An investigation of nitride precipitates in archaeological iron artefacts from Poland.

The paper describes the investigations of nitride precipitates in a spearhead and a sword found in the territory of Poland, in cremation graveyards of the Przeworsk Culture, dated to the Roman Period. Three different techniques of the examination of nitride precipitates were employed: optical microscope, scanning electron microscope (scanning electron microscope with energy dispersive X-ray spectrometer) and transmission electron microscope. Two types of precipitates have been observed, and their plate-like shape was demonstrated. The large precipitate has been confirmed to be gamma'-Fe(4)N, whereas the small one has been identified as alpha''-Fe(16)N(2). The origin of nitride precipitates in archaeological iron artefacts from Poland is probably a result of the manufacturing process or cremation as part of burial rites. An examination of available iron artefacts indicates that nitride precipitates (have only limited effect on mechanical properties) influence the hardness of metal only to a very limited degree.

Kinetics of C. elegans DcpS cap hydrolysis studied by fluorescence spectroscopy.

DcpS (scavenger decapping enzyme) from nematode C. elegans readily hydrolyzes both monomethyl- and trimethylguanosine cap analogues. The reaction was followed fluorimetrically. The marked increase of fluorescence intensity after the cleavage of pyrophosphate bond in dinucleotides was used to determine K(m) and V(max)values. Kinetic parameters were similar for both classes of substrates and only slightly dependent on pH. The hydrolysis was strongly inhibited by methylene cap analogues (m(7)Gp(CH(2))ppG and m(7)Gpp(CH(2))pG) and less potently by ARCA (m(7,3' O)GpppG).

The relationship between constitutive ATP release and its extracellular metabolism in isolated rat kidney glomeruli.

ATP and adenosine are important extracellular regulators of glomerular functions. In this study, ATP release from glomeruli suspension and its extracellular metabolism were investigated. Basal extraglomerular ATP concentration (1nM) increased several fold during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate. Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP added to glomeruli was almost completely degraded within 20 minutes. In that time, AMP was the main product of extracellular ATP metabolism. Significant accumulation of AMP was observed after 5 min (194 +/-16 microM) and 20 min (271 +/-11 microM), whereas at the same time concentration of adenosine was only 10 muM. A competitive inhibitor of ecto-5-nucleotidase alpha-beta-methylene-ADP (AOPCP), decreased extraglomerular ATP and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 microM) also markedly reduced extraglomerular ATP accumulation, whereas IMP, its deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP from extraglomerular media caused by AMP. These findings demonstrate that the decrease in extracellular ATP concentration observed after addition of AOPCP or AMP is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme catalyses reversible reaction 2ADP<->ATP+AMP, and a rise in the AMP concentration can lead to fall in ATP level. The present study provides evidence the extraglomerular accumulation of ATP reflects both release of ATP from glomeruli cells and its metabolism by ecto-enzymes. Our data suggest that AMP, produced from ATP in the Bowman's capsular space, might plays a dual role as a substrate for ecto-adenylate kinase and ecto-nucleotidase reactions being responsible for the regulation of intracapsular ATP and adenosine concentration. We conclude that AMP degrading and converting ecto-enzymes effectively determine the balance between ATP and adenosine concentration and thus the activation of P2 and/or adenosine receptors.

Regulation of cGMP synthesis in cultured podocytes by vasoactive hormones.

The podocytes are highly differentiated cells playing a key role in glomerular filtration. Vasoactive factors including angiotensin II (Ang II) and cyclic guanosine 5' monophosphate (cGMP) are synthesized by these cells upon stimulation as well as in the basal state. In this study we have tested whether angiotensin II affects the total synthesis of cGMP in primary culture of rat podocytes. The cells were stimulated with atrial natriuretic peptide (ANP) and/or a nitric oxide (NO) donor, S-nitroso-N-acetyl penicillamine (SNAP), in the absence or presence of Ang II. The cGMP synthesis was determined by radioimmunoassay (RIA). ANP or SNAP alone increased the cGMP synthesis in podocytes although the effects were not additive unless Ang II was present in the medium. Ang II suppressed the ANP-dependent cGMP synthesis whereas SNAP-dependent cGMP production remained unaffected. These effects were prevented by a non-specific antagonist of Ang II receptors (AT), saralasin. Adversely, PD123319, a specific inhibitor of AT2 receptors, augmented inhibition of ANP-dependent and enhanced the NO-dependent cGMP production. Probenecid, an inhibitor of cGMP extrusion from the cells, suppressed the cGMP generation by both ANP and SNAP. We conclude that cGMP synthesis in cultured podocytes is modulated by angiotensin II and that two adversely acting receptors, AT1 and AT2 are involved in this effect. Additionally, production of cGMP might be intrinsically inhibited by cGMP accumulating inside the cells.

Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG.

The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957-967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures were proven by 1H NMR. Transcripts produced with SP6 polymerase using "anti-reverse" cap analogs (ARCAs) were of the predicted length and indistinguishable in size and homogeneity from those produced with m7GpppG or GpppG. Analysis of the transcripts with RNase T2 and tobacco acid pyrophosphatase indicated that reverse caps were formed with m7GpppG but not with ARCAs. Both of the ARCAs inhibited cell-free translation with a K(I) similar to that of m7GpppG. Finally, the translational efficiency of ARCA-capped transcripts in a rabbit reticulocyte lysate was 2.3- to 2.6-fold higher than that of m7GpppG-capped transcripts. This suggests the presence of reverse caps in conventional in vitro-synthesized mRNAs reduces their translational efficiency.

Neural cell adhesion molecule in breast, colon and lung carcinomas.

Neural cell adhesion molecules (NCAM) play an important role in embryogenesis and in some tumors, especially of neuroectodermal origin. In this study, 18 cases of invasive breast carcinoma, 7 cases of sigmoid colon carcinomas and 17 cases of the non-small-cell lung carcinoma were immunostained for NCAM. NCAM expression, usually focal, was observed in some cases only. NCAM was expressed in the membranes, in a fine granular pattern. In 3 cases of breast cancer cytoplasmic localisation of NCAM was also observed, which may suggest its cytoplasmic formation. Furthermore, in 3 cases expression of NCAM in histologically normal ductal lobular units adjacent to invasive breast cancers without the presence of this antigen in cancer tissue was observed. The immunostaining was weak or absent in sigmoid colon carcinomas. In this study we confirm the observation of some authors that NCAM expression occurs in some cases of non-small-cell lung carcinomas.

Nitric oxide synthase type 1 expression in human lung cancer and its relation to p53.

BACKGROUND: The nitric oxide synthases (NOS) have been observed in human tumour cell lines as well as in solid tumours. Neuronal isoform of NOS (NOS1) was particularly abundant in low-differentiated gynaecological, breast and central nerve system tumours, suggesting that it may characterize poorly differentiated tumours. So far, little is known about expression of the neuronal NOS isoform in non-small cell lung cancer. Our aim was to compare NOS1 expression in non-small lung carcinomas with respect to tumor staging and p53 protein expression. MATERIAL AND METHODS: Thirty-two cases of non-small lung carcinomas of all grades of malignancy and ten control lung specimens with neoplastic lesions were examined. Paraffin-embedded tissues were stained with hematoxylin and eosin, or with antibodies to NOS1 and p53, and evaluated under light microscope. RESULTS: No statistical correlation between expression of p53, NOS1 and degree od tumour differentiation was observed. CONCLUSION: Expression of NOS1 can not serve as a marker for highly malignant tumours in the non-small cell lung carcinomas.

Correlation between liver damage and degree of gastric mucose colonization by Helicobacter pylori in subjects with parenchymatous liver damage.

BACKGROUND: Helicobacter pylori (H. pylori) is an important factor responsible for chronic inflammatory conditions of the gastric mucosa. It has been demonstrated in numerous animal studies that some Helicobacter species may cause parenchymatous liver damage. The aim of the study was to investigate whether there is any correlation between the incidence of parenchymatous liver damage, and the incidence and degree of colonization of the gastric mucosa by H. pylori. MATERIAL AND METHODS: The study was carried out in the group of 30 patients (14 females, 16 males) whose mean age was 37 years, hospitalized because of parenchymatous liver damage without clinical symptoms of cirrhosis. All the patients had gastroscopy and urease tests performed, and mucosal biopsies were taken for immunomorphological investigations. The patients were divided into groups, group I comprising those with positive, and group II with negative urease test results. RESULTS: Positive urease tests were obtained in 26/30 patients (group I), 18/26 of whom demonstrated macroscopic changes of the gastric mucosa visible in gastroscopy. Group II with negative urease test results comprised 4/30 patients, 2/4 of whom had detectable changes in the gastric mucosa. The presence of H. pylori antigens was demonstrated by gastric mucosa immunomorphology in all 30 patients. The degree of invasion of H. pylori was visualized by immunofluorescence, which allowed to differentiate deep mucosal invasion of H. pylori (bacterial antigens present in lymph follicles and at the base of muciferous glands) observed in group I in 14/26 and in group II in 1/4 cases and superficial invasion (epithelium and mucosal surface) observed in group I in 12/26, in group II in 3/4. CONCLUSIONS: The obtained results may suggest more frequent H. pylori infections in subjects with parenchymatous liver damage than in the population without liver damage. Immunofluorescence seems to be a highly sensitive method allowing for detection of even small degrees of gastric mucosa colonization by H. pylori.

Co-operation between particulate and soluble guanylyl cyclase systems in the rat renal glomeruli.

ANP and NO act via different receptors, although inducing the common intracellular messenger - cyclic GMP. However, interaction between both factors remains unclear. Our observations suggested that in the rat kidney glomeruli, activities of the ANP- and NO-dependent guanylyl cyclase systems may be mutually compensated. To check this, we have tested effects of ANP and sodium nitroprusside (SNP) on cGMP synthesis and relaxation of glomeruli contracted with angiotensin II. The glomeruli were isolated from Wistar rats receiving saline (Control), dexamethasone (DEX), deoxycorticosterone (DOCA) or N-c-nitro-L-arginine methyl ester (NAME) for 1 or 2 days. In the DEX glomeruli exposed to 100 microM SNP, rate of cGMP synthesis was significantly higher then in the Control (26.3 vs 16.0 pmol/mg.prot./2 min., P<0.05), while 1 microM ANP was markedly less effective (2.8 vs 16.7 pmol/mg.prot./2 min in Control, P<0.01). On the contrary, in NAME group 1 microM ANP stimulated cGMP synthesis up to 35.6 pmol/mg.prot./2 min whereas efficacy of SNP was slightly suppressed. High correlation coefficient (r = 0.979, p<0.01) indicates interrelationship between NO- and ANP-dependent cGMP synthesis. Ability of the glomeruli to relax in response to ANP or SNP was in accord to their ability to cGMP generation. This was confirmed by high correlation (r = 0.845, p<0.001) between degree of relaxation and rate of cGMP synthesis. Our results support strongly the hypothesis that both, ANP and NO dependent systems co-operate in regulation of the function of kidney glomeruli.

Molecular subtypes of the HLA-DR antigens in pulmonary tuberculosis.

OBJECTIVE: The aim of this study was to analyze association between HLA-DRB1 alleles and pulmonary tuberculosis (PTB) in the Polish population. METHODS: The HLA-DRB1 typing was performed using sequence-specific amplification (polymerase chain reaction with sequence specific primer [PCR-SSP] in 31 patients and 58 healthy volunteers. The DRB1 primers were supplied by DYNAL in the standard kit DYNAL DR "low-resolution"-SSP. RESULTS: The study showed that the DRB1*16 alleles frequency was higher in patients with PTB than in the tested group of healthy controls (P < 0.01). When HLA-DR2 alleles were combined (i.e., the DRB1*15 with DRB1*16 alleles), their frequency was comparable with that in the healthy individuals. The highest relative risk (RR) of tuberculosis was associated with DRB1*16 alleles (RR = 9.7). When HLA-DR6 alleles were combined (i.e., the DRB1*13 with DRB1*14 alleles), only a trend for higher frequency in patients with PTB was found. Frequency of DRB1*13 alleles of HLA-DR6 was significantly lower in PTB than in the healthy individuals (P < 0.001; RR = 0.04). CONCLUSIONS: Results suggest that the presence of HLA-DRB1*16 alleles may increase the risk of development of PTB, whereas HLA-DRB1*13 alleles may be resistant to tuberculosis.

Dendritic and cancer cells in the breast tumors--an immunohistochemical study: short communication.

Normal and dysplastic mammary glands express immunocompetent S-100 protein positive dendritic cells (DCs), which are located in a regular pattern, in the suprabasal cell layer of the ducts and alveolar nodules. The epithelial cells, however, are S-100 protein negative. Since some breast cancers also express the S-100 protein, our aim was to check the diagnostic and prognostic value of the S-100 protein distribution combined with the tumor grade and expression of synaptophysin (Syn), chromogranin A (Chg A), c-erbB-2 oncoprotein and p53 protein in infiltrating and metastatic breast tumors. Applying immunohistochemical methods, we show in paraffin- or frozen breast tissue sections that in some cases of the infiltrating breast carcinomas, S-100 protein positive cells do not appear, whereas in other cases, either S-100 protein positive DCs are closely associated with cancer cells, or the cancer cells themselves stain positive to S-100 protein. However, we found no correlation between the S-100 protein expression and other investigated parameters.

Hydrolysis of some mRNA 5'-cap analogs catalyzed by the human Fhit protein--and lupin ApppA hydrolases.

Hydrolysis of the following four cap analogs: m7G(5')ppp(5')A, m7G(5')ppp(5')m6A, m7G(5')ppp(5')m2'OG and m7G(5')ppp(5')2'dG catalyzed by homogeneous human Fhit protein and yellow lupin Ap3A hydrolase has been investigated. The hydrolysis products were identified by HPLC analysis and the K(m) and Vmax values calculated based on the data obtained by the fluorimetric method.

Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation.

Fifty-eight analogues of the 5'-terminal 7-methylguanosine-containing cap of eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extracting KI, the dissociation constant for the cap analogue.eIF4E complex, from protein synthesis data. The results indicated that addition of a methyl group to the N2 of guanine produced more inhibitory compounds, but addition of a second methyl group to N2 decreased the level of inhibition dramatically. Aryl substitution at N7 improved the efficacy of guanine nucleoside monophosphate analogues. Substitution of the aromatic ring at the para position with methyl or NO2 groups abolished this effect, but substitution with Cl or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di- or triphosphate analogues produced only minor effects, both positive and negative. By far the strongest determinants of inhibitory activity for cap analogues were phosphate residues. The beneficial effect of more phosphate residues was related more to anionic charge than to the number of phosphate groups per se. The second nucleotide residue in analogues of the form m7GpppN affected inhibitory activity in the order G > C > U > A, but there was no effect of 2'-O-modification. Opening the first ribose ring of m7GpppG analogues dramatically decreased activity, but alterations at the 2'-position of this ribose had no effect. Non-nucleotide-based cap analogues containing benzimidazole derivatives were inhibitory, though less so than those containing 7-methylguanine.

Fluorescence studies on association of human translation initiation factor eIF4E with mRNA cap-analogues.

Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5'-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence delta Fmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E-7-methyl-GDP complexes in crystal and solution.

Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme.

The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors. In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient. The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent Km values were 1.9 mM for GTP, 6.0 microM for S-adenosyl-L-methionine and 170 microM for Mg2+. NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (35%), GppppG (50%) and less efficiently GpppA (12%), m2GTP (9%), and m2,2GTP (25%), but not m7GppG. The most potent inhibitors for nsP1 methyltransferase were et2m7GMP (Ki value 42 microM), m2,7GMP, (64 microM), m2,7GpppG (82 microM), m2et7GMP (105 microM), m2(2-phet)7GMP (194 microM) and m2GMP (386 microM). Of these compounds, m2GMP, m2et7GMP and m2(2-phet)7GMP showed competitive inhibition, whereas the others showed mixed type inhibition. All compounds that inhibited the methyltransferase activity inhibited also the guanylyltransferase activity of nsP1.

Myoid cells and neuroendocrine markers in myasthenic thymuses.

We have studied myoid cells in normal and myasthenic thymuses as well as in thymomas. For the presence of neuroendocrine markers-producing cells and identification of synaptophysin (Syn) the immunohistochemical method and immunoblot analysis were used. Myoid cells can be demonstrated in the thymus of myasthenic patients in high number. These cells occur in the vicinity of Hassall's bodies but also within them. Some regenerated Hassall's bodies displayed majority of myoid cells with their concentric arrangement around the centrally situated lacunar-like cell with nuclei of monocytogenic origin. Such phenomenon may suggest cooperation of myoid cells and their epithelial transitional forms with monocytogenic cells in various thymic hormone production. It is likely that myoid cells are the source of some thymic epithelial cells. According to some authors, thymomatous epithelial cells and skeletal muscle share a common epitope defined by a monoclonal antibody (mAb), whereas thymic epithelial cells possess acetylocholine receptor (AChR) on their surface. The epithelial cells of some thymomas express also desmin. In normal thymuses of children, Syn and chromogranin A (Chg A) were demonstrated in some cells of Hassall's bodies by immunohistochemical method. In addition, antibodies to Syn stained nerve structures surrounding the thymic blood vessels. In myasthenic thymuses, Syn expression was in cortical and medullary epithelial cells, in myoid cells and only scanty and focal in keratinized epithelial cells of Hassall's bodies. The epithelial cells of some thymomas also express Syn. In some thymuses of all groups investigated in this study Chg A was seen in single cells of Hassall's bodies and focally in cortical epithelial cells. Our results show that in normal thymuses of cardiac surgery patients and in the adult myasthenic thymuses antibody raised against Syn recognized protein with molecular weight of 48,000 but not normal (38,000) Syn. It remains to be elucidated if the overexpression of synaptophysin-like protein in myasthenic thymuses is a compensatory phenomenon to the defect in normal synaptic function.

Epithelial-mesenchymal cell interactions and participation of the neuroendocrine markers in tumor development. Immunohistochemical study.

Existence of the organ stem cell population seems to decide on ability of the tissue to regenerate and, likewise, on carcinogenesis. The source of the organ specific stem cells may be perivascular mesenchyma of thin-walled vascular channels. Our previous study on the breast cancer indicates that the perivascular mesenchyma of thin-walled vessels appears to be source of myoid cells (myofibroblasts) from which cancer cells arise. Similar results have been observed in the cancers of lung, salivary gland and colon, investigated in the current study. The perivascular cells of thin-walled channels are the source of myoid cells with expression of synaptophysin (Syn) and/or chromogranin A (Chg A), and from these cells neoplastic cells could originate. Syn and/or Chg A positive neoplastic cells were particularly well visible in connection with the vascular channels on the peripheries of tumors while other parts of tumors were only weak positive or negative for those neuroendocrine markers. Similarly as in breast cancers, the S100-protein positive dendritic cells with various of distribution were seen, expressing intimate connection with neoplastic cells. The epithelial pearls especially abundant in non-small cell lung carcinomas demonstrated immunohistochemical analogy to Hassall's bodies: they had monocytogenic cell inside and they displayed thymosin alpha 1 (TA1), as well as mucin secretion and minute calcification. Some epithelial cells expressed desmin and Syn. All types of investigated cancers demonstrated TA1. Results of our present study suggest that the perivascular cells have a differentiation defect. Such defect may initiate abnormal stromal environment, commonly observed in neogenesis, however, the presence of thymic growth factors may favor tumor growth.

Fluorescence and NMR studies of intramolecular stacking of mRNA cap-analogues.

Intramolecular stacking of a series of new synthesized dinucleotide mRNA cap analogues has been investigated in aqueous buffers by means of fluorescence and 1H-NMR at various pH and temperatures, and compared with that for 7-methylguanosine(5')ppp(5')guanosine (m7GpppG), as well as its hypermethylated derivative m(3)2,2,7GpppG. Thermodynamic parameters for intramolecular self-association stabilized by stacking were established by temperature-dependent fluorescence quenching, taking into account collisional deactivation of the excited states. Relative orientations of the stacked bases in the cap analogues were determined with the aid of a program GEOSHIFT (Stolarski et al., Biochim. Biophys. Acta (1996) 1293, 97), based on ring-current anisotropy. 1D-soft-TOCSY experiments were applied to extract the exact values of vicinal coupling constants, and hence to resolve solution conformation of the cap molecules. Stacking interaction has been discussed in detail in terms of the cap structural features, e.g., types of bases and length of the 5',5'-phosphate bridges, and regarding the interactions stabilizing intramolecular stacking.

Inhibition of endogenous nitric oxide synthesis activates particulate guanylyl cyclase in the rat renal glomeruli.

Nitric oxide (NO) plays a crucial role in the regulation of kidney function and metabolism. Our previous study showed that dexamethasone, one of several known selective inhibitors of inducible nitric oxide synthase (NOS), had a stimulatory effect on soluble guanylyl cyclase in the glomeruli of rat kidney. However, in the presence of dexamethasone, the atrial natriuretic factor (ANF)-dependent system remained suppressed. The aim of the present study was to investigate whether inhibition of synthesis of endogenous NO modulates the activity of the guanylyl cyclase system(s) in glomeruli. In these studies, rats were injected with a non-selective NOS inhibitor, N-omega-nitro-L-arginine methyl ester (NAME; NAME-group), or saline solution (controls; C-group). Creatinine clearance (C(Cr)), and plasma and urinary nitrate/nitrite (NOx-) levels decreased in the NAME-group, but plasma and urinary guanosine 3',5'-cyclic monophosphate (cGMP) contents were unchanged. In the presence of 0.1 microM ANF, synthesis of cGMP in the NAME-group exceeded threefold the cGMP production in the C-group. In addition, the pre-contracted glomeruli of the NAME-group were fully relaxed at 0.1 microM ANF, but glomeruli obtained from the C-group were relaxed in the presence of a 10 times higher dose of ANF. The increased sensitivity of glomeruli to ANF was possibly due to the more than doubled activity of particulate guanylyl cyclase (pGC) in the NAME-group in comparison with the C-group. In the presence of 100 microM sodium nitroprusside (SNP), soluble guanylyl cyclase (sGC) generated significantly lower cGMP production in the NAME-group than in the C-group (1.61 +/- 0.33 vs. 2.91 +/- 0.69 nmol/mg protein/10 min, respectively). These results demonstrate that inhibition of the synthesis of endogenous NO may also have an inhibitory effect on the activity of sGC. In addition, increased activity of the pGC and ANF-dependent system appears to be compensatory to the altered activity of soluble guanylyl cyclase.

Dinucleoside 5', 5"'-P1, P3-triphosphate hydrolase from yellow lupin (Lupinus luteus) seeds: purification to homogeneity and hydrolysis of mRNA 5'-cap analogs.

Three separable forms of diadenosine 5',5"'-P1, P3-triphosphate (Ap3A)-degrading activity were revealed when proteins obtained from the meal of yellow lupin seeds by ammonium sulfate precipitation were chromatographed on a DEAE-Sephacel column. The major form, which eluted first at the lowest salt concentration (0.15 M KCl), was free of any activity converting the reaction products, ADP and AMP. Its further purification by gel filtration on Sephadex G-200 and by affinity elution from an AMP-agarose column yielded homogeneous protein as demonstrated on SDS-polyacrylamide gel electrophorograms. The enzyme is a single polypeptide chain of M(r) 41 kDa. Eleven guanosine-containing dinucleoside triphosphates, including analogs of the mRNA 5'-cap structure, have been tested as potential substrates of the lupin "Ap3A hydrolase." All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m7Gp3G, et7Gp3G, and bz7Gp3G were hydrolyzed randomly, whereas m7Gp3A, m7Gp3C, and m7Gp3U yielded at least 80% m7GMP plus corresponding NDP and 20% or less NMP plus m7GDP. Hydrolysis of the guanosine-containing hybrids, Ap3G, Cp3G, and Up3G, yielded at least 85% GMP plus corresponding NDP. All dinucleotides containing the m7G-moiety were hydrolyzed 2- to 4.5-fold faster than Ap3A. Thus a general name, "dinucleoside triphosphate hydrolase," is more appropriate for the enzymatic activity described.