Measures of physical functioning after hip fracture: construct validity and responsiveness of performance-based and self-reported measures.

OBJECTIVES: this study aimed to investigate the construct validity and responsiveness of performance-based and self-reported measures of strength, mobility and balance after hip fracture. DESIGN: secondary analysis of clinical trial data. SUBJECTS: a total of 148 older people undergoing hip fracture rehabilitation. METHODS: correlation coefficients assessed construct validity. Internal responsiveness was assessed by calculating effect sizes (ES) I and II. Area under the receiver operating characteristic curve (AUC) assessed external responsiveness with change in EuroQol as the reference. RESULTS: correlations between performance-based and self-reported measures were small to medium (strength r = 0.17, mobility r = 0.45 and balance r = 0.37). The most responsive performance-based measures included walking speed (ESI 1.7, ESII 1.2), Physical Performance and Mobility Examination (ESI 1.3, ESII 1.0) and chair-rise test (ESI 1.1, ESII 0.8). Self-reported mobility (ESI 0.8, ESII 0.6) and strength (ESI 0.8, ESII 0.6) were more responsive than self-reported balance (ESI 0.3, ESII 0.2). External responsiveness (AUC) was greatest for walking speed (0.72) and lowest for the measures of body sway (0.53). CONCLUSION: self-reported and performance-based indices appear to assess different constructs and may provide complementary information about physical functioning in people after hip fracture. Measures of strength and mobility showed greater ability to detect change than measures of balance.

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia.

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

Technical note: A rapid enzyme-linked immunosorbent assay blood test for pregnancy in dairy and beef cattle.

The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected in the blood of pregnant animals. The objective was to determine the accuracy of a rapid ELISA PAG-based test for the purpose of pregnancy detection in cattle. Blood was sampled from dairy cattle (539 Holstein cows, 173 Holstein heifers, 73 Guernsey cows, 22 Guernsey heifers, and 12 Jersey heifers) and crossbred beef cattle (145 cows and 46 heifers) that were >or=25 d after insemination (range = 25 to 45 d for dairy and 29 to 56 d for beef). Cattle were examined by ultrasonography for detection of pregnancy within 2 d of blood collection. Whole blood or plasma was incubated in a polystyrene tube coated with a monoclonal PAG antibody for 15 min. The tubes were then washed and subjected to sequential incubations with a biotinylated polyclonal PAG antibody (15 min, followed by wash), a horseradish peroxidase-streptavidin solution (15 min, followed by wash), and a peroxidase substrate. Tubes were visually assessed for color after 15 min (clear solution = PAG negative, not pregnant; blue solution = PAG positive, pregnant). Total assay time was approximately 90 min. The ultrasound examination was used as the standard for pregnancy diagnosis. The sensitivity (99.8 +/- 0.2%), specificity (91.7 +/- 1.4%), and negative predictive value (99.7 +/- 0.3%) for the PAG test used in dairy cattle were similar for different breeds and for cows and heifers. The positive predictive value for the test was greater in dairy heifers than in dairy cows (96.5 +/- 1.4% vs. 90.5 +/- 1.7%, respectively). In beef cattle, the sensitivity (100%), specificity (92.3 +/- 3.0%), positive predictive value (95.0 +/- 2.0%), and negative predictive value (100%) for the PAG test were similar for cows and heifers. The accuracy of the test was not different for dairy and beef cattle. In conclusion, the rapid ELISA pregnancy test based on PAG was highly sensitive and specific for pregnancy detection in dairy and beef cattle.

Fitness training for cardiorespiratory conditioning after traumatic brain injury.

BACKGROUND: Cardiorespiratory deconditioning is a common sequelae after traumatic brain injury (TBI). Clinically, fitness training is implemented to address this impairment, however this intervention has not been subject to rigorous review. OBJECTIVES: The primary objective was to evaluate whether fitness training improves cardiorespiratory fitness in people who have sustained a TBI. SEARCH STRATEGY: We searched ten electronic databases (Cochrane Injuries Group Trials Register; Cochrane Central Register of Controlled Trials (CENTRAL); EMBASE; PubMed (MEDLINE); CINAHL; AMED; SPORTDiscus; PsycINFO; PEDro and PsycBITE) and two clinical trials registers (TrialsCentral and Current Controlled Trials). The last search was August 2007. In addition we screened reference lists from included studies and contacted trialists to identify further studies. SELECTION CRITERIA: Randomised controlled studies with TBI participants were eligible if they compared an exercise programme incorporating cardiorespiratory fitness training to usual care, a non-exercise intervention or no intervention. DATA COLLECTION AND ANALYSIS: Two authors independently screened the search output, extracted data and assessed quality. All trialists were contacted for additional information. Mean difference and 95% confidence intervals (CI) were calculated for continuous data and risk difference or odds ratio and 95% CI were calculated for dichotomous data. Data were pooled when there were sufficient studies with clinical and statistical homogeneity. MAIN RESULTS: Six studies, incorporating 303 participants, were included. The participants were primarily males, in their mid thirties who had sustained a severe TBI. The studies were clinically diverse with regard to the interventions, time post-injury and the outcome measures used; therefore, the primary outcome could not be pooled. Three of the six studies indirectly assessed change in cardiorespiratory fitness after fitness training using the peak power output obtained during cycle ergometry (either at volitional fatigue or at a predetermined endpoint, that is, a percentage of predicted heart rate maximum). Cardiorespiratory fitness was improved after fitness training in one study (mean difference 59 watts, 95% CI 24 to 94), whilst there was no significant improvement in the other two studies. Four of the six studies had no drop-outs from their intervention group and no adverse events were reported in any study. AUTHORS' CONCLUSIONS: There is insufficient evidence to draw any definitive conclusions about the effects of fitness training on cardiorespiratory fitness. Whilst it appears to be a safe and accepted intervention for people with TBI, more adequately powered and well-designed studies are required to determine the effects across a range of outcome measures.

Reliability of bioimpedance spectroscopy and tonometry after breast conserving cancer treatment.

BACKGROUND: Measuring the female breast, especially after breast cancer treatment, is problematic due to breast size, texture, and patient positioning. However, being able to accurately measure changes in the breast is important, as it may help in the earlier diagnosis and treatment of early breast edema and later lymphedema. METHODS: 14 women who had undergone breast conserving surgery for breast cancer (> 12 months ago) were recruited to assess the between subject reproducibility of tonometry and bioimpedance spectroscopy (BIS). With the participant supine, two repeat measurements of the resistance of the tissues to compression (tonometry) and fluid levels (BIS) of the treated and normal breast were taken for each of the four quadrants of the breast. RESULTS: The between subject reproducibility for both measurement techniques was high, with covariance ranging from 1.29% to 3.25% for tonometry and 0.20-0.86% for BIS. CONCLUSIONS: The reliability of these two measurement techniques provides an opportunity for researchers and clinicians to easily quantify breast tissue and fluid changes which in turn may lead to the earlier diagnosis and targeted treatment of breast edema and lymphedema.

Endermologie (with and without compression bandaging)--a new treatment option for secondary arm lymphedema.

Two treatment protocols are presented using the LPG Endermologie system in combination with compression bandaging as a new treatment option for secondary arm lymphedema. Both protocols were applied 4 days a week for 4 weeks but differed in Trial II in time spent clearing the regions of the trunk adjacent to the swollen limb and the addition of a larger treatment head so that a greater area could be covered more quickly. The first protocol involved 24 women and the second involved 10 women. At the end of the treatment period, both protocols demonstrated overall reductions in limb volume (134mls; 18.3% p = 0.000 and 185mls; 28%, p = 0.002), limb fluid (182mls; 28%, p = 0.000 and 216mls; 33%. p = 0.014), truncal fluid (342mls; p = 0.002 and 290mls; p = 0.066), improvements in fibrotic induration in some lymphatic territories, and significant improvements in subject reporting of heaviness, tightness, tissue hardness and limb size. Trial II demonstrated additional benefits in terms of reduction in whole arm volume at 24 hours, improved fluid and arm volume reductions, and a significant improvement in subject reported arm range of movement. The additional time spent clearing the regions adjacent to the swollen limb in the second protocol appears to produce an increase in limb volume and limb fluid loss compared to the original treatment protocol.

Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo.

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the predominant isozyme of the Na(+)-pump that has previously been localized to the basolateral membranes of mammalian trophectoderm cells. The presence of multiple isoforms of Na/K-ATPase alpha- and beta-subunits at the time of cavitation suggests that there may be a degree of genetic redundancy amongst isoforms of the catalytic alpha-subunit that allows blastocyst formation to progress in the absence of the alpha1-subunit.

Treadmill training and body weight support for walking after stroke.

BACKGROUND: Treadmill training, with or without some body weight supported using a harness, is a method of treating walking after stroke. A systematic review is required to assess the cost, effectiveness, and acceptability of this treatment. OBJECTIVES: To assess the effectiveness of treadmill training and body weight support, individually or in combination, in the treatment of walking after stroke. The primary outcomes investigated were walking speed, endurance and dependency. SEARCH STRATEGY: We searched the Cochrane Stroke Group Trials Register (last searched 2 March 2005), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 4, 2004), MEDLINE (1966 to January 2005), EMBASE (1980 to February 2005), CINAHL (1982 to February 2005) and PEDro (last searched 2 March 2005). In addition, we handsearched relevant conference proceedings, screened reference lists and contacted trialists to identify further published and unpublished trials. SELECTION CRITERIA: Randomised or quasi-randomised controlled and cross-over trials of treadmill training and body weight support, individually or in combination, for the treatment of walking after stroke were eligible. DATA COLLECTION AND ANALYSIS: Two authors independently selected trials, extracted data, and assessed quality. We contacted trialists for additional information. We used a fixed-effect model for analysis, but if heterogeneity existed a random-effects model was used. We analysed the results as weighted mean differences (WMD) for continuous variables and relative risk (RR) for dichotomous variables. MAIN RESULTS: Fifteen trials (622 participants) were included. There were no statistically significant differences between treadmill training, with or without body weight support, and other interventions for walking speed or dependence. Among participants who could walk independently at the start of treatment, treadmill training with body weight support tended to produce higher walking speeds (WMD 0.09 m/s, 95% confidence interval (CI) -0.02 to 0.20 for speed; fixed-effect), but this result was not statistically significant. An individual trial tended to support the use of treadmill training with body weight support for dependent walkers as compared to treadmill training alone. One of three individual trials indicated that independent walkers may benefit from treadmill training combined with other task-orientated exercise. However, data are very limited. Adverse events occurred more frequently in participants receiving treadmill training but these were not judged to be clinically serious events. AUTHORS' CONCLUSIONS: Overall no statistically significant effect of treadmill training with or without body weight support was detected. Although individual studies suggested that treadmill training with body weight support may be more effective than treadmill training alone and that treadmill training plus task-oriented exercise may be more effective than sham exercises, further trials are required to confirm these findings.

The effect of gentle arm exercise and deep breathing on secondary arm lymphedema.

The aim of this study was to explore the benefits of gentle arm exercise combined with deep breathing for secondary arm lymphedema. 38 women participated in 10 minutes of standardized arm exercise and deep breathing and were measured every 10 minutes for 1 hour, then 24 hours and 1 week post regime. A smaller cohort of 24 women continued the 10 minute exercise regime morning and evening for 1 month, with measurements being repeated at the end of this time. Directly after performing the regime, there was a reduction in arm volume of 52 mls (5.8%), with the reduction being sustained at 30 minutes (50 mls, 5.3%). Even though participants were told not to further do the exercise, at 24 hours the volume reduction was 46 mls (4.3%) and at 1 week, 33 mls (3.5%). At the one month follow-up, the reduction was 101 mls (9.0%). All reductions were statistically significant. Reported arm heaviness and tightness also statistically significantly decreased directly after the regime with the reduction in tightness being sustained at 24 hours. The reduction in heaviness was sustained at 24 hours, 1 week, and even one month after the program. Perceived limb size was significantly reduced at 1 week and at the 1 month follow-up. There was also a significant improvement in the anterior thorax tonometry reading at the 1 month follow-up.

Mesenchymal stem cells: biology and potential clinical uses.

There has been an increasing interest in recent years in the stromal cell system functioning in the support of hematopoiesis. The stromal cell system has been proposed to consist of marrow mesenchymal stem cells that are capable of self-renewal and differentiation into various connective tissue lineages. Recent efforts demonstrated that the multiple mesenchymal lineages can be clonally derived from a single mesenchymal stem cell, supporting the proposed paradigm. Dexter demonstrated in 1982 that an adherent stromal-like culture was able to support maintenance of hematopoietic stem as well as early B lymphopoeisis. Recent data from in vitro models demonstrating the essential role of stromal support in hematopoiesis shaped the view that cell-cell interactions in the marrow microenvironment are critical for normal hematopoietic function and differentiation. Maintenance of the hematopoietic stem cell population has been used to increase the efficiency of hematopoietic stem cell gene transfer. High-dose chemotherapy and frequently cause stromal damage with resulting hematopoietic defects. Data from preclinical transplantation studies suggested that stromal cell infusions not only prevent the occurrence of graft failure, but they have an immunomodulatory effect. Preclinical and early clinical safety studies are paving the way for further applications of mesenchymal stem cells in the field of transplantation with respect to hematopoietic support, immunoregulation, and graft facilitation.

Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion.

OBJECTIVE: The human bone marrow contains mesenchymal stem cells capable of differentiating along multiple mesenchymal cell lineages. Using a non-human primate model, we sought to determine whether the systemic infusion of baboon-derived mesenchymal stem cells was associated with toxicity and whether these cells were capable of homing to and persisting within the bone marrow. MATERIALS AND METHODS: Five baboons (Papio anubis) were administered lethal irradiation followed by intravenous autologous hematopoietic progenitor cells combined with either autologous (n = 3) or allogeneic (n = 2) mesenchymal stem cells that had been expanded in culture. In four of these baboons, the mesenchymal stem cells were genetically modified with a retroviral vector encoding either the enhanced green fluorescent protein gene (n = 3) or the human placental alkaline phosphatase gene (n = 1) for tracking purposes. A sixth animal received only intravenous gene marked autologous mesenchymal stem cells but no hematopoietic stem cells or conditioning irradiation. RESULTS: Following culture, baboon mesenchymal stem cells appeared morphologically as a homogeneous population of spindle-shaped cells that were identified by the monoclonal antibodies SH-3 and SH-4. These cells did not express the hematopoietic markers CD34 or CD45. Baboon mesenchymal stem cells isolated from primary culture were capable of differentiating along both adipogenic and osteogenic lineages. There was no acute or chronic toxicity associated with the intravenous infusion of mesenchymal stem cells. In all five recipients of gene marked mesenchymal stem cells, transgene was detected in post-transplant bone marrow biopsies. In two animals receiving autologous mesenchymal stem cells, including the one non-conditioned recipient, transgene could be detected over 1 year following infusion. In one recipient of allogeneic gene marked mesenchymal stem cells, transgene was detected in the bone marrow at 76 days following infusion. CONCLUSION: These data demonstrate that baboon mesenchymal stem cells: 1) are not associated with significant toxicity when administered intravenously, 2) are capable of homing to the bone marrow following intravenous infusion, and 3) have the capacity to establish residence within the bone marrow for an extended duration following systemic administration.

Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo.

Human mesenchymal stem cells (MSCs) are capable of differentiating into multiple mesenchymal lineages including chondrocytes, osteocytes, adipocytes, and marrow stromal cells. Using a nonhuman primate model, we evaluated nonhuman primate MSCs as targets for gene therapy. Baboon MSCs (bMSCs) cultured from bone marrow aspirates appeared as a homogeneous population of spindle-shaped cells. bMSCs were capable of differentiating into adipocytes and osteocytes in vitro and chondrocytes in vivo. bMSCs were genetically modified with a bicistronic vector encoding the human erythropoietin (hEPO) gene and the green fluorescent protein (GFP) gene. Transduction efficiencies ranged from 72 to 99% after incubation of MSCs with retroviral supernatant. Transduced cells produced from 1.83 x 10(5) to 7.12 x 10(5) mIU of hEPO per 10(6) cells per 24 hr in vitro before implantation. To determine the capacity of bMSCs to express hEPO in vivo, transduced bMSCs were injected intramuscularly in NOD/SCID mice. In a separate experiment, transduced bMSCs were loaded into immunoisolatory devices (IIDs) and surgically implanted into either autologous or allogeneic baboon recipients. Human EPO was detected in the serum of NOD/SCID mice for up to 28 days and in the serum of five baboons for between 9 and 137 days. NOD/SCID mice experienced sharp rises in hematocrit after intramuscular injection of hEPO-transduced bMSCs. The baboon that expressed hEPO for 137 days experienced a statistically significant (p < 0.04) rise in its hematocrit. These data demonstrate that nonhuman primate MSCs can be engineered to deliver a secreted and biologically active gene product. Therefore, human MSCs may be an effective target for future human gene therapy trials.

Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach.

Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.

A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins.

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.

3-Acyl-4-hydroxyquinolin-2(1H)-ones. Systemically active anticonvulsants acting by antagonism at the glycine site of the N-methyl-D-aspartate receptor complex.

Most full antagonists at the glycine site of the NMDA receptor contain a carboxylic acid, which we believe to be detrimental to penetration of the blood-brain barrier. By consideration of a pharmacophore, novel antagonists at this site have been designed in which the anionic functionality is a vinylogous acid, in the form of a 4-hydroxyquinolin-2(1H)-one. In this series, a 3-substituent is necessary for binding, and correct manipulation of this group leads to compounds such as the 3-(3-hydroxyphenyl)propargyl ester 24 (L-701,273), with an IC50 for displacement of [3H]-L-689,560 binding of 0.17 microM and Kb against NMDA in the cortical slice of 1.39 microM. Compounds were tested for their ability to prevent audiogenic seizure in DBA/2 mice; the most potent compound in this series is the cyclopropyl ketone 42 (L-701,252), with an ED50 of 4.1 mg/kg ip. A model is proposed for binding to the glycine site, in which an important interaction is of a putative receptor cation with the pi-system of the 3-substituent.

2-Carboxytetrahydroquinolines. Conformational and stereochemical requirements for antagonism of the glycine site on the NMDA receptor.

2-Carboxy-1,2,3,4-tetrahydroquinoline derivatives, derived from kynurenic acid, have been synthesized and evaluated for in vitro antagonist activity at the glycine site on the NMDA receptor. 2,3-Dihydrokynurenic acids show reduced potency relative to the parent lead compounds (Table I) possibly as a result of conformational effects. Removal of the 4-oxo group results in further reduced potency, but introduction of a cis-carboxymethyl group to the 4-position restores antagonist activity (Tables III and IV). Replacement of the keto group of 5,7-dichloro-2,3-dihydrokynurenic acid with other alternative H-bonding groups, for example cis- and trans-benzyloxycarbonyl and cis- and trans-carboxamido (Table V), gives comparable activity, but there is negligible stereoselectivity. A significant increase in potency and stereoselectivity is seen within the 4-acetate series (Table VI). The trans-4-acetic acid is significantly more potent than the corresponding lead kynurenic acid and has 100-fold greater affinity than the cis isomer. The results are consistent with a requirement in binding for a pseudoequatorially placed 2-carboxylate and clearly demonstrate the importance for binding of a correctly positioned hydrogen-bond-accepting group at the 4-position. The high-affinity binding of an anionic group in the 4-substituent binding pocket suggests that the glycine site and the neurotransmitter recognition (NMDA) site may have some features in common.

4-Amido-2-carboxytetrahydroquinolines. Structure-activity relationships for antagonism at the glycine site of the NMDA receptor.

trans-2-Carboxy-5,7-dichloro-4-amidotetrahydroquinolines, evolved from the lead 5,7-dichlorokynurenic acid, have been synthesized and tested for in vitro antagonist activity at the glycine site on the N-methyl-D-aspartate (NMDA) receptor. Optimization of the 4-substituent has provided antagonists having nanomolar affinity, including the urea trans-2-carboxy-5,7-dichloro-4[[(phenylamino)carbonyl]amino]-1,2,3, 4-tetrahydroquinoline (35; IC50 = 7.4 nM vs [3H]glycine binding; Kb = 130 nM for block of NMDA responses in the rat cortical slice), which is one of the most potent NMDA antagonists yet found. The absolute stereochemical requirements for binding were found to be 2S,4R, showing that, in common with other glycine-site NMDA receptor ligands, the unnatural configuration at the alpha-amino acid center is required. The preferred conformation of the trans-2,4-disubstituted tetrahydroquinoline system, as shown by X-ray crystallography and 1H NMR studies, places the 2-carboxyl pseudoequatorial and the 4-substituent pseudoaxial. Modifications of the 4-amide show that bulky substituents are tolerated and reveal the critical importance for activity of correct positioning of the carbonyl group. The high affinity of trans-2-carboxy-5,7-dichloro-4-[1-(3-phenyl-2-oxoimidazolidinyl)]- 1,2,3,4-tetrahydroquinoline (55; IC50 = 6 nM) suggests that the Z,Z conformer of the phenyl urea moiety in 35 is recognized by the receptor. Molecular modeling studies show that the 4-carbonyl groups of the kynurenic acids, the tetrahydroquinolines, and related antagonists based on N-(chlorophenyl)glycine, can interact with a single putative H-bond donor on the receptor. The results allow the establishment of a three-dimensional pharmacophore of the glycine receptor antagonist site, incorporating a newly defined bulk tolerance/hydrophobic region.

Isoforms of Na,K-ATPase in rat lens epithelium and fiber cells.

PURPOSE: The lens epithelium is thought to conduct Na-K transport for the entire lens cell mass. Lens fibers have a poor ion transport capacity. The authors tested whether different Na,K-ATPase polypeptides are expressed in the two cell types and whether both cells have the machinery needed for ongoing Na,K-ATPase expression as judged by the presence of mRNA for the Na,K-ATPase alpha subunit. METHODS: Membranes were isolated from adult rat lens epithelium or fibers, and Western blot experiments were conducted for Na,K-ATPase alpha 1, alpha 2, and alpha 3 polypeptides. Total RNA was isolated from adult rat lens epithelium or fiber cells, and Northern analysis was conducted for Na,K-ATPase alpha 1, alpha 2, and alpha 3 mRNA. Some experiments were conducted using fiber cells from neonatal (3-day-old) rat lenses. RESULTS: Multiple isoforms of Na,K-ATPase were detected in adult rat lens epithelium. Judged by Northern blot band intensity, mRNA for Na,K-ATPase alpha 1 and alpha 2 was more abundant than for alpha 3 mRNA. By Western blot, Na,K-ATPase alpha 1, alpha 2, and alpha 3 polypeptides were observed as sharp bands at 100 to 108 kDa. In fiber cells, only Na,K-ATPase alpha 1 immunoreactive polypeptide was detected. Judged by immunoblot density, the amount of alpha 1 polypeptide was similar in both epithelium and fiber cell material. However, Na,K-ATPase alpha subunit mRNA was not found in adult lens fibers. To test whether Na,K-ATPase synthesis takes place during fiber cell growth, Northern blot analysis was conducted with RNA from neonatal (3-day-old) lens fibers; Na,K-ATPase alpha 1 mRNA was clearly visible. CONCLUSIONS: Adult rat lens epithelium expresses more than one isoform of Na,K-ATPase catalytic subunit, whereas only the alpha 1 isoform can be detected in fiber cells. In adult rat lens fiber cells, the observation of alpha 1 polypeptide, but no alpha 1 mRNA, suggests that ongoing alpha 1 synthesis is low. Based on the detection of alpha 1 mRNA in neonatal lens fibers, Na,K-ATPase synthesis by lens fibers may be higher during cell elongation and growth.

Na,K-ATPase polypeptide upregulation responses in lens epithelium.

PURPOSE: In a previous study, an increase in Na,K-ATPase alpha 2 expression was detected in the epithelium of porcine lenses exposed to amphotericin B, an ionophore that also increases lens sodium and stimulates active sodium transport. The purpose of the present study was to determine whether an increase of Na,K-ATPase alpha 2 synthesis is a response to an episode of rapid Na-K transport or whether the increase in lens sodium alone can initiate the response. METHODS: Western blot analyses were conducted to probe for Na,K-ATPase alpha polypeptides in membrane material isolated from porcine lens epithelium. Ouabain-sensitive adenosine triphosphate hydrolysis was used as an index of Na,K-ATPase activity, and lens ion content was determined by atomic absorption spectrophotometry. 86-Rubidium (86Rb) uptake was measured as an indicator for active potassium transport. RESULTS: 86Rb uptake was markedly diminished in lenses exposed to dihydro-ouabain (DHO), signifying inhibition of active sodium-potassium transport. Consistent with this, the sodium content of DHO-treated lenses increased. By western blot analysis, a marked increase of Na,K-ATPase alpha 2 polypeptide could be detected in the epithelium of DHO-treated lenses. To rule out the possibility that apparent stimulation of Na,K-ATPase alpha 2 synthesis stemmed from binding of DHO to Na,K-ATPase sites, experiments were conducted to confirm an increase of Na,K-ATPase alpha 2 polypeptide in the epithelium of lenses exposed to low-potassium medium to inhibit active sodium-potassium transport. Consistent with the apparent increase of Na,K-ATPase polypeptide, Na,K-ATPase activity was detectably increased in epithelial material isolated from lenses pretreated with DHO or low-potassium medium. CONCLUSIONS: An increase in Na,K-ATPase alpha 2 polypeptide can occur in the epithelium of lenses subjected to an episode of sodium pump inhibition. This suggests the response could be triggered by an increase in cell sodium and does not necessarily require a period of stimulated active sodium-potassium transport.