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cDNA cloning and differential gene expression of three catalases in pumpkin.

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.

Amino Acid Sequence↗

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse.

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca2+ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.

Adolescent↗

Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners.

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase delta assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.

Cells, Cultured↗

Delayed endocrine pancreas graft function after simultaneous pancreas-kidney transplantation. Incidence, risk factors, and impact on long-term outcome.

BACKGROUND: The incidence of delayed endocrine pancreas graft function and its impact on long-term outcome after simultaneous pancreas-kidney transplantation are unknown. METHODS: We studied 54 technically successful adult type I insulin-dependent diabetic recipients of cadaver, whole organ, bladder-drained simultaneous pancreas-kidney transplants (mean age, 37.6 years; 65% male, 35% female; 9% pancreas retransplants; 63% on chronic pretransplant dialysis; mean duration of diabetes, 25.1 years). Insulin was administered during the first 2 weeks after transplantation, as needed, to keep blood glucose < 150 mg/dl. Delayed endocrine pancreas graft function was defined as total, cumulative insulin requirement of > 30 U between day 5 and day 10, and/or > 15 U between day 11 and 15. Quadruple immunosuppression was used for all recipients. RESULTS: The incidence of delayed endocrine pancreas graft function was 69%. By univariate analysis, delayed endocrine graft function was associated with pretransplant recipient weight > 80 kg (P = 0.04), donor age > 45 years (P = 0.02), and cardiocerebrovascular (P = 0.06) and nontraumatic causes of donor death (P = 0.02). The incidence of acute pancreas rejection episodes was similar for recipients without and with delayed endocrine pancreas graft function. Pancreas graft survival at 1 and 3 years was 94% and 82% without versus 76% and 59% with delayed endocrine graft function (P = 0.03). CONCLUSIONS: Increased pancreas graft failure after delayed endocrine function was a consequence of insufficient functional reserve (e.g., older donors) rather than increased immunogenicity. Pretransplant reduction of recipient weight and careful donor selection are therefore crucial in order to decrease the incidence of delayed endocrine pancreas graft function and its negative impact on long-term outcome.

Adult↗

Ethnic differences in donation-related characteristics among potential hematopoietic stem cell donors.

BACKGROUND: Although the National Marrow Donor Program has been highly successful at recruiting ethnic minorities as potential hematopoietic stem cell donors, there have been no systematic investigations of whether donor characteristics that might be linked to the donation experience vary by ethnicity. METHODS: Questionnaires assessing four domains-demographic, volunteer-related, general psychosocial, and donation-related-were mailed to potential donors after they were contacted as a preliminary match for a patient and had agreed to donate. In all, 1,679 potential donors completed and returned a predonation questionnaire. Data from potential donors belonging to five major ethnic groups were analyzed; white, black, Asian/Pacific Islander, Hispanic, and Native American. RESULTS: Bivariate analyses indicated that virtually all factors in the four domains were associated with ethnicity. Direct discriminant function analysis identified three significant functions. The most striking of the three functions indicated that Asian Americans were more highly educated, more ambivalent (reluctant about donation), more concerned (medical, work/family), and more anxious and depressed than all other ethnic groups. Key differences among other ethnic group members were also identified. CONCLUSIONS: This study provides the first evidence of ethnic group differences in key predonation variables. Findings suggest that Asian/Pacific Islanders possess a number of characteristics that are known psychosocial risk factors for less positive postdonation outcomes and that more intensive pre and postdonation contact with this group may be necessary. Strategies for improving future research in this area are discussed.

Ethnicity↗

Delusional misidentification syndromes: a descriptive study.

Twenty-three patients with one or more delusional misidentification syndromes were studied. The majority of the subjects were females and the Capgras syndrome was the most common delusional misidentification syndrome in our sample. The Capgras syndrome cases were significant older than the Fregoli syndrome cases. Schizophrenia and schizo-affective psychosis were significantly associated with Fregoli syndrome. Discriminant function analysis was carried out on the sample and showed that age and symptoms of nuclear schizophrenia were the variables most predictive of the type of delusional misidentification. These findings are discussed in the light of current literature.

Adult↗

Early Reperfusion in Basilar Artery Occlusion Stroke Managed With Tenecteplase Versus Alteplase Before Endovascular Treatment.

BACKGROUND: Timely reperfusion is a critical determinant of favorable outcomes in basilar artery occlusion (BAO) stroke. We aimed to examine whether the choice of thrombolytic agent predicts early reperfusion (ER) in BAO stroke managed with tenecteplase versus alteplase before endovascular treatment. METHODS: This was a retrospective, multicenter US cohort of consecutive patients with BAO from 14 stroke centers treated with tenecteplase or alteplase within 4.5 hours of last known well before endovascular treatment. The primary end point was ER, defined as angiographic ER (expanded Thrombolysis in Cerebral Infarction grade 2b to 3 on the first diagnostic angiogram), or clinical ER, defined as substantial neurological improvement precluding endovascular treatment and a good functional outcome (modified Rankin Scale score 0-3). RESULTS: Among 163 patients with BAO and a median last known well-to-needle time of 135 minutes (interquartile range, 100-193) and last known well-to-puncture time of 228 minutes (interquartile range, 166-307), ER was observed in 27 (16.6%) patients. Rates of ER were comparable between tenecteplase (14/75, 18.7%) and alteplase (13/88, 14.8%; adjusted odds ratio, 1.082 [95% CI, 0.444-2.631]; P=0.862). In addition, rates of angiographic ER and clinical ER subgroups did not differ between thrombolytic agents. Higher Basilar Artery on Computed Tomography Angiography scores and a nonatherothrombotic cause independently predicted ER. In multivariable analysis, a good functional outcome was associated with younger age, lower stroke burden, and shorter last known well-to-puncture time. CONCLUSIONS: In BAO treated within 4.5 hours of last known well, tenecteplase and alteplase produced comparable early reperfusion rates. Achieving ER did not modify the association between thrombolytic agent and good functional outcome, consistent with rapid thrombectomy in patients with no ER.

basilar artery↗

Blockade of late-phase airway responses and airway hyperresponsiveness in allergic sheep with a small-molecule peptide inhibitor of VLA-4.

The leukocyte integrin very late antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) is an adhesion receptor predominantly expressed on lymphocytes, monocytes, and eosinophils, but not on neutrophils. Recent studies with monoclonal antibodies against VLA-4 suggest that antigen-induced late responses and airway hyperresponsiveness (AHR) may depend on the recruitment and/or activation of VLA-4-expressing leukocytes. To further test this hypothesis, we administered by aerosol either a potent small-molecule inhibitor of VLA-4, which prevents VLA-4-mediated binding to fibronectin (CS-1 ligand mimic), or an inactive control (30 mg twice daily for 3 d, and on the fourth day 0.5 h before and 4 h after antigen challenge) to six sheep with airway hypersensitivity to Ascaris suum antigen. Treatment with the small-molecule VLA-4 inhibitor resulted in a significant decrease in the early antigen-induced bronchial response (40%, p < 0.05), and almost complete blockade of the late-phase airway response (88%, p < 0.05). Moreover, at 24 h after antigen challenge, AHR to inhaled carbachol was not observed when the animals were dosed with the small-molecule VLA-4 inhibitor. In accord with protection against the functional abnormalities associated with antigen challenge, analysis of biopsy specimens taken 24 h after challenge indicated that the total numbers of VLA-4-positive cells (lymphocytes, eosinophils, and metachromatic-staining cells) in the group treated with the VLA-4 inhibitor did not increase, whereas these cells increased in the control group. The active agent, but not the inactive control, significantly blocked macrophage adherence to fibronectin (FN), indicating that the CS-1 ligand interfered with VLA-4-mediated adhesion in sheep cells. These results support our previous findings with a monoclonal antibody to VLA-4, and demonstrate that a small-molecule VLA-4 inhibitor, when given by aerosol, has a protective effect against antigen-induced late responses and AHR in allergic sheep.

Animals↗

The long-term results of the Liverpool Mark II knee prosthesis.

We have reviewed 105 Liverpool Mark II knee replacements in 71 patients to assess survival and long-term results. Assessment was both clinical and radiological, using a modification of the British Orthopaedic Association knee function assessment chart, and analysis was by the survivorship method as advocated by Tew and Waugh. The follow-up period was between 13 and 113 months with 42 prostheses being in situ for over six years. Eight knees (7.6%) have been revised or arthrodesed because of infection or loosening, giving a cumulative success rate of 89% after seven years based on prosthesis survival alone. Of the remaining knees, 71.1% were either free of pain or caused only minimal pain. The cumulative success rate as judged by the stricter criteria of the prosthesis being in situ and causing little or no pain suggested a 50% survival between 73 and 96 months. Most patients (77.3%) were enthusiastic or satisfied with their results. Complications included deep wound infection (8.6%), and loosening which needed further surgery (11.4%).

Consumer Behavior↗

A novel and functional interaction between cyclophilin A and prolactin receptor.

Precedent data have revealed that peptidyl isomerases can modulate the function of cell-surface receptors, but no such interactions have been previously shown for the members of the cytokine receptor superfamily. We demonstrate here that a functional interaction occurs between the prolactin receptor (PRLR) and peptidyl prolyl cis/trans isomerase cyclophilin A (CypA). CypA was co-immunoprecipitated with the PRLR in vivo from the breast epithelial cell line T47D and Chinese hamster ovary transfectants overexpressing transfected human PRLR. In addition, in vitro binding assays demonstrated a direct interaction of CypA with the PRLR, in the presence or absence of cyclosporine. Co-immunoprecipitation studies also showed an association of CypA with Jak2. Functional analysis revealed that overexpression of CypA inhibited PRL-induced Rac activation, while simultaneously prolonging Jak2 phosphorylation. These proximal actions had profound downstream effects: CypA overexpression significantly enhanced the basal and PRL-stimulated expression from a beta-casein reporter construct. Hence, the interaction between CypA and the PRLR plays a differential regulatory role in the various signaling pathways leading from the PRLR.

Animals↗

Investigation of the association between the presence of cytoplasmic residues on the human sperm midpiece and defective sperm function.

Defective sperm function has been identified as one of the most common causes of human infertility. The aim of this investigation was to identify whether the presence of retained cytoplasm on the human sperm midpiece is associated with defective sperm function. Statistical analysis of data demonstrated a strong negative correlation between the presence of residual cytoplasm on the midpiece of spermatozoa in the inseminate and fertilization rate during IVF. Significant negative correlations were also identified between the percentage of spermatozoa in the ejaculate bearing cytoplasmic residues and (i) spermatozoa having membrane integrity and (ii) sperm concentration. A highly significant positive correlation was also revealed between the percentage of spermatozoa in the ejaculate with membrane integrity and the percentage of motile spermatozoa. These correlations suggest that retained cytoplasm is a cause of subfertility. Measurements of the percentage of spermatozoa bearing residual cytoplasm in the IVF inseminate could provide the basis for a simple predictive test before IVF.

Cytoplasm↗

A His-155 to Tyr polymorphism confers gain-of-function to the human P2X7 receptor of human leukemic lymphocytes.

The P2X(7)R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X(7)R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X(7)R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X(7)R function was monitored by measuring ATP-induced Ca(2+) influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X(7) allelic variant. Ca(2+) influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca(2+) flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X(7)R confirmed an increased ATP-dependent activation of the P2X(7) 489T mutant with respect to the wild type receptor, as assessed by both by [Ca(2+)](i) influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X(7)R.

Adenosine Triphosphate↗

Cyclophosphamide pulse regimen in the treatment of alveolitis in systemic sclerosis.

OBJECTIVE: To evaluate, in a pilot study, the efficacy of a short term cyclophosphamide (CYC) pulse regimen on alveolitis in a cohort of patients with systemic sclerosis (SSc). METHODS: Twenty-three patients with SSc (17 diffuse SSc and 6 limited SSc) were selected in 5 centers in Italy, based on the findings of an abnormal bronchoalveolar lavage (BAL) cell analysis in association with altered pulmonary function tests (PFT) or recent deterioration in flow volume curve (FVC). Patients were also evaluated by skin score (Rodnan), esophageal manometry and barium swallow radiography, and electrocardiography and 2-mode echocardiography. The pre-enrolment pulmonary evaluation and after 6 months of therapy included evaluation of the clinical status, PFT (FVC, FEV1, DLCO), BAL. standard chest radiograph, and chest high resolution computed tomography. All patients received i.v. CYC (1000 mg/m2 of body surface monthly for 6 mo) and oral prednisone (25 mg daily for the first month and subsequently 5 mg daily of maintenance dosage for the remaining 5 mo). A complete blood count and urinalysis were obtained at monthly intervals. RESULTS: After 6 months of therapy the values for FVC did not change significantly. Individually, 8 of 23 patients showed an improvement (> 15% increase) in FVC after 6 months, while FVC in 13 cases remained stable. Only 2 patients had an important decline in FVC after 6 months of therapy (17 and 24% decrease). Improvement in DLCO was noted in 15 of 23 patients after 6 months of therapy. Four patients were stable and 4 patients had a worsened DLCO at the end of the study. After therapy the mean value of BAL fluid recovery did not change. There was a reduction in total cell number although this value did not reach statistical significance. The levels of neutrophils, eosinophils, lymphocytes, and macrophages did not change significantly. Scans for patients with grades 1, 2, and 3 did not differ significantly after 6 months of therapy, and 14 patients were stable. Changes in appearance, in relation to changes in extent of disease, were seen in 8 patients and consisted of an extension of reticular pattern and transformation from grade 1 to 2 (6/8 patients). All patients showed a ground-glass appearance indicating an acute alveolitis. Improvement in ground-glass was noted in 10 of 23 patients after 6 mo therapy. At the end of the study, 8 patients were stable and 5 patients had a diffusion of the ground-glass to other segments. No side effects were experienced during the treatment except for mild nausea in 4 patients; no patients discontinued therapy during the study. CONCLUSION: CYC pulse regimen seems to stabilize alveolitis in the majority of cases. The association of CYC pulsed modality with prednisone may be useful in SSc patients to control disease evolution in the lung.

Adult↗

[Transthoracic and transesophageal comparative echocardiography in mitral valve prolapse].

OBJECTIVE: Transthoracic and transesophageal comparative analysis of functional and morphological abnormalities associated to idiopathic mitral valve prolapse (MVP). DESIGN: Prospective study. SETTING: Outpatients with MVP diagnosis referred to echocardiographic laboratory of Cardiology Institute in Madrid, Spain. MATERIAL AND METHODS: In each case we analyzed by TTE and TEE, anterior, posterior and double localization of MVP, number of prolapsed mitral leaflets/patient, total area of MVP to mitral valve plane, mitral annulus diameter, total area and spatial distribution of mitral regurgitation. RESULTS: TEE diagnosed a greater number of prolapsed mitral leaflets and a greater percentage of double (80%) MVP. MPV area by TEE (50 +/- 31 mm2) was considerably larger (96 +/- 30 mm2) than TTE MVP area (50 +/- 31 mm3. Associated mitral valve regurgitation area calculated through TEE was larger (558 +/- 502 mm2) than the same parameter evaluated by TTE (450 +/- 515 mm2). CONCLUSIONS: TEE is an efficient technique in MVP non invasive diagnosis and particularly sensitive to posterior MVP. Our data could be helpful in MVP cases scheduled for mitral valve repairment.

Adolescent↗

[Relationship between the activity of the kallikrein, plasmin and thrombin systems of the blood during intense physical exertion].

The authors studied the changes in the level of precursors of kallikrein, plasmine, and their inhibitors with the aid of a complex method. Blood plasma of sportsmen was examined--of healthy persons and of those with the syndrome of myocardial overstraining under conditions of rest and intensive bicycle ergometric exercises. The results obtained and correlation analysis data pointed to the functional association of the "Hageman's factor system". Sportsmen with the overstrain syndrome displayed an aggravation of the functional possibilities of humoral systems of the hemovascular regulation, this being expressed in a reduced level of the proenzymatic activity and a reduction of the inhibitor values. Correlation of the mentioned indices was disturbed both at rest and during the exercises.

Adult↗

A study of loneliness among a national sample of Swedish elderly.

This study examined: 1) the loneliness experienced by a representative sample of persons from Sweden in 1986, and 2) the relative impact of age, gender, household size, subjective health assessments, and two indicators of income adequacy on loneliness. The results are based on the responses to a telephone survey, conducted by SIFO, of a weighted subsample of 212 persons aged 65+. The overall sample consisted of 1005 person 16 years of age and older. In general, only a minority (27%) of the Swedish elderly indicated loneliness was at least a somewhat serious problem. Cross-tabular, and discriminant function analysis showed that loneliness was associated with being older, being alone, and poor self-reported health. These findings are compared to those of other Swedish and American studies. Additionally, implications with regard to Swedish policy are suggested.

Aged↗

Chronic granulomatous disease. Molecular genetics.

Chronic granulomatous disease is an inherited disorder of microbial killing characterized by the failure of phagocytic cells to produce superoxide due to a lesion in a membrane-associated NADPH-oxidase. The components of the oxidase have been incompletely characterized and, therefore, a genetic approach has been used to identify the gene affected in the common X-linked form of CGD without reference to a specific protein product. The X-CGD gene was first mapped to Xp21.1. A phagocyte-specific RNA transcript derived from Xp21 was identified and shown to be deficient (or disrupted) in patients with X-CGD. Antisera directed toward the predicted protein product of the X-CGD gene have established its identity as a 90-kD membrane glycoprotein and a component of the phagocyte cytochrome b, recently purified as a heterodimer of a 90-kD species and a 22-kD polypeptide. The more recent genetic and biochemical findings now provide an explanation for the consistent absence of the phagocyte cytochrome b spectrum in X-CGD (now termed "X- -CGD"). Both subunits of the cytochrome b heterodimer are absent in X- -CGD, despite a genetic deficiency of only the larger polypeptide, which indicates that a complete understanding of cytochrome biosynthesis and function will require further characterization of the small subunit. We should anticipate that identification of other functionally associated proteins will aid in analysis of the phagocyte oxidase. Molecular reagents prepared from the cloned X-CGD cDNA or gene may prove to be clinically useful in prenatal diagnosis and may provide a basis for somatic gene therapy in the future.

Amino Acid Sequence↗

Determinants of no-show appointment behavior: the utility of multivariate analysis.

A multitude of determinants have been identified as predictive of broken appointments. The majority of prior studies have been limited to univariate analysis of the relationship between predictors and appointment keeping behavior. The present report studied 25 independent predictors of no-show behavior using both univariate and multivariate analyses. A total of 579 kept appointments and 84 failed appointments were analyzed. Results of univariate analysis indicated the following significant relationships with appointment behavior: age, ethnicity, marital status, mode of payment, chronic illness, telephone in house, type of care, prior visits to center, cost of care, transportation to center, physician ethnicity, and linguistic capability. However, multiple logistic function analysis revealed only six significant associations: type of care, chronic illness, linguistic capability, mode of payment, physician-patient sex differences, and marital status of the patient. Multivariate analysis may yield a more accurate and clinically useful model of no-show behavior. For example, language barrier may be more of a problem than the race of the patient. Prospective studies might benefit from these considerations.

Appointments and Schedules↗