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PubMed1996

Fertilization after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa.

OBJECTIVE: To assess the possibility of cryopreserving testicular tissue extracted sperm for intracytoplasmic sperm injection (ICSI). DESIGN: A report of two cases. Our study was approved by the Ethical Committee at the Instituto Valenciano de Infertilidad. SETTING: In vitro fertilization program at the Instituto Valenciano de Infertilidad. PATIENTS: Two azoospermic patients with severe spermatogenic failure but with focal spermatogenesis on testicular biopsies. In both cases, a first ICSI attempt with fresh testicular biopsy extracted sperm was unsuccessful. INTERVENTIONS: Cryopreservation of testicular spermatozoa in 100-micro L "pills." Intracytoplasmic sperm injection with thawed testicular spermatozoa. MAIN OUTCOME MEASUREMENTS: Fertilization rate, cleavage rate, embryo quality, clinical pregnancy. RESULTS: Fertilization rates were 36 percent and 100 percent after ICSI with fresh testicular spermatozoa, and 63 percent and 57 percent after ICSI with cryopreserved testicular sperm. In both cases, cleavage rates and embryo quality were similar when using fresh and cryopreserved testicular spermatozoa. No clinical pregnancies were achieved. CONCLUSION: High fertilization rates can be obtained after ICSI with frozen-thawed testicular tissue extracted spermatozoa. Cryopreservation of testicular sperm may avoid repetition of testicular biopsies in azoospermic patients in whom the only source of spermatozoa is the testicle.

Adult↗
PubMed2003

The cellular origins of the linear IgA disease target antigens: an indirect immunofluorescence study using cultured human keratinocytes and fibroblasts.

BACKGROUND: Linear IgA disease (LAD) is an IgA-mediated subepidermal immunobullous disease of adults and children, with heterogeneous immunopathology. Objectives To investigate to what extent the cellular origins of the target antigens account for the heterogeneity of the immune response in LAD. METHODS: Forty-nine adult and 33 childhood LAD sera were studied. Immunofluorescence was carried out to determine the expression of the LAD antigens by normal human keratinocytes, fibroblasts and mixed cultures of keratinocytes and fibroblasts. Immunoblotting was performed to determine the localization of the LAD target antigens in tissue extracts (48 adult and 31 childhood sera) and cell extracts (21 adult and 10 childhood sera). RESULTS: Thirty-one adult and 13 childhood LAD sera bound proteins expressed by human keratinocytes; of these sera, 15 adult and four childhood LAD sera also recognized proteins expressed by fibroblasts. A single adult serum was positive on fibroblasts alone. Seventeen adult and 20 childhood sera were negative on both cell types. There was a modest increase (9%) in the detection of the IgA autoantibodies on keratinocytes and fibroblasts grown together in mixed culture. Immunoblotting showed that the LAD target antigens could be detected in cell as well as in tissue extracts. CONCLUSIONS: Our results have shown that normal human keratinocytes and fibroblasts in culture express the LAD target antigens. LAD sera (with a single exception) bound antigens expressed by keratinocytes alone or by both keratinocytes and fibroblasts. The principal pattern of expression in keratinocytes was cytoplasmic, similar to that demonstrated by polyclonal antibodies to the 180-kDa bullous pemphigoid antigen (BP180). This reflects the pivotal role of BP180 in LAD. The finding that LAD antigens are expressed by both human keratinocytes and fibroblasts in culture may explain the heterogeneity of the target antigens, and may be a contributory factor in the immunopathology of the disease.

Adolescent↗
PubMed1999

Nitric oxide synthase expression in the opossum superior colliculus: a histochemical, immunohistochemical and biochemical study.

The expression of neuronal nitric oxide synthase (nNOS) in the superior colliculus (SC) of the opossum Didelphis marsupialis was studied by NADPH diaphorase (NADPH-d) histochemistry and nNOS immunohistochemistry. In addition, the activity of nNOS was quantified by measurement of [(3)H]-L-arginine conversion to [(3)H]-L-citrulline in tissue extracts from SC superficial layers in opossums and rats. Our results show that the number of NADPH-d stained cells was small and virtually identical in stratum opticum (SO) and stratum griseum superficiale (SGS) and their staining was very light, particularly in SGS. Neuropil staining was heavier in the stratum zonale (SZ) than in SGS or SO. The intermediate and deep layers contained heavily stained cells and moderate neuropil staining. Surprisingly, nNOS-immunoreactive cells were far more numerous than NADPH-d+ cells in every layer. The production of [(3)H]-L-citrulline from [(3)H]-L-arginine in tissue extracts enriched in superficial layers indicated that nNOS specific activity is as high in the opossum as in the rat. Our results suggest that the location of nNOS-expressing neurons in retino-receptive layers may be related to inter-specific differences in the processing of visual information.

Animals↗
PubMed1979

Determination of enzyme activity by chromatography and videodensitometry. I. Microassay of amino acid transforming enzymes in human tissue homogenates.

A chromatographic-videodensitometric assay was found to be appropriate for measuring the activity of glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, ornithine-2-oxoacid aminotransferase and histidine ammonia-lyase in human tissue homogenates. From the assay mixtures containing substrate(s), cofactor(s), buffer and tissue extract, five or ten microliters samples were taken at different time intervals and chromatographed on Dowex 50 X 8 type resin-coated chromatosheets. On each chromatoplate 50 nmoles of the amino acid to be measured were separately run as a reference for videodensitometric evaluation. By comparing the density of the reference amino acid to that of the individual samples the molar amount of amino acids formed or consumed in the reaction could be calculated. The present findings suggest that the chromatographic-videodensitometric microassay (CV-technique) is suitable for measuring the activity of amino acid transforming enzymes in minute amounts of tissue extracts.

Adult↗
PubMed1991

Identification of creatine kinase isoenzymes in the guinea-pig. Presence of mitochondrial creatine kinase in smooth muscle.

Isoenzymes of creatine kinase (CK, EC 2.7.3.2) in guinea-pig smooth, cardiac and skeletal muscles as well as in brain were analyzed by cellulose acetate electrophoresis and FPLC gel permeation chromatography. In crude tissue extracts of smooth muscles brain type BB-CK and the hybrid form MB-CK were detected, but in enriched mitochondrial fractions from different guinea-pig smooth muscles, mitochondrial type Mi-CK was unambiguously identified. Smooth muscle Mi-CK displayed the same electrophoretic mobility as Mi-CK from brain, which migrates slower than cardiac Mi-CK. Identical to parallel experiments with Mi-CK from cardiac muscle and brain, smooth muscle Mi-CK could be resolved into dimeric and octameric species, the latter being remarkably stable. In contrast to guinea-pig smooth muscles, Mi-CK was not detected in chicken gizzard tissue extracts nor in enriched mitochondrial fractions thereof. The presence of Mi-CK, predominantly in octameric form, in guinea-pig smooth muscles, but not in chicken gizzard, may represent a clue for the different physiological properties of these muscles and may provide the molecular basis for the dependence of the PCr production on oxidative metabolism observed in the guinea-pig taenia caeci.

Animals↗
PubMed1978

Distribution and homologies of subunits in the LDH isoenzyme pattern of urodeles.

1. The subunit distribution and homologies of LDH isoenzymes are investigated in the species Triturus vulgaris, cristatus, alpestris and helveticus, and Ambystoma mexicanum by means of immunoprecipitation and starch gel electrophoresis. 2. Fresh tissue extracts contain the following patterns: (a) Trit. vulgaris--Two isoenzymes: the M4 and H4 tetramers. No hybrid formation is observed between H and M subunits. (b) Trit. cristatus--Two isoenzymes: the M4 and H4 tetramers. Occasional hybrid formation between H and M subunits takes place. (c) Trit. alpestris--Six isoenzymes: the M4, H4, H3H', H2H'2, HH'3 and H'4 tetramers. No hybrid formation between the M and the H and H' subunits is detected. (d) Trit. helveticus--Six isoenzymes: the M4, H4, H3H', H2H'2, HH'3, and H'4 tetramers; the H' subunit is more positively charged than the M subunit, which leads to pattern reversal in heart and skeletal muscle tissues. No hybrid formation between the M and the H and H' subunits is observed. (e) Ambyst. mex.--Eleven isoenzymes in heart, eye and brain, six isoenzymes in all other tissues tested. The presence of two M subunits, which form hybrids with the H subunit in a restricted way, is suggested. 3. In tissue extracts of the tested species the tendency of all present LDH subunits to form hybrids with each other without restriction is increased after prolonged storage at 2 degrees C. 4. The most acidic of the major isoenzymes (LDH1) in Trit. vulgaris, cristatus and helveticus tends to split into a series of sub-bands which migrate faster to the anode than the original main band.

Ambystoma↗
PubMed1993

Retinoic acid enhances fibrinolytic activity in-vivo by enhancing tissue type plasminogen activator (t-PA) activity and inhibits venous thrombosis.

We studied the profibrinolytic effect and the anti-thrombotic potential of retinoic acid in-vivo. Male Wistar rats were treated with retinoic acid either acutely or twice daily for a period of 5 consecutive days in a dose range of 0.01 to 3.0 mg/kg. Fibrinolytic activity was measured ex-vivo using the diluted blood clot lysis test and net t-PA activity in tissue extracts was measured in a spectrophotometric rate assay. Clot lysis was dose dependently increased by retinoic acid up to about 170% (relative to vehicle treated rats) at a dose of 3 mg/kg. No tachyphylaxis could be detected. Ex-vivo measured fibrinolytic activity after single administration of 1 mg/kg of retinoic acid peaked at 3 h after ingestion. Even after 18 h a significantly higher clot lysis rate was measured. Lysis of blood clots from vehicle and retinoic acid treated rats could be completely blocked by addition of tranexamic acid or antibodies against rat t-PA before clot formation. t-PA activity in plasma was slightly increased after retinoic acid treatment; no effects were measured on plasma PAI-1, u-PA, plasminogen, and alpha 2-antiplasmin levels. t-PA activity in lung and kidney was marginally enhanced by retinoic acid but in heart and aortic tissue extracts t-PA activity was increased by about 50%. We confirmed this potential anti-thrombotic property in an in-vivo venous thrombosis model. A reduced clot size was observed after retinoic acid administration (35% reduction at a dose of 1 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells.

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 microg ml(-1) blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 microg ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 microg ml(-1). After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 microg ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.

Ascorbate Oxidase↗
PubMed2003

Human kallikrein 8, a novel biomarker for ovarian carcinoma.

Human kallikrein 8 (hK8; neuropsin) is a serine protease and new member of the hK family. The aim of this study was to examine if hK8 may serve as a novel cancer biomarker. An hk8-ELISA, developed in-house, was used to study the distribution of hK8 in various biological fluids and tissue extracts from healthy individuals and ovarian cancer patients of different stages of the disease (International Federation of Obstetrics and Gynecology II-IV). For ovarian cancer patients, very high levels in ascites fluid were observed (<or=1000 microg/liter; n = 85 samples). Elevated serum levels were seen in 24 of 40 (62%) of ovarian cancer patients. Higher ascites fluid hK8 concentration was associated with better ovarian cancer progression-free survival (P = 0.02). In both serum and ascites fluid, there is a significant correlation between hK8 and CA125 concentration (r = 0.51 and 0.58, respectively). The serum concentration of hK8 was an indicator of progression on regression on longitudinal monitoring of an ovarian cancer patient. These data suggest that hK8 protein is detectable in ovarian cancer tissue extracts, serum, and ascites fluid, indicating that it may serve as a new ovarian cancer marker.

Ascitic Fluid↗
PubMed1993

In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries.

We studied the ability of adenoviral vectors to achieve gene transfer into injured arteries. A recombinant adenoviral vector expressing a nuclear-targeted beta-galactosidase gene was constructed and infused into balloon-injured rat carotid arteries. Three days after gene transfer, recombinant gene expression was assessed quantitatively by (1) measuring beta-galactosidase antigen and activity in tissue extracts and (2) histochemical staining and counting of cells expressing beta-galactosidase. Exposure of injured carotid arteries to increasing concentrations of the vector (10(8) to 10(10) plaque-forming units per milliliter) resulted in a dose-responsive increase in beta-galactosidase expression, with peak expression of approximately 43 mU or 25 ng beta-galactosidase per vessel. Microscopic examination of histochemically stained arteries demonstrated gene transfer limited to the vascular media; transduced cells were identified immunohistochemically as smooth muscle cells. Counting of both histochemically stained and total nuclei in the media revealed that approximately 30% of the cells in the media of the injured vessels were transduced. Calculations based on both counting cells and on the level of beta-galactosidase expression in tissue extracts suggested the presence of 5000 to 10,000 transduced cells per 10 mm of vessel. Arteries infused with either vehicle only, a control adenoviral vector, or liposomes combined with the vector plasmid contained little or no evidence of beta-galactosidase expression. High levels of in vivo beta-galactosidase expression persisted for at least 7 days after gene transfer but declined significantly by day 14. We conclude that adenoviral vector-mediated gene transfer into the injured rat carotid artery results in efficient gene transfer into the vascular media, with levels of recombinant protein production significantly higher than any previously reported in arterial gene transfer studies. Adenoviral vectors appear to be particularly useful agents for in vivo arterial gene transfer.

Adenoviridae↗
PubMed1990

Determination of dexamethasone in bovine tissues by coupled-column normal-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry.

A new method for the determination of dexamethasone in bovine liver and muscle tissues has been developed. Crude tissue extracts were obtained by means of a three-phase liquid-liquid extraction scheme. The resulting residue was subjected to coupled-column normal-phase high-performance liquid chromatography which served to isolate the drug for the purpose of screening and quantification. Sample was injected onto the first column of the system, a phenyl column, from which a heart-cut was diverted to a short silica column which retained dexamethasone. The contents of this column were backflushed onto a cyanopropyl column which isolated dexamethasone. Mobile phases consisted of hexane modified with 2-propanol, acetic acid, and water. Analysis of each sample was completed in 15 min. Quantitation was performed by external standard calibration of ultraviolet response at 239 nm. Limits of detection were estimated to be 4 and 6 ppb in muscle and liver, respectively. In addition to screening and quantitation, the coupled-column system purified tissue extracts for gas chromatographic-mass spectrometric analysis which, in the selected-ion monitoring mode, confirmed the identity of the trimethylsilyl-enol-trimethylsilyl derivative of dexamethasone.

Animals↗
PubMed2000

New ELISA for quantitation of human urokinase receptor (CD87) in cancer.

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1), and its receptor (uPAR; CD87) facilitate cancer cell invasion and metastasis. Whereas uPA and PAI-1 antigen levels determined in tumor tissue extracts of breast cancer patients correlate with disease recurrence and overall survival, the prognostic relevance of uPAR is still a matter of debate. We established two new sandwich-type enzyme-linked immunosorbent assay (ELISA) formats (HU/IIIF10-ELISA and HU/HD13-ELISA) using the epitope-defined monoclonal antibody (mAb) IIIF10 or the conformation-dependent mAb HD13.1, a polyclonal chicken antibody (HU277), and recombinant soluble uPAR (CHO-suPAR) as the standard. The lower detection limit of the assays was at 0.16 ng/ml, with a linear dose-response up to 5 ng/ml of uPAR antigen. Both ELISA formats showed good reproducibility and recovery. The intra-assay and the inter-assay variation coefficients were respectively 4.3% and 11.7% (HU/IIIF10-ELISA) and 4.0% and 10.7% (HU/HD13-ELISA). The recovery rate of uPAR in cell lysates spiked with CHO-suPAR was above 82% and 88%, respectively. With these new ELISA formats, uPAR antigen content in breast cancer tissue extracts and tumor cell lysates was determined and compared to a commercially available ELISA (ADI-ELISA). By all of the three uPAR ELISA formats CHO-suPAR and uPAR present in lysates of non-malignant epithelial cells and stimulated monocytes were quantified with similar sensitivity. Interestingly, in breast cancer cell lines of epitheloid origin a higher uPAR antigen content was determined by the HU/IIIF10-ELISA than the HU/HD13- or ADI-ELISA formats. In lysates of fibroblastic breast cancer cell lines similar uPAR values were obtained with the HU/IIIF10- and ADI-ELISA formats, whereas with the HU/HD13-ELISA significantly lower uPAR concentrations were determined. The prognostic relevance of tumor uPAR antigen was evaluated in 199 primary breast cancer patients with a median follow-up of 24 months. uPAR antigen values above the cut-off of 3.33 ng/mg protein as determined by the HU/IIIF10-ELISA were significantly correlated with short disease-free survival (p=0.025). Results obtained by the other two ELISA formats (HU/HD13-ELISA and ADI-ELISA) were not associated with prognosis. Our findings stress the need of well-characterized antibodies, which detect both uPAR of non-malignant and tumor cells, in setting up a uPAR-ELISA useful for assessing breast cancer patient prognosis.

Animals↗
PubMed1988

Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein.

A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development.

Acid Phosphatase↗
PubMed1990

Measurement of myocardial glycogen synthesis in diabetic and fasted rats.

Diabetes and fasting provoke an increase in heart glycogen content, despite a decline in the amount of active glycogen synthase present. To determine if the activity of glycogen synthase i is still rate limiting for glycogen synthesis, we used 13C-nuclear magnetic resonance to measure the in vivo rate of glycogen synthesis and compared this with the activity of glycogen synthase and phosphorylase measured in tissue extracts using physiological concentrations of substrates and activators. In the basal state the activity of glycogen synthase i was depressed in the diabetic and fasted hearts (P less than 0.01). The rate of heart glycogen synthesis was measured during a 50-min infusion of D-[1-13C]-glucose (10 mg/min) and insulin (1 U/min) and averaged 0.32 +/- 0.04 mumol.min-1.g wet wt-1 in controls and was diminished in both the diabetic (0.18 +/- 0.04 mumol.min-1.g wet wt-1) and fasted (0.16 +/- 0.03 mumol.min-1.g wet wt-1) and fasted (0.16 +/- 0.03 mumol.min-1.g wet wt-1) rats (P less than 0.05 for each). During the glucose and insulin infusion the average activity of glycogen synthase i was greater in control than diabetic or fasted hearts (P less than 0.01 for each) and approximated the rates of net glycogen synthesis in each group. In contrast, there were no significant differences in phosphorylase alpha activity, measured in tissue extracts, among the three groups. Furthermore, although this phosphorylase alpha activity greatly exceeded synthase activity, it did not appear to be expressed in vivo. We conclude that in normal, diabetic, and fasted rats, glycogen synthase is rate limiting for glycogen synthesis.

Animals↗
PubMed1984

In vivo and in vitro binding of platinum to metallothionein.

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammineplatinum(II]. Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl2 significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl2 pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.

Animals↗
PubMed2005

Plasma cardiotrophin-1 is elevated in human hypertension and stimulated by ventricular stretch.

OBJECTIVE: Cardiotrophin-1 (CT-1) is an interleukin-6-related cytokine with known hypertrophic and protective actions upon cardiac myocytes. We provide here the first report of cardiac tissue and plasma levels of CT-1 in human and experimental hypertension, demonstrate cardiac CT-1 secretion stimulated by ventricular stretch, and characterise molecular forms of CT-1 in tissue and plasma. METHODS: CT-1 levels in human and rat plasma and in rat cardiac tissue extracts were determined by specific radioimmunoassay (RIA). Cardiac CT-1 secretion during ventricular stretch was studied in isolated, perfused hearts. Molecular forms of CT-1 were identified using RIA coupled with high performance liquid chromatography (HPLC). Results are given as mean+/-SEM. RESULTS: Plasma levels of CT-1 in patients with untreated hypertension (UTH, 606+/-18 pmol/L, n=24) were significantly higher than those in age-and BMI-matched normotensive volunteers (NT, 546+/-12 pmol/L, n=31, P<0.01 vs. UTH). CT-1 levels in matched patients with treated hypertension (THT, 618+/-10 pmol/L, n=35) were similar to those in UTH patients, but higher than in NT controls (P<0.01). Plasma CT-1 demonstrated a weak but significant correlation with systolic blood pressure in all patients (r=0.241, P<0.05, n=90). In contrast, CT-1 levels in male, 40-week-old, NT-WKY rats (1295+/-98 pmol/L) were significantly higher than those in matched UTH-SHR (937+/-31 pmol/L, P<0.01). In both WKY and SHR rats, atrial tissue concentrations of CT-1 were 8-fold higher than ventricular levels. Left ventricular tissue CT-1 protein concentrations were significantly higher in 40-week-old SHR compared with age-matched WKY (SHR 12.6+/-0.5 fmol/g vs. WKY 9.5+/-0.8 fmol/g, P<0.01). Ventricular stretch of Langendorff perfused, isolated WKY/SHR hearts resulted in significant, acute release of CT-1 and BNP. HPLC coupled with specific RIA revealed CT-1 in human/rat plasma, isolated rat heart perfusate, and rat heart tissue extracts to consist of complex, high molecular weight forms. CONCLUSIONS: This is the first report to show increased levels of plasma CT-1 in hypertensive disease. CT-1 is a unique cardiac cytokine whose release is stimulated by ventricular stretch. The atrium contains the highest levels of the protein. The stored and circulating molecular form of CT-1 is complex, which may modulate its in vivo role in cardiovascular disease.

Aged↗
PubMed1991

Expression of parathyroid hormone-related protein in abnormal human parathyroids.

The expression of parathyroid hormone-related protein (PTHrP) in abnormal human parathyroids was investigated. Northern blot analysis of RNA extracted from human benign parathyroid adenomata (n = 4) revealed multiple PTHrP mRNA species ranging in size from 1.8 to 4 kb. The relative abundance of PTHrP mRNA expressed in two of the adenomata was similar to that of a tumour (DAF) associated with humoral hypercalcaemia of malignancy, whereas PTHrP mRNA was of low abundance in a third and was undetectable in the fourth. PTHrP-like immunoreactivity was detected in extracts of abnormal parathyroid tissue (benign adenoma (n = 7), hyperplasia (n = 5) and parathyroid carcinoma (n = 2] using a sensitive specific two-site immunoradiometric assay for human (h) PTHrP(1-86) and a radioimmunoassay for hPTHrP(1-34). Ratios of hPTHrP(1-86)- and hPTHrP(1-34)-like immunoreactivities relative to hPTH(1-84)-like immunoreactivity in the parathyroid tissue extracts were, on average, less than 1%. PTHrP bioactivity in the extracts could not be distinguished from that of PTH, by an osteosarcoma cell bioassay. We conclude that, despite reports of over-expression of PTHrP mRNA in parathyroid adenomata, the potential contribution of PTHrP to the total PTH-like activity of adenomata and other abnormal parathyroid tissue may be insignificant relative to PTH.

Adenoma↗
PubMed1993

Immunofluorescent analysis of plasma fibronectin incorporation into the lung during acute inflammatory vascular injury.

Incorporation of plasma fibronectin into tissues is believed to influence endothelial cell-cell interaction, as well as endothelial cell adhesion to matrix. We used immunofluorescent microscopy coupled with tissue extraction of noncovalently incorporated fibronectin to delineate the time course for matrix incorporation of soluble plasma-derived fibronectin into the lung of sheep during postoperative bacteremia. Adult sheep were surgically prepared with both lung and peripheral lymph fistulas. Sheep were anesthetized 2 days following surgery and injected intravenously with a sublethal dose of live Pseudomonas aeruginosa, which consisted of 5 x 10(8) live organisms suspended in 0.9% saline. Bacterial infusion elicited a 300% increase in lung transvascular protein clearance but no increase in peripheral transvascular protein clearance. Purified dimeric human plasma fibronectin (hFn), used as an "immunologic marker," was then infused intravenously (100 mg/sheep) into two additional groups of sheep (nonbacteremic control group and bacteremic experimental group) and allowed to mix with the plasma pool of endogenous soluble sheep fibronectin (sFn). Incorporation of the plasma-derived hFn into the lung matrix and its distribution in relation to endogenous sheep fibronectin in the matrix was assessed by dual-label immunofluorescence using antibodies specific to either sFn or hFn. Human fibronectin from the vascular compartment codistributed with endogenous sheep fibronectin in the lung matrix. Moreover, its deposition into the lung was markedly increased in postoperative bacteremic sheep compared with nonbacteremic control sheep. Increased hFn deposition in the lung with bacteremia was clearly apparent within 2 h. The hFn deposited in the lung was nonextractable using a heparin-urea tissue extraction buffer, suggesting its rapid covalent cross-linking and incorporation into the lung matrix. Microscopic analysis of serial lung biopsies revealed focal areas of inflammation with an intense mononuclear infiltrate into the lungs by 2 h in the bacteremic sheep. Interstitial edema and vascular endothelial injury were observed by 4 h, with alveolar edema apparent over 6 to 8 h. Thus, postoperative bacteremia results in a rapid incorporation of plasma fibronectin into the lung matrix. This may be a physiologic mechanisms to stabilize the integrity of the lung vascular barrier.

Animals↗