Concentrations In Tissue Extracts Research Articles

NT113, a pan-ERBB inhibitor with high brain penetrance, inhibits the growth of glioblastoma xenografts with EGFR amplification.

This report describes results from our analysis of the activity and biodistribution of a novel pan-ERBB inhibitor, NT113, when used in treating mice with intracranial glioblastoma (GBM) xenografts. Approaches used in this investigation include: bioluminescence imaging (BLI) for monitoring intracranial tumor growth and response to therapy; determination of survival benefit from treatment; analysis of tumor IHC reactivity for indication of treatment effect on proliferation and apoptotic response; Western blot analysis for determination of effects of treatment on ERBB and ERBB signaling mediator activation; and high-performance liquid chromatography for determination of NT113 concentration in tissue extracts from animals receiving oral administration of inhibitor. Our results show that NT113 is active against GBM xenografts in which wild-type EGFR or EGFRvIII is highly expressed. In experiments including lapatinib and/or erlotinib, NT113 treatment was associated with the most substantial improvement in survival, as well as the most substantial tumor growth inhibition, as indicated by BLI and IHC results. Western blot analysis results indicated that NT113 has inhibitory activity, both in vivo and in vitro, on ERBB family member phosphorylation, as well as on the phosphorylation of downstream signaling mediator Akt. Results from the analysis of animal tissues revealed significantly higher NT113 normal brain-to-plasma and intracranial tumor-to-plasma ratios for NT113, relative to erlotinib, indicating superior NT113 partitioning to intracranial tissue compartments. These data provide a strong rationale for the clinical investigation of NT113, a novel ERBB inhibitor, in treating patients with GBM.

CCR5 Signaling Suppresses Inflammation and Reduces Adverse Remodeling of the Infarcted Heart, Mediating Recruitment of Regulatory T Cells

Myocardial infarction triggers an inflammatory reaction that is involved in cardiac remodeling. Cardiac repair is dependent on regulatory mechanisms that suppress inflammation and prevent excessive matrix degradation. Chemokine induction in the infarcted heart mediates recruitment of leukocyte subsets with distinct properties. We demonstrate that signaling through the CC chemokine receptor 5 (CCR5) prevents uncontrolled postinfarction inflammation and protects from adverse remodeling by recruiting suppressive mononuclear cells. CCR5 and its ligands macrophage inflammatory protein (MIP)−1α and MIP-1β were markedly induced in the infarcted mouse myocardium. In addition, almost 40% of the mononuclear cells infiltrating the infarct expressed CCR5. CCR5−/− mice exhibited marked upregulation of proinflammatory cytokine and chemokine expression in the infarct. In wild-type infarcts CCR5+ mononuclear cells had anti-inflammatory properties, expressing higher levels of IL-10 than CCR5− cells. In contrast, mononuclear cells isolated from CCR5−/− infarcts had reduced IL-10 expression. Moreover, enhanced inflammation in the absence of CCR5 was associated with impaired recruitment of CD4+/foxp3+ regulatory T cells (Tregs). The CCR5+ Treg subset exhibited increased IL-10 expression, reflecting potent anti-inflammatory activity. Accentuated inflammation in CCR5−/− infarcts was associated with increased matrix metalloproteinase (MMP) expression, reduced TIMP levels, and enhanced MMP-2 and MMP-9 activity, resulting in worse cardiac dilation. These results suggest that CCR5-mediated Treg recruitment may restrain postinfarction inflammation, preventing excessive matrix degradation and attenuating adverse remodeling.

Heterologous Expression of Dehydroascorbate Reductase from Rice and Its Application to Determination of Dehydroascorbate Concentrations

We constructed an expression vector for rice dehydroascorbate reductase (DHAR) (EC 1.8.5.4) with a polyhistidine tag at the amino terminus and introduced the vector into several strains of Escherichia coli. On conventional induction treatment with isopropylthiol-beta-D-galactoside, E. coli harboring rice DHAR cDNA produced doublet polypeptides of about 27 kDa. Induction duration or growth temperature did not affect the ratio of these polypeptides. Only the larger polypeptide, corresponding to full-length recombinant DHAR, was produced in E. coli supplemented with tRNAs for several minor codons. Most of the enzymatic characteristics of the recombinant DHAR were similar to those of the native one, although the recombinant protein showed increased heat susceptibility. Using recombinant DHAR, we developed a method for simple and precise determination of dehydroascorbate concentrations in tissue extracts by spectrophotometry, and we successfully applied the method to several fruit juices and vegetables.

A Functional Analysis of Mouse Models of Cardiac Disease through Metabolic Profiling

Since the completion of the human and mouse genomes, the focus in mammalian biology has been on assessing gene function. Tools are needed for assessing the phenotypes of the many mouse models that are now being generated, where genes have been "knocked out," "knocked in," or mutated, so that gene expression can be understood in its biological context. Metabolic profiling of cardiac tissue through high resolution NMR spectroscopy in conjunction with multivariate statistics has been used to classify mouse models of cardiac disease. The data sets included metabolic profiles from mouse models of Duchenne muscular dystrophy, two models of cardiac arrhythmia, and one of cardiac hypertrophy. The metabolic profiles demonstrate that the strain background is an important component of the global metabolic phenotype of a mouse, providing insight into how a given gene deletion may result in very different responses in diverse populations. Despite these differences associated with strain, multivariate statistics were capable of separating each mouse model from its control strain, demonstrating that metabolic profiles could be generated for each disease. Thus, this approach is a rapid method of phenotyping mouse models of disease.

Defining a metabolic phenotype in the brain of a transgenic mouse model of spinocerebellar ataxia 3.

Many of the spinocerebellar ataxias (SCAs) are caused by expansions of CAG trinucleotide repeats encoding abnormal stretches of polyglutamine. SCA3 or Machado-Joseph disease (MJD) is the commonest dominant inherited ataxia disease, with pathological phenotypes apparent with a CAG triplet repeat length of 61-84. In this study a mouse model of SCA3 has been examined which was produced using a human yeast artificial chromosome containing the MJD gene with a CAG triplet expansion of 84 repeats. These mice have previously been shown to possess a mild progressive cerebellar deficit. NMR-based metabolomics/metabonomics in conjunction with multivariate pattern recognition identified a number of metabolic perturbations in SCA3 mice. These changes included a consistent increase in glutamine concentration in tissue extracts of the cerebellum and cerebrum and spectra obtained from intact tissue using magic angle spinning (1)H-NMR spectroscopy. Furthermore, these profiles demonstrated metabolic abnormalities were present in the cerebrum, a region not previously implicated in SCA3. As well as an increase in glutamine both brain regions demonstrated decreases in GABA, choline, phosphocholine and lactate (representing the summation of lactate in vivo, and postmortem glycolysis of glucose and glycogen). The metabolic changes are discussed in terms of the formation of neuronal intranuclear inclusions associated with SCA3. This study suggests high-resolution (1)H-NMR spectroscopy coupled with pattern recognition may provide a rapid method for assessing the phenotype of animal models of human disease.

Substance P in the Gastrointestinal Tract of Non-Obese Diabetic Mice

Background: Gut dysmotility occurs in most diabetics. Substance P is a neurotransmitter that plays an important role in regulating gastrointestinal motility. The present investigation was carried out to evaluate the possible role of this neurotransmitter in the pathogenesis of dysmotility in diabetics. Methods: Pre-diabetic and diabetic female non-obese diabetic (NOD) mice aged 22-24 weeks were studied. As controls, BALB/CJ mice of the same age and sex were used. Substance P concentrations in tissue extracts from the antrum, duodenum, and colon were determined with radioimmunoassay. Substance P-immunoreactive nerve elements and endocrine cells were identified by immunocytochemistry and quantified with computerized image analysis. Results: Substance P levels in the antrum of both pre-diabetic and diabetic NOD mice were significantly lower than those of controls. In the duodenum and colon substance P levels were higher than those of the controls in both pre-diabetics and diabetic NOD mice. The relative volume density of substance P-immunoreactive nerve fibres in the colon of diabetic NOD mice was significantly decreased. There was no statistically significant difference between pre-diabetic and diabetic NOD mice and controls with regard to the relative volume density of substance P immunoreactive nerve fibres in the antrum and duodenum. In the antrum the number of substance P-immunoreactive cells decreased significantly in both pre-diabetic and diabetic NOD mice. In the duodenum and colon the numbers of these cells in NOD mice did not differ from those of controls. Conclusions: The changes in substance P contents in various parts of the gastrointestinal tract of NOD mice seem to be primary to the onset of diabetes. The decreased antral substance P contents in NOD mice seems to be caused by structural change in the mucosal endocrine cells. In the small and large intestine the increase in substance P levels appears to be caused by change in the physiologic activities of the nerve element and/or endocrine cells rather than by structure changes. The abnormalities observed here in an animal model for diabetes type I might have relevance for the gastrointestinal dysmotility displayed in human diabetes.

Mucosal tenascin C content in inflammatory and neoplastic diseases of the large bowel.

Tenascin C is a glycoprotein of the extracellular matrix. It is upregulated during embryologic development, wound healing, and under conditions of normal and neoplastic growth. Most available data on tenascin C expression in tissues is based on immunohistologic studies. The present study was designed to quantify tissue concentrations in patients with inflammatory and neoplastic diseases of the large bowel. Fifty patients with ulcerative colitis, 19 patients suffering from familiar adenomatous polyposis without malignant transformation, and 69 patients with colorectal carcinoma were investigated. Tenascin C concentrations in tissue extracts were determined by semiquantitative Western blotting. The tenascin C tissue concentration of normal mucosa was 2.6 +/- 3.4 microg/mg (n = 55), 2.9 +/- 2.1 microg/mg in colorectal adenomas (n = 19), 7.5 +/- 4.7 microg/mg in ulcerative colitis (n = 50), and 18 +/- 15 microg/mg in colorectal carcinomas (n = 69; mean +/- standard deviation). In ulcerative colitis, the mucosal tenascin C content correlated with histopathologic disease activity. No differences were found between subgroups of adenomas or carcinomas. Tenascin C tissue concentrations were not altered in adenomas, slightly elevated in ulcerative colitis, and substantially increased in colorectal carcinomas. Although less useful as a diagnostic parameter, tenascin C tissue levels serve as an instrument for assessing the activity of stromal remodeling in large-bowel diseases generally. Specifically, they may reflect disease activity in ulcerative colitis.

Measurement of Total and Bioactive Interleukin-2 in Tissue Samples by Immunoaffinity-Receptor Affinity Chromatography

The detection and measurement of cytokines is an important issue in the clinico-pathological diagnosis of several clinical entities, including organ transplant rejection. Existing techniques, although sensitive, measure only total cytokine concentrations and cannot measure bioactivity. A chromatographic system combining immunoaffinity chromatography with an immobilized receptor detection cartridge has been developed for measuring total and bioactive interleukin (IL)-2 concentrations in tissue extracts prepared from biopsy materials taken from renal transplant recipients during both rejection and drug-induced nephrotoxic episodes. The technique employs a short high-pressure chromatography column packed with antibody-coated glass beads for the initial analyte separation and concentration, followed by detection of bioactive molecules through their interactions with specific, immobilized receptors. This system compares favourably with both conventional bioassays and immunoassays for measuring IL-2 in tissue samples.

Neuropeptide Y in the human prenatal and mature gonads

In the present paper we have examined the distribution and identity of neuropeptide Y (NPY) immunoreactivity in the prenatal and mature human gonads. Tissue specimens were obtained from prenatal gonads (gestational age < 42 weeks) of induced abortions, premature birth and stillbirths. Specimens were also taken from infants who died aged less than 4 days. Tissue from mature gonads was obtained during surgery. The distribution and concentration in tissue extracts were determined by immunohistochemistry and radioimmunoassay. NPY-immunoreactivity (NPY-IR) was demonstrated in both the prenatal and mature human gonads. In the gonads from fetuses at 16–25 weeks the NPY-IR was scarce and could only be demonstrated by radioimmunoassay. In the female gonads from fetuses at 40 weeks of gestation, distinct nerve fibres were detected in relation to blood vessels and primordial follicles. In the prenatal male gonads at the same gestational length as well as in the mature testis, the NPY-IR nerves were detected in relation to blood vessels and seminiferous tubules. The gel filtration profile from the prenatal gonads and from the mature ovary revealed a single peak corresponding to the elution position of bioactive synthetic human NPY. The gel filtration profile from the mature testis demonstrated an additional peak at K d of 0.17. The presence of NPY-IR in relation to the developing and mature gonads suggests that NPY participates in gonadal functions from an early point in life.

Overexpression of group II phospholipase A2 in human breast cancer tissues is closely associated with their malignant potency

Membrane-associated phospholipase A2 (M-PLA2) is an enzyme that hydrolyses the sn-2 fatty acyl ester bond of phosphoglycerides. We measured M-PLA2 concentration in tissue extracts from 325 human breast cancers using a specific radioimmunoassay recently developed. Correlation analyses between the tissue concentration of M-PLA2 and clinicopathological factors showed that the enzyme level was significantly higher in patients with distant metastasis than in those without. In addition, M-PLA2 concentration was significantly higher in scirrhous carcinoma than in other histological types. No significant association was found between M-PLA2 concentration and age, menstrual status, tumour size, histological grade, vessel involvement or oestrogen receptor (ER) and progesterone receptor (PR) status. The expression of M-PLA2 mRNA was examined in a fibroadenoma, a stage IV breast cancer and its metastatic site of skin. Northern blot analysis showed a clear hybridisation band corresponding to M-PLA2 mRNA in both primary breast cancer and its metastatic site, while the fibroadenoma expressed a faint band corresponding to M-PLA2 mRNA. Breast cancer patients with high M-PLA2 concentrations exhibited significantly shorter disease-free and overall survival than those with low M-PLA2 concentration at the cut-off point of 5 ng 100 mg-1 protein, which was determined in a separate study. In multivariate analysis, M-PLA2 was found to be an independent prognostic factor for disease recurrence and death in human breast cancer. The possible significance of M-PLA2 expression in human breast cancer tissue is discussed.

Autoimmunity to collagen in adult periodontal disease: immunoglobulin classes in sera and tissue.

The immunoglobulin class distribution of antibody to human collagen type I has been examined in sera and gingival extracts from patients with adult chronic periodontitis. Tissue extracts were made either by simple washing or ultrasonication. With either method, IgG and IgA antibodies to collagen were present in higher concentration in tissue extracts than in autologous serum when adjustment was made for dilution differences. No significant differences were found for IgM antibodies. Antibodies to human collagen type I are usually "natural antibodies" of the IgM class and, therefore, our findings suggest a class switch to IgG in inflamed gingivae, presumably due to prolonged antigenic stimulation.

Measurement of the concentration of amphotericin B in brain tissue of scrapie-infected hamsters with a simple and sensitive method

A simple, sensitive, and reproducible assay for the measurement of the amphotericin B concentration in tissue extracts was developed by using the fourth derivative of the absorption spectrum of amphotericin B between wavelengths of 330 and 430 nm. The amphotericin B concentration in spleen and brain was proportional to the total amount administered. The amphotericin B concentration in the brain was highly correlated with the increase in the mean incubation period of intracerebrally scrapie-infected hamsters.

Isolation of digoxin-like immunoreactive factors from mammalian adrenal cortex

Endogenous digoxin-like immunoreactive factors (DLIF) are present in serum and tissues of humans and animals. To date, a tissue source for these factors has not been rigorously defined nor have these factors been isolated to identifiable homogeneity. In this study, we define the distribution of DLIF in mammalian tissues, demonstrate the adrenal cortex to be the principal source of this factor in bovine, and isolate DLIF to chromatographic homogeneity using high performance liquid chromatography (HPLC). DLIF concentrations in tissue extracts from rats measured as follows: adrenal glands, 44.3; serum, 6.3; liver, 5.2; kidney, 1.2; heart, brain, or lungs, less than 1.4 ng of digoxin-equivalent per g of protein. Human tissues showed similar results. In dogs, the ratio of the DLIF concentration in lumbar vein serum to that in infrarenal inferior vena cava serum was 3.3 +/- 0.4 (mean +/- S.E., n = 4). Bovine adrenal cortex contained 7 times more DLIF per g of tissue than the adrenal medulla. 70 +/- 4% (n = 7) of the total bovine cortical DLIF activity (6,159 pg of digoxin-equivalent) applied to a reverse phase HPLC column eluted as one definitive fraction. 60% of the digoxin-like immunoreactivity extracted from bovine serum also co-eluted with DLIF from adrenal. None of the 14 steroid molecules or 7 cardiac glycoside congeners co-eluted with the major DLIF activity. Our data indicate that 947 pmol of DLIF is equivalent to 1 pmol of digoxin-equivalent immunoreactivity. Preliminary mass spectral analysis suggests that purified DLIF has a molecular mass of 780 daltons comprised of one 390-dalton aglycone component plus several sugar moieties. This study establishes a definitive link between DLIF in serum and the adrenal cortex as a source tissue. We also demonstrate a method for purifying DLIF to chromatographic homogeneity with an extraction capacity of 1.2 nmol of DLIF per g of adrenal cortex.

Heterogeneity of [3H]inositol 1,4,5-trisphosphate binding sites in adrenal-cortical membranes. Characterization and validation of a radioreceptor assay

1. The characterization of a radioreceptor assay for determining Ins(1,4,5)P3 concentration in tissue extracts is described which utilizes the binding of [3H]Ins(1,4,5)P3 to an adrenal-cortex membrane fraction. 2. Analysis of [3H]Ins(1,4,5)P3 binding by isotope dilution demonstrated an apparent single population of binding sites (KD 3.65 +/- 0.18 nM, Bmax. 872 +/- 70 fmol/mg of protein). Specific binding of [3H]Ins(1,4,5)P3 was enhanced at alkaline pH values (maximum at pH 8.5), with complete loss of specific binding at pH less than 6. These binding sites displayed strict stereo- and positional specificity for Ins(1,4,5)P3, with L-Ins(1,4,5)P3, Ins(1,3,4)P3 and DL-Ins(1,3,4,5)P4 causing 50% displacement of specific [3H]Ins(1,4,5)P3 binding (IC50 values) at concentrations of 14 +/- 3 microM, 3.0 +/- 0.3 microM and 0.53 +/- 0.03 microM respectively. 3. Kinetic analysis of binding data, however, revealed a high-affinity [3H]Ins(1,4,5)P3 binding site (KD 0.052 nM) in addition to the lower-affinity site (KD 2.53 nM) already demonstrated in displacement studies. 4. It is shown that the presence of the high-affinity site can be exploited to increase the sensitivity of the [3H]Ins(1,4,5)P3 radioreceptor assay, allowing accurate detection of 20 fmol of Ins(1,4,5)P3 in 300 microliters of tissue extract. 5. Further validation of the specificity of the above assay for Ins(1,4,5)P3 was provided by incubating tissue extracts with either a 5-phosphatase or 3-kinase preparation. It was shown that identical loss occurred of both Ins(1,4,5)P3 mass and [3H]Ins(1,4,5)P3, added to parallel incubations. 6. The ability of the assay to measure basal and agonist-stimulated increases in Ins(1,4,5)P3 concentration has been demonstrated with rat cerebral cortex and bovine tracheal smooth-muscle slices and a range of cultured and isolated cell preparations.

Occurrence and distribution of neuropeptides in the human skin. An immunocytochemical and immunochemical study on normal skin and blister fluid from inflamed skin

The content and distribution of substance P (SP), somatostatin, vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) in human skin were investigated. Radioimmunoassay was performed on pooled tissue samples from several regions (fingers, toes, axillas and thighs) and on tissue fluid from spontaneous blisters on inflamed skin. Immunocytochemical localization showed all peptides examined except somatostatin to be present in nerve fibers. Nerve fibers storing SP and CGRP, which were found to coexist, were mostly present as free nerve endings in the superficial part of dermis and in epidermis. SP/CGRP fibers were most abundant in fingers and toes. VIP fibers and NPY fibers were localized in the deeper parts of dermis around blood vessels and acini of sweat glands. Also fibers containing these neuropeptides were most common in fingertips and toes. VIP occurred in relatively high amounts also in skin from axilla whereas NPY in this region was below detection limit. Immunoreactive somatostatin was found in low concentrations in tissue extracts and was not present in amounts sufficient for reliable immunostaining. Fluid from spontaneous blisters on inflamed skin contained detectable amounts of all neuropeptides.

Low molecular weight FSH binding inhibitor in bovine testis.

Purification of low Mr (less than 5000) inhibitors of FSH binding to receptors has been hampered by their low concentration in tissue extracts and physiologic fluids. The calf testis represents an accessible and economic source of large quantities of tissue, and was therefore studied as a source of FSH binding inhibitors. Supernatants (27,000 X g) of calf testis homogenates inhibited binding of [125I]-hFSH as well as [125I]-hCG to membrane-bound receptor from the same source. FSH binding inhibitor was concentrated from large volumes of testis supernatant by precipitation of inert material with metaphosphoric acid, concentration/desalting of the resulting supernatant by ultrafiltration (Amicon UM-05 membranes) and lyophilization. Separation of FSH binding inhibitor and LH(hCG) binding inhibitor activities could be achieved by molecular sieving on Sephadex G-25. A partially purified fraction with inhibitors of FSH binding activity (ED50 = 44 micrograms protein) and free of LH(hCG) binding inhibitor activity (no activity at 800 micrograms protein) emerged with a Ve/Vo of 1.9, reflecting an apparent Mr of about 1500. Inhibitors of LH(hCG) binding activity emerged with the column outer volume. Rechromatography of the FSH binding inhibitor fraction on G-25 indicated two closely associated peaks of activity. These could be further resolved by gel filtration through BioGel P2, to give a salt-free fraction with an FSH binding inhibitor activity (ED50) of 24 micrograms protein. The inhibitor was heat-labile, losing 80% of its activity after 2 hours at 100 C. The testicular low molecular weight FSH binding inhibitor is similar to bovine follicular fluid and serum FSH binding inhibitor by several parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

34] Monoclonal antibodies against PGH synthase: An immunoradiometric assay for quantitating the enzyme

The observations from several laboratories indicate that changes in PGH synthase levels can play a role in regulating the rates of prostaglandin formation in both normal and pathological situations. There are a number of methods for assaying PGH synthase enzyme activity including polarographic assays, use of radioactive fatty acid substrates and measurement of labeled products, and the use of unlabeled fatty acids and measurement of products by radioimmunoassays. There does, however, exist a need for methods to quantitate changes in enzyme protein levels. This chapter describes the preparation of four different monoclonal antibodies against the PGH synthase and the use of iodinated monoclonal antibodies for quantitating PGH synthase protein concentrations in tissue extracts by immunoradiometric assay. This immunoradiometric assay is 103-104 times as sensitive as the most accurate and sensitive polarographic assay for enzyme activity.

The effect of dimethyl 3,3'-dithiobispropionimidate on the adenylate cyclase activity of bovine corpus luteum; stimulation of particulate enzyme activity and the stabilization of activity to dispersion in detergent [proceedings

In a previous report (Young, 1979) it was shown that dimethyl 3,3'-dithiobispropionimidate inhibited p[NHlppG-stimulated adenylate cyclase of the washed 600g sediment of bovine corpus-luteum homogenate in a dose-dependent manner at concentrations above 0.02m~. This initial series of experiments did not show any statistically signscant effect of dimethyl 3,3'-dithiobispropionimidate concentrations below 0.02 m~ on the activity of the particulate enzyme, although some individual experiments indicated a slight enhancement of activity by 0.0 1 mwdimethyl 3,3'-dithiobispropionimidate. The accumulation of more data has revealed a small, but statistically significant, enhancement of the particulate enzyme activity by dimethyl 3,3'-dithiobispropionimidate at concentrations around 0.0 1 mM, and we now report the biphasic effect of dimethyl 3,3'-dithiobispropionimidate on p[NHlppG-stimulated adenylate cyclase activity. The washed 600g sediment of bovine corpus-luteum homogenate was prepared as described previously (Young & Stansfield, 1977). The adenylate cyclase of the 600g sediment was activated by incubation with 0.1 m~-p{NH]ppG in 4 0 m ~ Tris/HCl (pH7.5)/6rnM-MgSO4 at 37OC and for the times indicated in the Figures, followed by washing twice with p[NHJppG-free buffer at 4OC. Portions of the washed p[NH]ppG-activated 600g sediment and of the native nontreated 600g sediment were resuspended in 40 m~-Tficine/ NaOH buffer, pH 7.5, containing various concentrations of dimethyl 3,3'-dithiobispropionirnidate (see Figure legends). Cross-linking was carried out at 4OC for 18h, followed by washing the sediment twice with buffer (40 mM-Tricine/NaOH, pH 7.5, followed by 40m~-Tris/HCI, pH 7.5). The sediments were resuspended in 40 mM-Tris/HCl buffer, pH 7.5, containing 6mwMgS0, and, where indicated, Lubrol 12A9 (lOg/litre). All tissueextract concentrations were eqiuvalent to 75 mg wet wt. of corpus luteum/ml. Adenylate cyclase was assayed as described previously (Young & Stansfield, 1977, 1978a,b,c). The assay incubation (1 ml) contained 40m~-Tris/HCI, pH 7.5, ~ ~ M M ~ S O , , 1 mMATP, 6.7mlul-catTeine and 5 0 0 ~ 1 of enzyme preparation. Incubation was at 37OC for l0min. The cyclic AMP produced was measured by competitive protein binding (Young & Stansfield, 1977). Assays were performed in duplicate and the mean and range of duplicate values are shown. Fig. 1 demonstrates that there is a biphasic response of p[NHlppG-activated cyclase with respect to dimethyl 3,3'dithiobispropionimidate concentration. Concentrations around 5 p ~ enhanced the activity of the pH1ppG-treated enzyme, whereas higher concentrations (>0.05 mM) inhibited the enzyme. A paired t-test on the normalized data obtained in 12 separate experiments showed that 0.01 mM-dimethyl 3,3'-dithiobispropionimidate causes a significant increase in the activity of p[NH]ppG-treated cyclase (P < 0.01). The mean percentage increase in activity due to treatment with 0.01 mwdimethyl 3,3'-dithiobispropionirnidate was 23.5 (range -9 to 78; S.D. 2 1.7). Dimethyl 3,3'dithiobispropionimidate was without

Creatine kinase isoenzyme MB assay by electrophoresis.

A method for determination of the creatine kinase isoenzyme MB (CK-MB) is reported: separation of the isoenzymes was done by electrophoresis and the activity of the isoenzyme bands quantitated by scanning fluorometry. Total CK activity was used for calculation of CK-MB level. The precision of the method was satisfactory: coefficient of variation 5-10%. Its accuracy good: CK-MB was consistently found in high concentrations in tissue extracts of myocardium, but was virtually absent in skeletal muscle and could not be demonstrated in serum from patients with skeletal muscle damage. The sensitivity of the method fitted its clinical use: CK-MB was undetectable (less than 5 U/l) in normal sera, below 30 U/l in seventy-six out of seventy-seven patients in whom the diagnosis of acute myocardial infarction (AMI) was disproved, and above 30 U/l in all seventy-two patients with AMI according to WHO criteria. The CK-MB concentration in serum rises to a maximum about 20 h after onset of clinical symptoms of AMI and reaches baseline levels 20-30 h later. The electrophoretic CK-MB method is easy, fast and reliable and is considered as an important diagnostic test for AMI.

New enzyme cycling method for determination of oxidized and reduced nicotinamide-adenine dinucleotide

A highly sensitive enzyme cycling method for the measurement of NAD +(H) concentration in tissue extracts has been described. The assay measured propanediol formed during the coupled oxidation-reduction reactions between ethanol and lactaldehyde as catalyzed by liver alcohol dehydrogenase in the presence of catalytic concentrations of NAD +(H). The assay can be used to determine NAD +(H) concentrations in the range of 0.4 to 4 pmoles of coenzyme in a 0.1 ml reaction volume.