST. The FAST binding sites function as Nodal-responsive elements and are sufficient for the initiation but not for the maintenance of asymmetric expression. The maintenance requires an Nkx2-5 binding site also present within the ASE. These results suggest that the left-sided expression of Pitx2 is directly initiated by Nodal signaling and is subsequently maintained by Nkx2. Such two-step control may represent a general mechanism for gene regulation during development.

Digital subtraction angiogram registration method with local distortion vectors to decrease motion artifact.

We have been investigating registration methods for improving digital subtraction angiography (DSA) images to extract blood vessels by reducing artifacts due to body motion, such as rotation, contraction, and dilation. In this paper, we propose a new and simple DSA registration algorithm with local distortion vectors to reduce artifacts. According to the results, the proposed method works well for vascular system around the nasal cavity and the orbit of the head and neck DSA images, which cannot be observed clearly by conventional methods. Additionally, we have applied the proposed method to abdominal and leg DSA images.