Isolation and some properties of DNA coding for tRNA1met from Xenopus laevis

DNA containing the reiterated genes for tRNA₁^met has been partially purified from Xenopus laevis by centrifugation in actinomycin C₁-CsCl and Ag⁺-Cs₂SO₄ gradients. These gradients separate the tRNA₁^met genes from those coding for tRNA₂^met and tRNA^val, thus confirming our earlier suggestion that these genes are not intermingled with each other (Clarkson, Birnstiel, and Purdom, 1973). The gradients also demonstrate the existence of a minor 5S DNA fraction which appears to differ from that previously isolated by Brown, Wensink, and Jordan (1971).When the enriched tDNA₁^met is digested to completion with either of the restriction endonucleases EcoRI or Hpa I, the tRNA₁^met genes are predominantly found within DNA fragments that are about 3100 base pairs long. A partial digestion with EcoRI shows that these fragments arise from the regular spacing of the enzyme restriction sites. The 3100 base pair EcoRI fragments are cleaved by Hpa I into fragments of two size classes, one of which is about 2200 base pairs long and contains the tRNA₁^met genes. The shorter fragments are about 700 base pairs long, and they appear to contain genes coding for at least one other kind of tRNA species. X. laevis tDNA₁^met thus comprises tandemly repeated DNA whose component parts show little if any length heterogeneity.     

Determination of [d-Ala2, d-Leu5]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Autoradiographic Visualization of Kappa Opioid Receptors with Labelled Dynorphins in Guinea Pig Brain

Abstract

The distribution of kappa opioid receptors in guinea pig brain was measured by in vitro receptor autoradiography using [³H]dynorphin A_1–9, [³H]dynorphin A_1–8 and [³H]bremazocine as ligands. The sites labelled by the two dynorphins had identical, heterogeneous distributions in brain sections. High levels of kappa receptors were seen in striatum, claustrum, nucleus accumbens and laminae V and VI of the cerebral cortex. The substantia nigra and superior colliculus also had high dynorphin binding levels. The [³H]dynorphin autoradiographs were closely similar to those obtained using [³H]bremazocine in the presence of mu and delta receptor displacers. It is concluded that tritiated dynorphin A fragments can be used for autoradiographic studies of kappa opioid receptors in brain.     

Sequence organization of a cloned tDNA1met fragment from xenopus laevis

3.18 kb fragments of X. laevis DNA coding for tRNA₁^met have been inserted into a λ vector via Hind III termini and cloned in E. coli. The organization of one cloned fragment has been analyzed by restriction endonuclease digestion and RNA-DNA hybridization. From the distribution of sites for three enzymes, this fragment appears to be typical of the majority of λ. laevis tandem tDNA₁^met repeat units. Evidence is presented to suggest that it contains two genes coding for tRNA₁^met and at least one gene coding for a second as yet unidentified 4S RNA species. The two tRNA₁^met genes are located on the same DNA strand 0.96 and 1.38 kb from one end of the repeat unit. A detailed restriction map for 19 enzymes reveals that the spacers between these genes are not identical, and it provides no indication of short repetitive sequence elements within the spacers.     

Determination of [d-Ala, d-Leu]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Sequence comparison of two codominant RAPD markers in Brassica nigra: deletions,substitutions and microsatellites

Summary Two RAPD fragments segregating codominantly were investigated in a F₂ population of Brassica nigra. Southern hybridization of these DNA fragments to genomic B. nigra DNA digested with several endonucleases revealed similar restriction profiles. Sequencing of the two fragments disclosed 93% homology. The differences were due mainly to an internal 41 nucleotide deletion in one of the fragments. Minor deletions of one to three bases, including a microsatellite of CTT motif were also observed. In addition, base substitutions, mostly transitions were detected. These relatively small differences suggested that the two RAPD products were indeed different versions of the same sequence. The larger fragment of 1154 bp was denominated A05¹ whereas the shorter one, denominated A05², had 1116 bp.     

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice

The clearance of human fibrinogen fragments D₁, D₂, D₃ and fibrin fragment D₁ dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D₁ and its dimer were essentially identical. Fragments D₂ and D₃ cleared at a progressively slower rate. Competition studies were performed between ¹²⁵I-labeled fragment D₁ and large molar excesses of unlabelled human fragments D₁, D₂, D₃, D₁ dimer, fragment E, fibrinogen, macroalbumin, mannan and asialooroscomucoid. Of these ligands only the fragment D variants competed for the clearance of ¹²⁵I-labeled fragment D₁. Cross-competition was observed when ¹²⁵I-labeled fragment D₁ dimer was cleared in the presence of large molar excesses of fragment D₁. Autopsies demonstrated that injected fragments D₁, D₂, D₃ and D₁ dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D₁. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cell, respectively). It is concluded that fragments D₁, D₂, D₃ and D₁ dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D₁, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.     

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of ^99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed ^99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the ^99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The ^99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the ^99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between ^99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N₂S₂) compound. The Fab′ with IgG2a isotype displayed tighter binding to ^99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N₂S₂ challenge and incubation with human plasma. The in vivo biodistribution of five ^99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable ^99mTc labeling of antibody fragments from three of the major murine isotypes.     

The isochore patterns of mammalian genomes and their phylogenetic implications

The compositional distributions of high molecular weight DNA fragments from 20 species belonging to 9 out of the 17 eutherian orders were investigated by analytical CsCl density gradient centrifugation and by preparative fractionation in Cs₂SO₄/BAMD density gradients followed by analysis of the fractions in CsCl. These compositional distributions reflect those of the isochores making up the corresponding genomes. A “general distribution” was found in species belonging to eight mammalian orders. A “myomorph distribution” was found in Myomorpha, but not in the other rodent infraorders Sciuromorpha and Histricomorpha, which share the general distribution. Two other distributions were found in a megachiropteran (but not in microchiropteran, which, again, shares the general distribution) and in pangolin (a species from the only genus of the order Pholidota), respectively. The main difference between the general distribution and all other distributions is that the former contains sizable amounts (6–10%) of GC-rich isochores (detected as DNA fragments equal to, or higher than, 1.710 g/cm³ in modal buoyant density), which are scarce, or absent, in the other distributions. This difference is remarkable because gene concentrations in mammalian genomes are paralleled by GC levels, the highest gene concentrations being present in the GC-richest isochores. The compositional distributions of mammalian genomes reported here shed light on mammalian phylogeny. Indeed, all orders investigated, with the exception of Pholidota, seem to share a common ancestor. The compositional patterns of the megachiropteran and of Myomorpha may be derived from the general pattern or have independent origins.     

Asymmetric orientation of amino groups in the α-subunit and the β-subunit of (Na+ + K+)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla

The orientation of amino groups in the membrane in the α- and β-subunits of (Na⁺ + K⁺)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[¹²⁵I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na⁺ + K⁺)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of α-subunit, β-subunit and proteolytic fragments of α-subunit. Both the α- and the β-subunit of (Na⁺ + K⁺)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the β-subunit are located on the extracellular surface. In the α-subunit, 65–80% of modified groups are localized to the cytoplasmic surface and 20–35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of ¹²⁵I-labeling within the α-subunit. The preservation of (Na⁺ + K⁺)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E₁- and E₂-forms of the α-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the β-subunit being on the extracellular surface, while the α-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.     

Photosynthesis in the Higher Plant Vicia faba: V. Role of Malate as a Precursor of the Tricarboxylic Acid Cycle

In the higher plant Vicia faba, anomalous labeling patterns in the organic acids and related amino acids of the tricarboxylic acid cycle which result from photosynthetic ¹⁴CO₂ fixation (in conjunction with an enzyme localization pattern unique to plant mitochondria) suggest that the tricarboxylic acid cycle functions primarily as a pathway leading to glutamic acid biosynthesis during autotrophic growth. The distribution of isotope in citrate indicates little recycling of oxaloacetate for the resynthesis of citrate. Rather, malate appears to provide both the C₂ and C₄ fragments for the synthesis of citrate, and [³H]formate and ¹⁴CO₂-labeling patterns implicate serine as the ultimate C₃ precursor of malate.     

Primary photochemistry of Photosystem I in chloroplasts. Dynamics of reversible charge separation in open reaction centers at 25 K

The reduction rate of oxidized reaction center chlorophyll of Photosystem I after laser-flash excitation at 25 K has been determined for D-144 subchloroplast fragments and chloroplasts. A maximum of 40% of Photosystem I reaction centers undergo irreversible charge separation (P-700, Cluster A: P-700⁺, Cluster A^?) at 25 K, a percentage which is independent of laser-flash intensity. The remaining reaction centers in chloroplasts and D-144 fragments undergo reversible charge separation with biphasic recombination. Similar amplitudes and time constants (chloroplasts, 49 μs (61%); D-144 fragments, 90 μs (67%)) were obtained for the fast component, while the slower component differed considerably in time (chloroplasts, 2.9 ms; D-144 fragments, 170 ms). It is known that Fe-S Cluster A is photoreduced in less than 1 ms at 25 K. Data obtained support a model for Photosystem I involving a single intermediate in the decay path between the reduced primary electron acceptor (A^?₁) and P-700⁺ and a second intermediate in the decay path between a reduced secondary electron acceptor and P-700⁺. Dual laser-flash experiments to determine rate constants for these processes are included.     

Time course localization of immunoglobulin M monoclonal antibody and its fragments in leukemic tumor-bearing mice

Summary In vivo localization of a mouse monoclonal antibody (F₂-10.23 IgM) binding leukemic L 1210 cells was studied in DBA/2 mice bearing an L 1210 tumor. F(ab)₂ fragments were prepared and their specific binding to L 1210 cells was analyzed by flow cytofluorometry. Radiolocalization studies were performed by using ¹²⁵I- or ¹³¹I-labeled IgM monoclonal antibody or its F(ab')₂ fragments to ascertain their capacity to visualize the L 1210 tumor. F(ab)₂ fragments were cleared more rapidly than the whole IgM; the clearance was as fast in healthy as in tumor-bearing mice. The tumor-to-muscle ratio observed 24 h after injection of ¹²⁵I-radiolabeled F(ab)₂ fragments and ¹²⁵I-radiolabeled IgM was 10; the radioactivity level in the blood with F(ab)₂ fragments was lower than with IgM, and so -camera imaging was workable with F(ab)₂ fragments without background substraction. The tumor localization was studied over a period of 5 days by recording the distribution of the iodinated fragments in the tumor-bearing leg compared with that in the normal leg, and by computer analysis of the region of interest. F(ab)₂ fragments gave better results than intact IgM in tumor visualization. Nevertheless, the rapid clearance of this antibody or its F(ab)₂ fragments make them hardly suitable as carriers of toxic drugs. Abbreviations used are: MEM Minimum essential medium; SDS sodium dodecylsulfate; PAGE polyacrylamide gel electrophoresis     

Computational study of the NO,SO<Subscript>2</Subscript>, and NH<Subscript>3</Subscript> adsorptions on fragments of 3N-graphene and Al/3N graphene

The adsorption properties of common gas molecules (NO, NH₃, and SO₂) on the surface of 3N-graphene and Al/3N graphene fragments are investigated using density functional theory. The adsorption energies have been calculated for the most stable configurations of the molecules on the surface of 3N-graphene and Al/3N graphene fragments. The adsorption energies of Al/3N graphene-gas systems are ?220.5 kJ mol^?1 for Al/3NG-NO, ?111.9 kJ mol^?1 for Al/3NG-NH₃, and ?347.7 kJ mol^?1 for Al/3NG-SO₂, respectively. Compared with the 3N-graphene fragment, the Al/3N graphene fragment has significant adsorption energy. Furthermore, the molecular orbital, density of states, and electron densities distribution were used to explore the interaction between these molecules and the surface. We found that orbital hybridization exists between these molecules and the Al/3N graphene surface, which indicates that doping Al significantly increases the interaction between the gas molecules and Al/3N graphene. In addition, compared with Li, Al can more powerfully enhance adsorption of the 3N-graphene fragment. The results indicate that Al/3N graphene can be viewed as a new nanomaterial adsorbent for NO, NH₃, and SO₂.     

Technetium-99m labeled 50H.19 antibody fragments: Interaction of the antibody with platelets

The monoclonal antibody 50H.19 recognized three antigens (M_r = 31-, 40-, 45-K) on normal and thromboasthenic platelets, but only one (M_r = 31-K) on Bernard-Soulier platelets. The intact antibody and its F(ab′)₂ fragments, had direct platelet-aggregating activity, and induced the platelet release reaction. The intact antibody potentiated platelet aggregation induced by platelet-activating factor or thrombin. Additions of indomethacin did not inhibit aggregation: addition of PGI₂, or a calcium channel blocker completely inhibited aggregation. A reduced amount of platelet-aggregating activity was observed with antibody fragments prepared for labeling with ^99mTc by pre-exposure to stannous ions, and herein used in biodistribution studies and elsewhere in thrombus imagining studies (J. Nucl. Med. 27: 1315; 1986). Antibody fragments radiolabeled with ^99mTc bound to isolated platelets and to clots containing platelets.     

Association of β-amyloid peptide fragments with neuronal nitric oxide synthase: Implications in the etiology of Alzheimers disease

Neuronal nitric oxide synthase (nNOS) was purified on DEAE-Sepharose anion-exchange in a 38% yield, with 3-fold recovery and specific activity of 5 µmol.min^?1.mg^?1. The enzyme was a heterogeneous dimer of molecular mass 225?kDa having a temperature and pH optima of 40°C and 6.5, K_m and V_max of 2.6 μM and 996 nmol.min^?1.ml^?1, respectively and was relatively stable at the optimum conditions (t_½?=?3?h). β-Amyloid peptide fragments Aβ_17–28 was the better inhibitor for nNOS (K_i?=?0.81 µM). After extended incubation of nNOS (96?h) with each of the peptide fragments, Congo Red, turbidity and thioflavin-T assays detected the presence of soluble and insoluble fibrils that had formed at a rate of 5?nM.min^?1. A hydrophobic fragment Aβ_17–21 [Leu₁₇ – Val₁₈ – Phe₁₉ – Phe₂₀ – Ala₂₁] and glycine zipper motifs within the peptide fragment Aβ_17–35 were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst.     

Spectrofluorimetric analysis of the interaction of amyloid peptides with neuronal nitric oxide synthase: Implications in Alzheimer's disease

Background

The deposition of aggregated β-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides.

Methods

Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aβ_1–40 and be catalytic towards amyloid fibril formation.

Results

Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aβ_1–40 (Aβ_17–28 and Aβ_25–35) had Stern–Volmer values (K_SV) of 0.111 μM⁻¹ and 0.135 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aβ_17–28 and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aβ_29–37] and bind to the enzyme at a site remote to the active region.

Conclusions

The interaction and binding of Aβ_17–28 and Aβ_25–35 to nNOS causes the movement of two critical tryptophan residues of 0.77 nm and 0.57 nm respectively towards the surface of the enzyme.

General significance

The binding of Aβ-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aβ-peptides and fibrillogenesis in senile plaques in an AD brain.     

Unique nucleotide sequence adjacent to the poly (A) region in yeast RNA

Yeast cells growing in a low phosphate medium were labeled with a pulse of ³²P_i or [³H]adenine and harvested after 15 minutes. Total RNA was extracted and digested with ribonuclease T₁. Poly(A)-rich fragments were isolated from the digest by hybridization to poly(U) impregnated fiberglass filters. Gel filtration showed the fragments to have a uniform chain length of about sixteen. Analysis of the composition gave (A₁₁, C₄, U). Complete pancreatic ribonuclease and partial spleen phosphodiesterase digests gave the sequence of the 5′ end of the fragment as CpApApUp-. Since the fragment was a ribonuclease T₁ product, the data points to a unique sequence of at least five residues, -GpCpApApUp-, adjacent to the poly(A)-rich terminus of pulse-labeled yeast mRNA. The remainder of the poly(A)-rich fragment consists of A residues with a few randomly interspaced C residues. The known specificity of yeast poly(A) polymerase can account for the presence of C residues in poly(A) tracts.     

Cooperativity in the calcium ion-induced quenching of the intrinsic fluorescence of a series of normal and GLA-deficient bovine prothrombin fragment 1 molecules

Ca²⁺ titrations of the intrinsic fluorescence of a series of -carboxyglutamic acid (GLA)-deficient bovine prothrombin fragments 1 yield response Hill plot parameters useful for characterization of the metal ion-binding process. 11-, 10-, and 9-GLA fragments 1 exhibitT _m (the (Ca²⁺)_total concentration at which ln (B/F)=0 in the response Hill plot) values between 0.2 and 0.3 mM. A 22-fold increase inT _m to 5.4 mM is observed for 8-GLA fragment 1.T _m decreases to 3.8 mM for the 7- and 6-GLA proteins. The value ofh, about 2.8±0.2 for 11-, 10-, and 9-GLA fragments 1, abruptly decreases to 1.2–1.3 for 8-, 7-, and 6-GLA fragments 1. The observed degree of quenching induced by saturating levels of calcium ions is affected by both changes in the intrinsic fluorescence of the metal ion-free proteins and in the maximum possible degree of quenching in the presence of calcium. The kinetic characteristics of the calcium ion-induced quenching of the intrinsic fluorescence of 6-GLA fragment 1 are identical to those observed in 10-GLA fragment 1, suggesting that the fluorescence quenching observed in the 6- and 10-GLA fragments 1, while different in magnitude, involves similar processes. Observation of an abrupt change in the relative electrophoretic mobilities of 11- to 9-GLA fragments 1 compared to 8- to 6-GLA fragments 1, in the absence or presence of Ca²⁺, suggests the existence of a major protein conformation change which occurs concomitantly with the noted changes inT _m andh response Hill plot parameters. Molecular mechanics calculations suggest a structural hypothesis unifying these observations. Central to this model is the presumption of the existence of hydrogen bond-mediated interactions between metal ion-binding sites.