Catalytic site-specific cleavage of a DNA-target by an oligonucleotide carrying bleomycin A5.

Oligonucleotide reagents have been created which are capable of catalytic site-specific cleavage of DNA-targets. The oligonucleotide reagent Blm-R-pd(CCAAACA) bearing the bleomycin A5 (Blm-RH) residue was used to degrade the DNA-target pd(TGTTTGGCGAAGGA). It has been shown that at equimolar reagent: target concentration the bleomycin oligonucleotide derivative can repeatedly cleave the target at G9, G7, T5, T4 and T3 in site-specific manner. This paper demonstrates that with a 10-fold excess of the DNA-target relative to the reagent 30% degradation of the target was observed primarily at a single position G7. The paper also shows that one reagent molecule containing bleomycin A5 residue was capable to degrade three molecules of the DNA-target. The catalytic activity of Blm-R-pd(CCAAACA) was the highest in the temperature range close to the melting temperature of the reagent-target complex, that is under conditions where the oligonucleotide reagent can form a complementary complex and easily dissociate to interact with the next molecule of the target. The number of target molecules degraded by the bleomycin reagent is limited by the degradation of the antibiotic residue itself.     

Sequence-specific modification of nucleic acids by oligonucleotide derivative containing alkylating groups in the C-5-position of deoxyuridine]

A new type of alkylating derivatives of oligonucleotides with 4(N-methyl-N-2-chloroethylamino)benzyl (RCl) group at C-5 of deoxyuridine with a high extent of the target modification was prepared. The synthesized reagents d(ULNHRClCCACTT), where L = CH2 (Ia), CH2OCH2CH2 (Ib) and CH2NHCOCH2CH2 (Ic), proved to effectively (80-90%) modify the oligonucleotide d(TAAGTGGAGTTTGGC). The reagents (Ia) and (Ib) alkylate G6, G7 and G9 positions, while the reagent (Ic) modifies predominantly G9.     

Characterization of polyriboadenylate polymerase from Tetrahymena pyriformis.

Poly(A) polymerase [polyadenylate nucleotidyltransferase, EC 2.7.7.19] was extracted from Tetrahymena pyriformis. The enzyme was demonstrated to be present in three forms by column chromatography on DEAE-cellulose, and they were termed poly(A) polymerase Ia, Ib, and II in order of increasing affinity to the column. The properties of enzymes Ia and Ib were similar except that Ia utilizes poly(A) as a primer rather efficiently. Enzyme II differed from enzymes Ia and Ib not only in elution profile on DEAE-cellulose column chromatography but also in pH and temperature preferences, molecular weight, requirement for divalent cations, sensitivity to salts at high ionic strength, optimal primer concentration, and subcellular localization. The molecular weights of enzymes Ia and Ib measured by gel filtration were both 43,000, and that of enzyme II was 95,000. All three enzymes required Mn2+ for maximal activity; Mg2+ could replace Mn2+ in the reaction of enzyme II, but only partially. In the presence of 0.1 M ammonium sulfate the activities of enzymes Ia and Ib were both completely inhibited, whereas enzyme II still showed 42% of its original activity. These findings suggest that there are two distinct types of poly(A) polymerase in Tetrahymena pyriformis.     

Direct cleavage of a DNA fragment by a bleomycin-oligonucleotide derivative

Bicomycin A₁ oligonucleotide derivative was used for direct cleavage of a DNA target. In the presence of Fe²⁺ ions and 2-mercaptoethanol, Blm-R-pd(CCAAACA) (I) damaged the target, pd(TGTTTGGCGAAGGA), with the yield of 80%, without affecting its own oligonucleotide tail. The sites of the cleavage were T^?-T^? and G^?-G^?. Unbound bleomycin A₅, damaged the G⁶-G⁷-G⁸ site. Reagent I formed more stable complementary complexes with the target than parent oligonucleotide (ΔT_m=11°C)     

Studies on thymocyte subpopulations in guinea pigs. III. Physical and functional characterization of six subpopulations separated by density gradient centrifugation and PNA binding

Thymocytes were separated according to increasing buoyant density into the three subpopulations Ia (25% of recovered cells), Ib (20%) and II (55%), and according to binding to peanut agglutinin (PNA)into PNA+ (65%) and PNA- cells (35%). The frequency of PNA+ was 56% in Ia, 60% in Ib and 66% in population II. Electronic cell volume determinations disclosed mean volumes of 160 fl for Ia, 130 fl for Ib and 100 fl for population II. PNA+ and PNA- cells were very similar as regards cell volume. Thus, PNA+ and PNA- cells are remarkably uniformly distributed among cell categories of different density and cell volume. The rapidly cycling thymocytes, regarded as the most immature cells in the thymus, and the target cells for a thymocyte growth factor both belonged to the PNA+ cells of population Ia. The mitogen-responsive thymocytes also belonged to population Ia, but were PNA-. The largest subpopulation of thymocytes, apparently corresponding to the small, non-cycling cortical cells, were recovered as PNA+ cells of population II.     

Isolation and identification of fetuin-B-like protein from rainbow trout seminal plasma and its localization in the reproductive system

Seminal plasma of rainbow trout (Oncorhynchus mykiss, Salmonidae) contains an inhibitory system consisting of three fractions (I-III) characterized by different electrophoretic migration rates. Using a two-step isolation procedure we purified (20- and 43-fold to homogeneity) and characterized the two subforms of inhibitor I (Ia and Ib). On the basis of the homology alignment of the amino acid sequences, inhibitor I was classified to the family of cysteine proteinase inhibitors - fetuins. The molecular masses were determined to be 61,146.5Da and 63,096.0Da, and the isoelectric points were estimated to be 6.04 and 6.22 for inhibitor Ia and Ib. Both inhibitors were glycoproteins with a carbohydrate content about 13% for inhibitor Ia and 19% for inhibitor Ib. The equilibrium association constant of inhibitor Ib with cod trypsin was determined to be 7.1×10(8)M(-1). Except for the cod trypsin inhibition, the inhibitor Ib effectively inhibited papain belonging to the cysteine proteainases. Comparative studies of the distribution of inhibitor I and the previously described inhibitor II were performed. The presence of inhibitor I in the seminal plasma was a common feature of several Salmoniformes, which was contrary to inhibitor II detected in seminal plasma of other fish families. Inhibitors I and II showed different expression patterns in the testes and spermatic duct of the rainbow trout.     

Copper(II).bleomycin, iron(III).bleomycin, and copper(II).phleomycin: comparative study of deoxyribonucleic acid binding

The kinetics and mechanism of binding of Cu-(II).bleomycin, Fe(III).bleomycin, and Cu(II).phleomycin to DNA were studied by using fluorometry, equilibrium dialysis, electric dichroism, and temperature-jump and stopped-flow spectrophotometry. The affinity of Cu(II).bleomycin for DNA was greater than that of metal-free bleomycin but less than that of Fe(III).bleomycin. Cu(II).bleomycin exhibited a two-step binding process, with the slow step indicating a lifetime of 0.1 s for the Cu(II).bleomycin.DNA complex. Fe(III).bleomycin binding kinetics indicated the presence of complexes having lifetimes of up to 22 s. DNA was lengthened by 4.6 A/molecule of bound Cu(II).bleomycin and by 3.2 A/bound Fe(III).bleomycin but not at all by Cu(II).phleomycin, suggesting that both bleomycin complexes intercalate while the phleomycin complex does not. However, phleomycin exhibited nearly the same specificity of DNA base release as bleomycin. These results suggest that the coordinated metal ion plays a major role in the binding of metal-bleomycin complexes to DNA but that intercalation is neither essential for DNA binding and degradation nor primarily responsible for the specificity of DNA base release by these drugs.     

Electrical properties and gustatory responses of various taste disk cells of frog fungiform papillae

We compared the electrical properties and gustatory response profiles of types Ia cell (mucus cell), Ib cell (wing cell), and II/III cell (receptor cell) in the taste disks of the frog fungiform papillae. The large depolarizing responses of all types of cell induced by 1 M NaCl were accompanied by a large decrease in the membrane resistance and had the same reversal potential of approximately +5 mV. The large depolarizing responses of all cell types for 1 mM acetic acid were accompanied by a small decrease in the membrane resistance. The small depolarizing responses of all cell types for 10 mM quinine-HCl (Q-HCl) were accompanied by an increase in the membrane resistance, but those for 1 M sucrose were accompanied by a decrease in the membrane resistance. The reversal potential of sucrose responses in all cell types were approximately +12 mV. Taken together, depolarizing responses of Ia, Ib, and II/III cells for each taste stimulus are likely to be generated by the same mechanisms. Gustatory depolarizing response profiles indicated that 1) each of Ia, Ib, and II/III cells responded 100% to 1 M NaCl and 1 mM acetic acid with depolarizing responses, 2) approximately 50% of each cell type responded to 10 mM Q-HCl with depolarizations, and 3) each approximately 40% of Ia and Ib cells and approximately 90% of II/III cells responded to 1 M sucrose with depolarizations. These results suggest that the receptor molecules for NaCl, acid, and Q-HCl stimuli are equivalently distributed on all cell types, but the receptor molecules for sugar stimuli are richer on II/III cells than on Ia and Ib cells. Type III cells having afferent synapses may play a main role in gustatory transduction and transmission.     

Cyclic nucleotide phosphodiesterase activities from pig coronary arteries. Lack of interconvertibility of major forms

DEAE-cellulose chromatography, with or without dithiothreitol and over a pH range of 6.0 to 8.5, resolved two phosphodiesterase activities (peaks I and II) from the soluble fraction of pig coronary arteries. The activity of peak I was increased by calmodulin (3-7-fold), whereas that of peak II was not. Chromatography of peak I on Biol-Gel A-0.5 m columns resolved two peaks of phosphodiesterase activity (peaks Ia and Ib). Peak Ia was eluted in the presence or absence of 0.1 M KCl and was relatively insensitive to calmodulin. Peak Ib was eluted only in the presence of KCl and was sensitive to calmodulin. The substrate specificity and kinetic behavior were the same for peaks I, Ia, and Ib. Repeated gel chromatography of either peak Ia or Ib, under appropriate conditions, yielded a mixture of peaks Ia and Ib. Peak Ia appears to be a reversible aggregate of peak Ib. Gel chromatography of peak II resolved only one phosphodiesterase activity, which was eluted without KCl, was highly specific for cyclic AMP, was not sensitive to calmodulin and migrated differently on the gel column than either peak Ia or Ib. Sucrose density gradient centrifugation of the soluble fraction from pig coronary arteries in the presence or absence of dithiothreitol resolved two peaks of phosphodiesterase activity (6.6 S and 3.6 S) which were similar to peaks I and II separated by DEAE-cellulose chromatography with regard to their substrate specificity and their sensitivity to calmodulin. Upon recentrifugation, each of the two peaks of phosphodiesterase activity gave a single peak of activity which migrated with the same S value as did its parent. These results indicate that the two major forms of phosphodiesterase of pig coronary arteries, which are representative of those found in many tissues, are not interconvertible in cell-free systems.     

Making DNA without iron - induction of a manganese-dependent ribonucleotide reductase in response to iron starvation

Ribonucleotide reductases supply cells with their deoxyribonucleotides. Three enzyme types are known, classes I, II and III. Class II enzymes are anaerobic whereas class I enzymes are aerobic, and so class I and II enzymes are often produced by the same organism under opposing oxygen regimes. Escherichia coli contains two types of class I enzyme (Ia and Ib) with the Fe-dependent Ia enzyme (NrdAB) performing the major role aerobically, leaving the purpose of the Ib enzyme (NrdEF) unclear. Several papers have recently focused on the class Ib enzymes showing that they are Mn (rather than Fe) dependent and suggesting that the E. coli NrdEF may function under redox-stress conditions. A paper published in this issue of Molecular Microbiology from James Imlay's group confirms that this unexplained NrdEF Ib enzyme is Mn-dependent, but shows that it does not substitute for NrdAB during redox stress. Instead, a role during iron restriction is demonstrated. Thus, the purpose of NrdEF (and possibly other class Ib enzymes) is to enhance growth under aerobic, low-iron conditions, and to functionally replace the Fe-dependent NrdAB when iron is unavailable. This finding reveals a new mechanism by which bacteria adjust to life under iron deprivation.     

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain I. Identification, purification, and structural comparisons.

Two Ca(2+)-calmodulin (CaM)-dependent protein kinases were purified from rat brain using as substrate a synthetic peptide based on site 1 (site 1 peptide) of the synaptic vesicle-associated protein, synapsin I. One of the purified enzymes was an approximately 89% pure protein of M(r) = 43,000 which bound CaM in a Ca(2+)-dependent fashion. The other purified enzyme was an apparently homogenous protein of M(r) = 39,000 accompanied by a small amount of a M(r) = 37,000 form which may represent a proteolytic product of the 39-kDa enzyme. The 39-kDa protein bound CaM in a Ca(2+)-dependent fashion. Gel filtration analysis indicated that both enzymes are monomers. The 43- and 39-kDa enzymes are named Ca(2+)-CaM-dependent protein kinases Ia and Ib (CaM kinases Ia, Ib), respectively. The specific activities of CaM kinases Ia and Ib were similar (5-8 mumol/min/mg protein). CaM kinase Ia (but not CaM kinase Ib) activity was enhanced by addition of a CaM-Sepharose column wash (non-binding) fraction suggesting the existence of an "activator" of CaM kinase Ia. Both kinases phosphorylated exogenous substrates (site 1 peptide and synapsin I) in a Ca(2+)-CaM-dependent fashion and both kinases underwent autophosphorylation. CaM kinase Ia autophosphorylation was Ca(2+)-CaM-dependent and occurred exclusively on threonine while CaM kinase Ib autophosphorylation showed Ca(2+)-CaM independence and occurred on both serine and threonine. Proteolytic digestion of autophosphorylated CaM kinases Ia and Ib yielded phosphopeptides of differing M(r). These characteristics, as well as enzymatic and regulatory properties (DeRemer, M. F., Saeli, R. J. Brautigen, D. L., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13466-13471), indicate that CaM kinases Ia and Ib are distinct and possibly previously unrecognized enzymes.     

NMR studies of the interaction of bleomycin with (dC-dG)3.

The interaction of bleomycin A2 and Zn(II)-bleomycin A2 with the oligonucleotide (dC-dG)3 has been monitored by nuclear magnetic resonance spectroscopy. Binding of the drug to the oligonucleotide is indicated by an upfield shift of the bithiazole proton resonances consistent with partial intercalation of this group between base pairs. The effect of temperature and ionic strength on the binding of both free bleomycin and the Zn(II) complex has been studied. Consistent with earlier studies on polynucleotides, the rate of exchange between the free drug and the drug-oligonucleotide complex is rapid on the 1H NMR chemical shift time scale. Binding of the oligonucleotide induced changes in resonances assigned to protons in the metal-binding region of Zn(II)-bleomycin. Intermolecular nuclear Overhauser effect enhancements between bleomycin and the oligonucleotide have not been detected.     

Hydrochlorothiazide, allopurinol and magnesium oxide in the treatment of recurrent calcium urolithiasis]

The study aimed at presenting own experience in prevention of new urinary calculi in 18 patients with metabolically active calcium urolithiasis treated with hydrochlorothiazide in daily doses of 100 mg (group I) for 2 years, and in 6 patients with the same disease treated with magnesium oxide in daily doses 300 mg twice a day (group II) for average period of 10 months. In 9 patients a new calculus was formed during the treatment with hydrochlorothiazide, in 7 patients no recurrence was noted, and in 2 remaining patients the results were controversial (coral calculus). Therefore, patients were subdivided into group Ia (failure of hydrochlorothiazide therapy), and group Ib (no recurrence noted). Hydrochlorothiazide did not lead to the stable decrease in the saturation of the urine with calcium oxalate in group Ia whereas in group Ib (without recurrence of urolithiasis) the content of calcium oxalate in the urine was significantly lower than that in group Ia after a 2-year treatment with hydrochlorothiazide (p < 0.01) Recurrence of the disease was seen only in one patient of group II, i.e. treated with magnesium oxide. The treatment of the recurrent calcium urolithiasis is justified and efficient in those patients in whom therapy decreases the content of calcium oxalate in the urine.     

Identification of Group B Streptococci by Immunofluorescence Staining

Gamma globulin fractions of rabbit antisera prepared with whole cell vaccines of group B types Ia, Ib, II, and III and labeled with fluorescein isothiocyanate stained group B streptococci type specifically. Type Ic cells, which contain the Ia polysaccharide antigen of type Ia and the Ic protein antigen of type Ib, were specifically stained by both Ia and Ib conjugates. A group B conjugate pool (B pool) that contained one conjugate specific for each group B type at its predetermined titer gave positive fluorescent-antibody (FA) reactions (4+ intensity) with group B stock strains and negative FA reactions (less than 2+ intensity) with stock strains of streptococcal groups A, C through H, and K through U, viridans streptococci, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae, and representative Enterobacteriaceae. Examination of 883 clinical isolates submitted to the Streptococcus Laboratory (Center for Disease Control, Atlanta, Ga.) for identification revealed a 99.1% agreement between FA and culture-precipitin methods. All 305 group B streptococci identified by culture-precipitin and six nonhemolytic group B streptococci missed initially by culture tests were identified correctly by FA. Results of cultural and FA methods in a double-blind study of 99 vaginal swabs agreed on 96 of 99 strains. Three nonhemolytic group B streptococci were identified first by FA and later confirmed by culture-precipitin tests.     

The phospholipid composition of embryonic chick liver microsomes

1. The phospholipid composition of hepatic microsomal fractions from different developmental stages of embryonic chick was established. The major components were phosphatidylcholine (approx. 66%), phosphatidylethanolamine plus phosphatidylserine (approx. 21%) and sphingomyelin (approx. 9%). 2. There were no significant changes in the phospholipid composition during embryonic development from 9 to 20 days. 3. When microsomal subfractions were prepared it was found that the smooth-microsomal fractions (Ia and Ib) had a significantly greater sphingomyelin content than the rough-microsomal fraction (II). This was compensated by a lower phosphatidylcholine content in fractions Ia and Ib and an increase of phosphatidylcholine in fraction II. 4. The significance of the differences in the phospholipid composition of smooth and rough microsomes is discussed with particular reference to the origin and interrelation of smooth and rough endoplasmic reticulum.     

External morphology of sensilla in the sacculus of an antennal flagellum of the fruit fly Drosophila melanogaster Meigen (Diptera : Drosophilidae)

Sensilla in the sacculus of an antennal 3rd segment, a funiculus, of the fruit fly, Drosophila melanogaster (Diptera : Drosophilidae) have been examined by scanning electron microscopy. The sacculus was divided into 3 cavities in its interior. A morphologically distinct group of sensilla was present in each cavity. Grooved sensillum (GS), found in the largest cavity, was further subclassified on the basis of the side wall sculpture into 3 subgroups: GS Ia, GS Ib and GS II. GS Ia was 4 μm long and had 10–12 grooves (0.25 μm wide) and GS Ib was 3.8 μm long and had 6–9 grooves (0.25-0.4 μm wide). GS 1a and GS Ib were inferred to be olfactory and thermoreceptive, and olfactory, respectively. GS II was 3.2 μm long, and had 4–5 grooves. Basiconic sensillum (BS), found in the smallest cavity, was 4.5 μm long and had an irregularly sculptured side wall, suggesting the presence of numerous irregular-shaped olfactory pores. Blunt-tipped sensillum (BTS), found in the middle-sized cavity, was 1.9 μm long and had a smooth-surfaced side wall and a button-like structure on its apex. These features suggested that BTS was hygro- and thermoreceptive.     

Studies on Metabolites of Alternaria kikuchiana Tanaka,a Phytopathogenic Fungus of Japanese Pear

The structure of a metabolite, C₁₀H₁₀O₅ (I), obtained as a weakly toxic substance from the culture filtrate of Alternaria kikuchiana, was determined as 3,6,8-trihydroxy-3-methyl-3,4- dihydroisocoumarin (Ia). Though three tautomeric forms—lactol form (Ia), keto form (Ib) and enol form (Ic) were possible for (I), it was found that the compound (I) existed as the lactol form (Ia) both in a crystalline state and in acetone, chloroform and methanol solutions. This metabolite gave weak necrotic symptom to pear leaves, but stimulated the root elongation of rice and radish seedlings (ca. 20~30%) at 5 ppm and also showed synergistic activity with gibberellin A₃ on the stem elongation of rice seedlings (ca. 30%).     

Characterization of Alder Phytophthora Isolates from Wallonia and Development of SCAR Primers for their Specific Detection

Isolates of alder Phytophthora were collected in the southern part of Belgium on riverbanks planted with Alnus glutinosa and A. incana. They were compared with strains isolated in other European countries in terms of maximum temperature for growth, oogonia shape, pathogenicity on Alnus seedlings and genetic traits. Using both molecular techniques [random amplified polymorphic DNA (RAPD) and random amplified microsatellite (RAMS)], two groups of isolates were identified, the first group being further divided into two subgroups, Ia and Ib, using RAPD. Most of the Walloon alder Phytophthora isolates as well as the standard type from UK (formally designated P. alni subsp. alni) fell into group Ia. One isolate was classified in group Ib with the German and Dutch variants (P. alni subsp. multiformis), while three isolates were placed with the Swedish variant (P. alni subsp. uniformis) in group II. In terms of morphological properties, isolates from groups Ia and Ib developed colonies with a felt‐like appearance and usually produced numerous oogonia, varying from wavy to warty after 1 week (group Ia) or 2–3 weeks (Ib) in darkness. In contrast, colonies from group II isolates were generally irregular, and smooth oogonia were produced in low quantities after approximately 1 month in culture. A polymerase chain reaction (PCR) using sequence‐characterized amplification region (SCAR) primers derived from a polymorphic amplification product generated with a RAPD primer was developed for the specific detection of alder Phytophthora. The specificity and sensitivity of this test are discussed here.     

Resolution and some properties of enzymes involved in enantioselective transformation of 1,3-dichloro-2-propanol to (R)-3-chloro-1,2-propanediol by Corynebacterium sp. strain N-1074.

During the course of the transformation of 1,3-dichloro-2-propanol (DCP) into (R)-3-chloro-1,2-propanediol [(R)-MCP] with the cell extract of Corynebacterium sp. strain N-1074, epichlorohydrin (ECH) was transiently formed. The cell extract was fractionated into two DCP-dechlorinating activities (fractions Ia and Ib) and two ECH-hydrolyzing activities (fractions IIa and IIb) by TSKgel DEAE-5PW column chromatography. Fractions Ia and Ib catalyzed the interconversion of DCP to ECH, and fractions IIa and IIb catalyzed the transformation of ECH into MCP. Fractions Ia and IIa showed only low enantioselectivity for each reaction, whereas fractions Ib and IIb exhibited considerable enantioselectivity, yielding R-rich ECH and MCP, respectively. Enzymes Ia and Ib were isolated from fractions Ia and Ib, respectively. Enzyme Ia had a molecular mass of about 108 kDa and consisted of four subunits identical in molecular mass (about 28 kDa). Enzyme Ib was a protein of 115 kDa, composed of two different polypeptides (about 35 and 32 kDa). The specific activity of enzyme Ib for DCP was about 30-fold higher than that of enzyme Ia. Both enzymes catalyzed the transformation of several halohydrins into the corresponding epoxides with liberation of halides and its reverse reaction. Their substrate specificities and immunological properties differed from each other. Enzyme Ia seemed to be halohydrin hydrogen-halide-lyase which was already purified from Escherichia coli carrying a gene from Corynebacterium sp. strain N-1074.