Gelatinases, endonuclease and Vascular Endothelial Growth Factor during development and regression of swine luteal tissue

Background  

The development and regression of corpus luteum (CL) is characterized by an intense angiogenesis and angioregression accompanied by luteal tissue and extracellular matrix (ECM) remodelling. Vascular Endothelial Growth Factor (VEGF) is the main regulator of angiogenesis, promoting endothelial cell mitosis and differentiation. After the formation of neovascular tubes, the remodelling of ECM is essential for the correct development of CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). During luteal regression, characterized by an apoptotic process and successively by an intense ECM and luteal degradation, the activation of Ca⁺⁺/Mg⁺⁺-dependent endonucleases and MMPs activity are required. The levels of expression and activity of VEGF, MMP-2 and -9, and Ca⁺⁺/Mg⁺⁺-dependent endonucleases throughout the oestrous cycle and at pregnancy were analyzed.     

Production of discrete high specific activity DNA probes using the polymerase chain reaction

Conditions are described for the synthesis of discrete DNA probes with high specific activity using the polymerase chain reaction. This method enables the production of DNA probes between any two oligonucleotide sequences from cloned or uncloned templates. These probes are uniform in length and their specific activity (1 × 10⁹ cpm/μg) is comparable to probes produced by other methods.     

Convenient methods to determine the specific radioactivity of (gamma-32P)ATP of high specific activity.

A satisfactory method for the determination of the specific activity of highly labeled [γ-³²P]ATP has not been reported previously. Yields of high specific activity ³²P labeled material usually are too small to be detected by ultraviolet spectrophotometry or phosphate analysis. Recent reports describing the assay of ATP by enzyme catalyzed phosphate transfer to ³H labeled glucose (1) or galactose (2) are not suitable for use with highly labeled ³²P material since the crossover into the ³H channel will greatly exceed the radioactivity of the ³H labeled phosphate acceptor. Recently Schendel and Wells reported the preparation of essentially carrier free [γ-³²P]ATP. They indicated, however, that the specific activity of the labeled product could not be determined by conventional methods (3). We have developed and now routinely use an expedient method for the determination of the specific activity of picomole quantities of highly labeled [γ-³²P]ATP. This procedure measures the phosphate transfer from [γ-³²P]ATP to oligothymidylic acid [dT(pT)₁₀] catalyzed by bacteriophage T4 induced polynucleotide kinase. The specific activity is determined by measuring the radioactivity present in d-³²pT(pT)₁₀, and can be verified by an isotope dilution method employing the same assay. Specific activities as high as 240 Ci/mmole have been determined.     

Proliferative capacity of embryonic chick notochord: a comparaison between PCNA positivity versus 3H-thymidine incorporation.

The proliferation rate of the embryonic chick notochord is measured during its functional period. Chick embryos of 4 different age groups respectively, 3.5, 4.5, 5.5 and 6.5 days of development, are selected. To measure proliferation activity in the notochords, two different methods are used. 1. Notochords are immunostained for proliferating cell nuclear antigen (PCNA). 2. After in vivo incorporation of ³H-thymidine (³H-TdR), DNA synthesis is visualized by autoradiography. After embedding in paraffin, 5 μm sections are cut and stained with PC10 monoclonal antibody and avidine biotin peroxidase complex (ABC) technique. Corresponding chick embryos of the same age groups are injected in ovo with ³H-thymidine. After fixation 2 μm sections are made and exposed for autoradiography. Results obtained by PCNA and autoradiography labelling techniques for the evaluation of the proliferative capacity are different. Both however show a regression of the proliferative activity. The regression of the number of cells that went through S-phase, as shown by autoradiography, is lower than that of PCNA positive cells. So, a number of notochordal cells stops in G1 phase. Our results moreover, indicate that the proliferating activity of the notochord of chick embryos is higher at young developmental stages than at later stages.     

Importance of 3-Hydroxy Fatty Acid Composition of Lipopeptides for Biosurfactant Activity

Biosurfactant production may be an economic approach to improving oil recovery. To obtain candidates most suitable for oil recovery, 207 strains, mostly belonging to the genus Bacillus, were tested for growth and biosurfactant production in medium with 5% NaCl under aerobic and anaerobic conditions. All strains grew aerobically with 5% NaCl, and 147 strains produced a biosurfactant. Thirty-five strains grew anaerobically with 5% NaCl, and two produced a biosurfactant. In order to relate structural differences to activity, eight lipopeptide biosurfactants with different specific activities produced by various Bacillus species were purified by a new protocol. The amino acid compositions of the eight lipopeptides were the same (Glu/Gln:Asp/Asn:Val:Leu, 1:1:1:4), but the fatty acid compositions differed. Multiple regression analysis showed that the specific biosurfactant activity depended on the ratios of both iso to normal even-numbered fatty acids and anteiso to iso odd-numbered fatty acids. A multiple regression model accurately predicted the specific biosurfactant activities of four newly purified biosurfactants (r² = 0.91). The fatty acid composition of the biosurfactant produced by Bacillus subtilis subsp. subtilis strain T89-42 was altered by the addition of branched-chain amino acids to the growth medium. The specific activities of biosurfactants produced in cultures with different amino acid additions were accurately predicted by the multiple regression model derived from the fatty acid compositions (r² = 0.95). Our work shows that many strains of Bacillus mojavensis and Bacillus subtilis produce biosurfactants and that the fatty acid composition is important for biosurfactant activity.     

Isotopic studies of urea metabolism in rabbits

1. The half-life of [¹⁵N]urea was found to be significantly longer than that of [¹⁴C]urea injected at the same time, the differences being due to endogenous catabolism of urea, which is accompanied by little or no reutilization of ¹⁴C but is approx. 20% for ¹⁵N. [¹⁵N]Urea therefore appears to be valueless as an indicator of nitrogen metabolism unless the extents of endogenous catabolism of urea and of fractional reutilization of ¹⁵N can be separately estimated. 2. Though measurements of the radioactivity of expired ¹⁴CO₂ confirmed the existence of considerable urea catabolism these could not be used for quantitative assessments. 3. Alternative graphical methods based on [¹⁴C]urea specific activities in plasma and urine samples were used to calculate the fraction of urea production that is excreted. Values by the two methods were in good agreement and showed that some animals excrete less than half the urea that they produce. 4. Specific activity differences between simultaneous samples of urinary and plasma urea reflect the presence of a pool of urea in the kidney that is not in equilibrium with the body urea pool. Calculations indicate the presence of urea in the kidney that in some cases may represent as much as 15% of the body pool, and in two animals in which post-mortem renal analyses were performed the masses of urea found agreed closely with the calculated values. 5. A model for urea metabolism is proposed that includes this pool in the excretory pathway. The related theory is shown to be adequate to explain the shape of the specific activity curves of urinary urea from the time of injection and the constant delay of the specific activity of urinary urea, relative to that of plasma urea, that is observed after a short preliminary equilibration period. 6. The body urea pool was calculated from the activity retained at 1·5hr. by excluding renal activity and the corrected specific activity of plasma urea at the same time. The urea pool was calculated to be distributed at the plasma concentration in a substantially smaller water volume than that found by injecting tritiated water in five animals. Reasons for this are discussed. 7. Urea synthesis rates calculated from the pool values are in close agreement with rates calculated from the mass of urea recovered in the urine and the fraction of newly synthesized urea that is excreted.     

Oligolabeling DNA probes to high specific activity with sequenase

In this protocol we present a reproducible method of preparing DNA probes of high specific activity using Sequenase. The probes produced by this method had a specific activity of 2.8×10⁹ cpm/μg with 69% of the total radioactivity incorporated into the TCA-precipitable materials. Probes with 5 to 10-fold lower specific activity were obtained using commercially available kits or using currently empolyed methods.     

Synthesis of [3,5-125I]triiodo-l-thyronine of high specific activity

Two methods for the synthesis of [3,5-¹²⁵I]triiodo-l-thyronine of high specific activity are described. This triiodthyronine which carries the iodine label exclusively in the nonphenolic ring has not been available so far. Both methods start from [3,5-¹²⁵I]diiodo-l-thyronine which is iodinated either with iodine in potassium iodide or with iodide and chloramine T. The concentration of the iodinating agent is critical in both methods and the pH of the reaction mixture must be high enough (～11) to cause complete ionization of the phenolic group of the substrate. The triiodothyronine obtained in over 70% yield is purified by ion-exchange chromatography.     

Studies on the specific activity of [γ-32P]ATP in adipose and other tissue preparations incubated with medium containing [32P]phosphate

1. The specific activity of the γ-³²P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717–720). The HPLC method also allowed the incorporation of ³²P into the (α + β)-positions of ATP to be determined. 2. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [γ-³²P]ATP attained a steady-state value after 1–2 h incubation in medium containing 0.2 mM [³²P]phosphate. Addition of insulin or the β-agonist isoprenaline increased this value by 5–10% within 15 min. 3. Under these conditions the steady-state specific activity of [γ-³²P]ATP was 30–40% of the initial specific activity of the medium [³²P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the γ-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [³²P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. 4. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.     

Assignment of secondary structure from Cα coordinates

A multiple regression analysis has established a nonlinear relationship between the backbone dihedral angles and the C^α coordinates obtained from the x-ray crystal structures of 14 proteins. The regression equations have been applied to predict specific dihedral angles of each residue in the backbone of 24 proteins. Overall this method (Nonlinear Regression Distance Torsion) predicts values of ϕ and ψ within a ±45° window of those found in the x-ray structure with an accuracy of 94 and 91% and within a ±30° window of 88 and 81%. Two methods for the assignment of motif from C^α coordinates are reported. For the first method, motif is assigned from the dihedral angles predicted using the regression equations. By the second method, motif of the ith residue is assigned from the distance C^α_i-1 to C^α_i+2 (v₆) and torsional angle C^α_i-1, C^α_i, C^α_i+1, C^α_i+2 (v₁₃). For the 24 proteins, 23.7% of the residues by the former method and 19.6% by the latter method are assigned differently than in the Protein Data Bank. © 1997 John Wiley & Sons, Inc.     

Improvements in steroid receptor assays including rapid computer analysis of data

Improvements on the steroid receptor assay have been developed which eliminate tedious procedures and give faster and more accurate results. A protein assay (Waddell method) is discussed which directly measures peptide bonds in cytosolic material. It is more rapid and more sensitive than the Lowry method. Programs for the analysis of data obtained by either the dextran-coated charcoal method or the sucrose density gradient have been written. These are superior to previously published programs and can be incorporated into any laboratory with a minimum of cost. The program for the dextran-coated charcoal method converts the counts per minute from both total and nonspecific ³H-steroid binding activity into information needed for a Scatchard analysis. These data are plotted and a linear regression and determination coefficient for best fit of the data are performed. In the sucrose density gradient method program, the counts per minute from both total and nonspecific ³H-steroid binding activity is converted to disintegrations per minute and plotted versus the corresponding fraction number. The specific binding activity in femtomoles/milligram of protein in the various molecular forms can then be obtained through set programmed steps.     

A simplified procedure for the preparation of tritiated GM1 ganglioside and other glycosphingolipids

The ganglioside II³NeuAc-GgOse₄Cer and other glycosphingolipids can be radiolabeled to high specific activity by the galactose oxidase-NaB³H₄ procedure, by purifying the oxidized compounds prior to reductive labeling. The oxidized products are separated from nonoxidized compounds and detergents (Triton X-100 and sodium taurocholate) present during the enzymatic oxidation. Since the oxidized derivatives are separated, the final specific activity depends solely upon the specific activity of the NaB³H₄ and the reduction conditions.     

A regression model predicting isometric shoulder muscle activities from arm postures and shoulder joint moments

Tissue overloading is a major contributor to shoulder musculoskeletal injuries. Previous studies attempted to use regression-based methods to predict muscle activities from shoulder kinematics and shoulder kinetics. While a regression-based method can address co-contraction of the antagonist muscles as opposed to the optimization method, most of these regression models were based on limited shoulder postures. The purpose of this study was to develop a set of regression equations to predict the 10th percentile, the median, and the 90th percentile of normalized electromyography (nEMG) activities from shoulder postures and net shoulder moments. Forty participants generated various 3-D shoulder moments at 96 static postures. The nEMG of 16 shoulder muscles was measured and the 3-D net shoulder moment was calculated using a static biomechanical model. A stepwise regression was used to derive the regression equations. The results indicated the measured range of the 3-D shoulder moment in this study was similar to those observed during work requiring light physical capacity. The r² of all the regression equations ranged between 0.228 and 0.818. For the median of the nEMG, the average r² among all 16 muscles was 0.645, and the five muscles with the greatest r² were the three deltoids, supraspinatus, and infraspinatus. The results can be used by practitioners to estimate the range of the shoulder muscle activities given a specific arm posture and net shoulder moment.     

A sensitive method for the separation and quantitation of (14C)proline and (14C)hydroxyproline from the collagen of rat uterus

A specific and sensitive method is described for the isolation and quantitation of [¹⁴C]proline and [¹⁴C]hydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a protease-free bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment with collagenase. There is a consistent recovery of [¹⁴C]proline and [¹⁴C]hydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [¹⁴C]proline into collagen of the rat uterus and to quantitate its conversion to [¹⁴C]hydroxyproline. Coupled with the colorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.     

Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP): determination of cellular PRPP pools and assay of PRPP synthetase activity.

Conditions are described where 5-phosphoribosyl-α-1-pyrophosphate (PRPP) can be determined by thin-layer chromatographic methods commonly used for the determination of nucleoside triphosphate pools in ³²P-labeled bacteria. A two-dimensional chromatographic system is described where very small pools of PRPP (about 0.03 μmol per gram dry weight bacteria) can be determined. In a uni-dimensional chromatographic system the lower limit for detection of PRPP pools is about 0.3 μmol per gram dry weight bacteria. This uni-dimensional system offers an assay also for PRPP synthetase activity even in crude extracts using [γ-³²P]ATP as a substrate. The assay is highly specific due to the chromatographic isolation of PRPP and is very sensitive due to the use of ³²P labeling.The chromatographic methods for determination of PRPP pools and of activities of PRPP synthetase have been applied to the analysis of some mutants of Salmonella typhimurium and have provided results that agree well with the results obtained by conventional methods of PRPP analysis.     

Xanthine dehydrogenase AtXDH1 from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a potent producer of superoxide anions via its NADH oxidase activity

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD⁺ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD⁺ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD⁺ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD⁺-dependent xanthine oxidation indicates that both NAD⁺ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.     

A comparison of methods for determining compartmental analysis parameters

The traditional method for determining compartmental analysis parameters relies on a visual selection of data points to be used for regression of data from each cellular compartment. This method is appropriate when the compartments are kinetically discrete and are easily discernible. However, where treatment effects on compartment parameters are being evaluated, a more objective method for determining initial parameters is desirable.

Three methods were examined for determining initial isotopic contents and half-times of ⁸⁶Rb elution from cellular compartments using theoretical data with known parameters. Experimental data from roots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) and barley (Hordeum vulgare L.) intact seedlings were also used. The three methods were a visually assisted, linear regression on data of semilog plot of isotope elution versus time, a microcomputer-assisted, linear regression on semilog plot where maximization of the square of the correlation coefficient (r²) was the criterion to determine data points needed for each regression and a mainframe computer-assisted, direct nonlinear regression on elution data using a model of the sum of three exponential decay functions. The visual method resulted in the least accurate estimates of compartmental analysis parameters. The microcomputer-assisted and nonlinear regression methods calculated the parameters equally well.

     

Fibroblast procollagen production rates in vitro based on [H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [³H]proline and measuring the subsequent formation of [³H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [³H]proline, and [³H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [¹⁴C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45–55]. Procollagen production rates were readily determined by this method using 5 to 20 μCi [³H]proline and approximately 10⁶ cells. It was found that ³H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/μg DNA · h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

Direct determination of the specific activity of RNA uniformly labeled with 32P

A simple method for the direct determination of the specific activity of RNA uniformly labeled with ³²P is described. The procedure is based on the premise that upon disintegration of ³²P to ³²S, the phosphodiester bond is broken. Analysis of the rate of decay of the full-length molecule by gel electrophoresis and autoradiography can accurately determine the “intramolecular specific activity” of the RNA. An equation that predicts the relative intensity of the intact RNA molecules remaining as a function of time is presented. These predictions are confirmed using in vitro-synthesized RNA labeled at a known specific activity. This procedure has been used to determine the intramolecular specific activity of RNA labeled in vivo in yeast. It can also be employed to choose the best conditions for experiments utilizing uniformly labeled RNA or single-stranded DNA and requiring the detection of intact molecules.