bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteolytic modification of human complement protein C9: loss of poly(C9) and circular lesion formation without impairment of function.
Authors: Dankert J R , Esser A F
Published: May 2006

We have compared the ability of thrombin-cleaved C9 (C9n) with that of native C9 to produce tubular or ring-like poly(C9) and to express the classical complement lesion on target membranes. Three procedures were used to produce poly(C9): (i) limited proteolysis with trypsin, (ii) interaction with small unilamellar lipid vesicles, and (iii) incubation with a 2- to 4-fold molar excess of ZnCl2. In contrast to C9, which could be converted to tubular poly(C9), C9n was converted to smaller peptides by the first procedure and was aggregated into string-like poly(C9) by the other two methods. C9-depleted human serum (R-9 serum) was reconstituted with either C9 or C9n and these sera were then used to lyse sensitized sheep erythrocytes. Numerous classical complement lesions could be detected on ghost membranes obtained from cells lysed by C9-reconstituted R-9 serum but only a few on ghost membranes produced by C9n-reconstituted R-9 serum. C9n was shown to be hemolytically as active as C9 even when tested under "single-hit" conditions and it was about twice as efficient when compared with C9 in releasing sucrose and inulin from resealed ghosts. These results are interpreted to indicate that formation of the classical complement lesion is only incidental to lysis and not an obligatory event and that enlargement of the "functional pore size" of the complement lesion is not linked to formation of a circular membrane attack complex.
Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644.
Authors: Nowycky M C , Fox A P , Tsien R W
Published: May 2006

A large-conductance calcium channel in chicken dorsal root ganglion neurons was studied with patch-clamp recordings of unitary currents. In addition to the conventional pattern of Ca-channel gating previously described in neurons ("mode 1"), we observed a different form of gating behavior ("mode 2"). Unlike the brief (approximately equal to 1 ms) openings in mode 1, mode 2 openings tend to be longer (greater than 10 ms) and often outlast the test pulse. In mode 2, the probability of channel openness (P) is high at relatively negative potentials where P in mode 1 is low. Mode 2 activity appears much less often than mode 1 activity in the absence of drug. However, the balance is strongly shifted in favor of mode 2 by the dihydropyridine Ca agonist Bay K 8644, an effect that underlies a marked enhancement of Ca-channel activity. This is the first evidence for dihydropyridine control of neuronal Ca-channel function at the single-channel level. Sweeps showing mode 1 or mode 2 gating appeared interspersed with sweeps with no openings, during which the channel was unavailable for opening ("null mode" or "mode 0"). Two approaches showed that switching between all three modes occurred on a time scale of seconds: (i) channels tended to remain in the same mode from one sweep to the next, with pulses at 0.25 Hz; and (ii) steady depolarizations in Bay K 8644 produced clusters of mode 2 openings lasting several seconds. Changes in the rates of switching might be important in neurochemical modulation of Ca channels. Bay K 8644 and other dihydropyridine Ca agonists might be useful experimental tools for manipulating transmitter release, neurite extension, and other neuronal functions dependent on intracellular Ca.
Cell-cycle dependence and properties of the HeLa cell DNA polymerase system.
Authors: Delfini C , Alfani E , De Venezia V , Oberholtzer G , Tomasello C , Eremenko T , Volpe P
Published: May 2006

Analysis of the properties of the DNA polymerase (pol) system as a function of fundamental factors of the assay environment allowed a rather accurate estimation of its dependence on the HeLa cell cycle. For pol alpha, the temperature and pH optima were 38.1 degrees C and 8.0, respectively; for pol beta, these optima were 36.2 degrees C and pH 7.4. Pol gamma showed a pH optimum at 7.7. Optimum activity for both the alpha and beta enzymes was observed at 60 mM Tris. The maximal activity at 36.2 degrees C and pH 7.4 was associated with resistance to N-ethylmaleimide (MalNEt), whereas that at 38.1 degrees C and pH 8.0 was sensitive to MalNEt. Incorporation of [3H]dTTP was maximal after 1 hr of incubation for the former activity and after 4 hr, for the latter. In extracts from cells in early S phase, the pol activity decreased after 1 hr of incubation, was MalNEt-resistant, and was characterized by temperature and pH optima at 36.2 degrees C and 7.4, respectively. In extracts of late S-phase cells, the pol-catalyzed incorporation of [3H]dTTP continued after 4 hr of incubation, was MalNEt-sensitive, and was characterized by temperature and pH optima at 38.1 degrees C and 8.0, respectively. Thus, a pol beta-type activity appeared in early S phase, whereas a pol alpha-type activity appeared in late S. During the G1, M, and G2 phases, a background level of pol activity was observed that showed intermediate kinetic properties.
Differential efficiencies of in vitro translation of mouse c-myc transcripts differing in the 5' untranslated region.
Authors: Darveau A , Pelletier J , Sonenberg N
Published: May 2006

We have studied the in vitro translational efficiencies of two murine c-myc transcripts synthesized in vitro that differ in the lengths of their 5' noncoding regions (448 and 83 nucleotides) and also in their 3' noncoding regions. When translated in a reticulocyte translation system, the shorter transcript was translated 10-fold more efficiently. These results are consistent with the hypothesis of Saito et al. [Saito, H., Hayday, A. C., Wiman, K., Hayward, W. S. & Tonegawa, S. (1983) Proc. Natl. Acad. Sci. USA 80, 7476-7480] that translation of full-length human c-myc mRNA is normally repressed, whereas in several Burkitt lymphomas that have deletions of the mRNA 5' noncoding region (resulting from translocation of the c-myc gene), translation of the c-myc mRNA is more efficient. Our results suggest that activation of murine c-myc expression by production of a more efficient mRNA might in some cases play a role in neoplastic transformation.
Protein kinase C regulates ionic conductance in hippocampal pyramidal neurons: electrophysiological effects of phorbol esters.
Authors: Baraban J M , Snyder S H , Alger B E
Published: May 2006

The vertebrate central nervous system contains very high concentrations of protein kinase C, a calcium- and phospholipid-stimulated phosphorylating enzyme. Phorbol esters, compounds with inflammatory and tumor-promoting properties, bind to and activate this enzyme. To clarify the role of protein kinase C in neuronal function, we have localized phorbol ester receptors in the rat hippocampus by autoradiography and examined the electrophysiological effects of phorbol esters on hippocampal pyramidal neurons in vitro. Phorbol esters blocked a calcium-dependent potassium conductance. In addition, phorbol esters blocked the late hyperpolarization elicited by synaptic stimulation even though other synaptic potentials were not affected. The potencies of several phorbol esters in exerting these actions paralleled their affinities for protein kinase C, suggesting that protein kinase C regulates membrane ionic conductance.
Effect of regional DNA methylation on gene expression.
Authors: Keshet I , Yisraeli J , Cedar H
Published: May 2006

The effect of DNA methylation on the transcriptional activity of the hamster adenine phosphoribosyltransferase (aprt) and the herpes thymidine kinase (tk) genes has been investigated. By using M13 constructs containing these gene sequences, specific segments of each gene were methylated in vitro by restriction fragment primer-directed second-strand synthesis using the substrate 2'-deoxy-5-methyl-cytidine triphosphate (dmCTP). These hybrid-methylated molecules were inserted into mouse Ltk- cells by DNA-mediated cotransfer. In all cases, the integrated sequences retained the in vitro-directed methylation pattern. The aprt gene was inhibited by CpG methylation in the 5' region but was unaffected by methylation at the 3' end or in adjacent M13 sequences. In contrast to this, DNA methylation in both the 5' promoter region and the 3' structural region of the tk gene had a strong inhibitory effect. This suggests that this modification may affect transcription by mechanisms that do not involve the direct alteration of recognition sequences for RNA polymerase.
Initiation of transcription from each of the two human mitochondrial promoters requires unique nucleotides at the transcriptional start sites.
Authors: Hixson J E , Clayton D A
Published: May 2006

Promoters for transcriptional initiation on each strand of human mtDNA lie in close proximity in the displacement-loop region. Transcription initiates within these promoter elements, providing an opportunity to analyze the effects of nucleotide sequence changes on the efficiency and fidelity of this event within two delimited regions. Each promoter was individually altered by a site-directed mutagenesis approach and assayed in vitro for the ability to support transcription. The data clearly confirm the previous assignment of promoter functions for these elements and show that single nucleotide substitutions immediately upstream of the transcriptional start sites can abolish transcription. In all cases of decreased transcription, the correct site of initiation is maintained. Although the heavy-strand promoter and light-strand promoter are similar in primary sequence, they exhibit remarkably different sensitivities to base substitutions.
Evidence that polymorphism in the murine major histocompatibility complex may be generated by the assortment of subgene sequences.
Authors: Miyada C G , Klofelt C , Reyes A A , McLaughlin-Taylor E , Wallace R B
Published: May 2006

The high degree of polymorphism found among the class I genes of the murine major histocompatibility complex (H-2) has led to the postulation that specific genetic mechanisms are responsible for their diversity. These same genetic mechanisms are probably responsible for the high spontaneous mutation frequency seen in H-2 alleles. The bml mutation of the H-2Kb gene has been shown to be 7 base pair changes over a 13 base pair region that result in three amino acid substitutions in the C1 domain of the protein product. The clustering of base-pair changes has suggested that the bm1 mutation resulted from a recombinational event analogous to gene conversion between the H-2Kb gene and a "donor" gene sequence. A 23-base oligonucleotide complementary to the bm1 mutant sequences was synthesized and used to probe genomic DNA restriction digests of the parental H-2b haplotype as well as other H-2 haplotypes. Our results indicate that a potential donor gene sequence is present in the genomes of all of the five mouse strains studied. Of eight tissues that were tested by blot-hybridization analysis, the potential donor gene sequences are transcribed only in the liver. Models for the generation of polymorphism among the H-2 class I genes via subgene rearrangements are proposed.
Complete primary structures of two major murine serum amyloid A proteins deduced from cDNA sequences.
Authors: Yamamoto K , Migita S
Published: May 2006

cDNA clones encoding two major mouse serum amyloid A proteins, SAA1 and SAA2, were isolated from a liver cDNA library of the lipopolysaccharide-stimulated BALB/c mouse, and their nucleotide sequences were determined. The insert of the SAA2 cDNA clone contained 607 nucleotides with a 5' untranslated region of 36 nucleotides, a signal peptide region corresponding to 19 amino acids, a mature protein region corresponding to 103 amino acids, and a 3' untranslated region of 202 nucleotides. The SAA1 cDNA insert contained 549 nucleotides specifying a part of a signal peptide region, a mature protein region, and a 3' untranslated region. A comparison of the nucleotide and deduced amino acid sequences of SAA1 cDNA with that of SAA2 cDNA showed a high degree of homology: 95% nucleotide sequence homology in the coding region (91% amino acid sequence homology) and 90% homology in the 3' untranslated region. One of nine amino acid differences between SAA1 and SAA2 predicted from the cDNA sequences was located in a putative proteolytic cleavage site for amyloid A protein formation: SAA2 had the Thr-Met sequence in this site, while SAA1 had the Thr-Ile sequence. This suggests that SAA1, which does not deposit as amyloid A protein, is also potentially susceptible to putative proteolytic enzymes. In addition, as compared with mouse SAA2, human SAA1, monkey and mink amyloid A protein, mouse SAA1 had two unique substitutions, which may play a role in differential deposition of mouse SAA isotypes in amyloid tissues.
Distribution of UDPglucuronosyltransferase in rat tissue.
Authors: Chowdhury J R , Novikoff P M , Chowdhury N R , Novikoff A B
Published: May 2006

UDPglucuronosyltransferase [UDPglucuronate beta-D-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17] is a group of enzymes with distinct but partially overlapping substrate specificity. A rabbit antiserum raised against one purified rat liver UDPglycuronosyltransferase isoform was specific for UDPglucuronosyltransferase and recognized all transferase isoforms by immunodiffusion or immunotransblot analysis. The transferase activity toward all substrates was immunoabsorbed from solubilized rat liver microsomes by IgG purified from the antiserum. The purified IgG was used for immunocytochemical localization of UDP-glucuronosyltransferase in rat liver, jejunum, kidney, and adrenal gland. In the liver, UDPglucuronosyltransferase was present exclusively in hepatocytes and was uniformly distributed within all zones of the hepatic lobule. In the jejunum, the transferase was present exclusively in the epithelial cells and showed a progressive increase in concentration from the crypt to the villar tip. In the kidney, the greatest concentration of the transferase was observed in the epithelial cells of the proximal convoluted tubule. Adrenal medullary cells showed intense immunocytochemical staining; the zona glomerulosa and the zona reticularis of the adrenal cortex were more intensely stained than the zona fasciculata. By light microscopy, UDPglucuronosyltransferase was found in the endoplasmic reticulum and nuclear envelope of all the four organs; this was confirmed in the hepatocyte by electron microscopy. The transferase was not observed in mitochondria, Golgi apparatus, lysosomes, peroxisomes, and plasma membrane, even after 3- to 4-fold induction of various substrate-specific UDPglucuronosyltransferase activities.
Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12.
Authors: Donovan W P , Kushner S R
Published: May 2006

The isolation of a temperature-sensitive allele of RNase II (rnb) by in vitro mutagenesis has permitted the demonstration that RNase II and polynucleotide phosphorylase (PNPase) are required for cell viability and mRNA turnover in Escherichia coli. Double-mutant strains carrying the pnp-7 and rnb-500 alleles (PNPase deficient and RNase II thermolabile) ceased growing in Luria broth within 30 min after shift to the nonpermissive temperature. Cessation of growth was accompanied by an accumulation of mRNA fragments 100-1500 nucleotides long. In contrast, single-mutant and wild-type control strains grew normally at the nonpermissive temperature and did not accumulate mRNA. No significant changes in rRNA patterns were observed in any of the strains.
O4-Methyl, -ethyl, or -isopropyl substituents on thymidine in poly(dA-dT) all lead to transitions upon replication.
Authors: Singer B , Spengler S J , Fraenkel-Conrat H , Kuśmierek J T
Published: May 2006

In a previous paper, we reported that O4-methyl dTTP can be incorporated into poly(dA-dT) in place of thymidine without distortion of the helical structure, but on replication it could behave as deoxycytidine and misincorporate dGTP. Only weak interactions are possible for any O4-modified T X A pair. While O4-alkyl T X G pairing should be favored, experiments to detect the ability of Escherichia coli DNA polymerase I (pol I) to utilize the triphosphate as dCTP were ambiguous. dTTPs with larger alkyl groups (ethyl, isopropyl) have now been synthesized and tested for their recognition as dTTP by pol I. Enhanced steric hindrance could be expected, particularly for O4-isopropyl dTTP, which has a three-carbon branched chain. However, both compounds behaved qualitatively like O4-methyl dTTP, being incorporated into poly(dA-dT) and then directing deoxyguanosine misincorporation by pol I. Quantitative comparisons of mutagenicity were not possible because of the finding that, unlike polymers made with O4-methyl dTTP, those made with ethyl or isopropyl dTTP were resistant to hydrolysis by using a variety of nucleases. The frequent misincorporations of dGTP would be expected to produce transitions in vivo. O4-ethyldeoxythymidine is very poorly repaired in vivo, which would also be expected for repair of O4-isopropyldeoxythymidine. Therefore, under suitable conditions, these particular carcinogen products are likely to be initiators of carcinogenesis.
Glucocorticoid-mediated induction of glucocortin: a rapid primary response common to major target tissues.
Authors: Colbert R A , Young D A
Published: May 2006

Glucocorticoids are known to rapidly induce four proteins in rat thymus cells in time to account for the earliest known metabolic hormone effects. We report here an additional protein, which we refer to as "glucocortin" (Mr = 17,000, pI = 4.7). It is of special interest since it is the only protein rapidly induced in all of the glucocorticoid target cells we have examined. We have characterized the kinetics of glucocortin mRNA induction in isolated thymus cells. Giant two-dimensional gel electrophoretic analysis of in vitro translation products reveals a 2-fold increase in the level of translatable mRNA within 15 min of dexamethasone addition, with maximal stimulation (approximately equal to 7-fold) by 45 min. Cycloheximide does not reduce the hormone-mediated increase in glucocortin mRNA, suggesting that the induction represents a primary response to glucocorticoids. This protein is induced by dexamethasone in a dose-dependent manner, with maximal induction at 10(-6) M and partial inductions at concentrations as low as 10(-10) M. It is strongly induced by cortisol at 10(-6) M, but it is not induced by estradiol or testosterone or by thyroid hormone, even at concentrations as high as 10(-6) M. Deoxycorticosterone has no effect at 10(-8) M but does generate a half-maximal effect at 10(-6) M, a finding consistent with its status as a partial glucocorticoid agonist. In summary, glucocortin appears to be a primary glucocorticoid-induced protein that represents the most rapid induction so far detected, and it appears to be the only one that may be common to all glucocorticoid target cells.
A model-based approach for estimating the mean incubation period of transfusion-associated acquired immunodeficiency syndrome.
Authors: Lui K J , Lawrence D N , Morgan W M , Peterman T A , Haverkos H W , Bregman D J
Published: May 2006

The incubation period, representing the interval between the date of exposure and the date of diagnosis, can be firmly ascertained in transfusion-associated cases of acquired immunodeficiency syndrome (AIDS). However, because the observation period of all transfusion-infected persons may be short compared with the average incubation period for AIDS, many cases with long incubation periods have not yet been diagnosed. Thus, the simple average of 2.6 years tends to underestimate the true mean. To correct for this underestimation bias, we assumed that the underlying distribution of the incubation periods is a member of a broad class of probability densities. Then, by maximum likelihood techniques, the mean incubation period for transfusion-associated AIDS was estimated to be 4.5 years, with the 90% confidence interval ranging from 2.6 to 14.2 years. The long incubation period has important consequences for infected individuals and implications for public health intervention and prevention policy.
Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells.
Authors: Gerdes K , Rasmussen P B , Molin S
Published: May 2006

The stability locus parB+ of plasmid R1 has been found to specify a unique type of plasmid maintenance function. Two genes, hok (host killing) and sok (suppressor of killing), are required for the stabilizing activity. The hok gene encodes a highly toxic gene product, whose overexpression causes a rapid killing and a concomitant dramatic change in morphology of the host cell. The other gene, sok, was found to encode a product that counteracts the hok gene-mediated killing. The parB+ region was inserted in a plasmid with a temperature-sensitive replication system. At nonpermissive temperature, the parB+ plasmid was maintained in the population for a significantly longer period than the corresponding parB- plasmid. Coupled to this extended maintenance, a large fraction of the population was shown to be nonviable plasmid-free cells with the characteristic hok-induced change in morphology. Based on these findings, we propose that the parB+ locus mediates plasmid stability by killing cells that have lost the parB+ plasmid during the preceding cell division, thereby ensuring that a growing bacterial culture predominantly consists of plasmid-containing cells.
Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues.
Authors: Sheves M , Albeck A , Friedman N , Ottolenghi M
Published: May 2006

Artificial bacteriorhodopsin pigments based on synthetic retinal analogues carrying an electron-withdrawing CF3 substituent group were prepared. The effects of CF3 on the spectra, photocycles, and Schiff base pKa values of the pigments were analyzed. A reduction of 5 units in the pKa of the Schiff base is observed when the CF3 substituent is located at the C-13 polyene position, in the vicinity of the protonated Schiff base nitrogen. The results lead to the unambiguous characterization of the (direct) titration of the Schiff base in bacteriorhodopsin and to the conclusion that the deprotonation rate of the Schiff base during the photocycle (i.e., the generation of the M412 intermediate) is determined by a structural change in the protein.
A defined system for in vitro packaging of DNA-gp3 of the Bacillus subtilis bacteriophage phi 29.
Authors: Guo P , Grimes S , Anderson D
Published: May 2006

The bacteriophage phi 29 DNA-gene product 3 complex (DNA-gp3) has been efficiently packaged into proheads in a completely defined in vitro system. The phi 29 DNA packaging protein gp16, the product of gene 16, was overproduced in Escherichia coli and purified to near homogeneity. The purified gp16 packaged 23% of the DNA-gp3 added to purified proheads in the defined mixture, while gp16 in an extract of phage-infected cells packaged 26% of the DNA-gp3. No host proteins were required in the defined system. ATP-dependent packaging of DNA-gp3 in the defined system was optimal with approximately equal to 100 copies of gp16 per DNA-gp3, an amount similar to the production of gp16 per DNA-gp3 in phi 29-infected cells.
Short- and long-term interactions of endothelium and vascular smooth muscle in coculture: effects on cyclic GMP production.
Authors: Ganz P , Davies P F , Leopold J A , Gimbrone M A , Alexander R W
Published: May 2006

In intact blood vessels, many vasodilators act by stimulating the release from endothelium of factor(s) that relax vascular smooth muscle and stimulate increases in cGMP. To investigate how endothelium regulates cGMP production in vascular smooth muscle, bovine aortic endothelial cells and rat aortic smooth muscle cells were cultured both separately and together in cocultures for 48 hr. Nitroprusside (1 mM) increased intracellular cGMP concentration 30-fold in smooth muscle cells (from a basal level of 103 +/- 54 fmol/mg of cell protein to 2920 +/- 1800 fmol/mg) but only 2-fold in endothelial cells (from 41 +/- 7 fmol/mg to 93 +/- 23 fmol/mg). When endothelial and smooth muscle cells were cocultured as a mixed cell population (1:1 cell ratio), both basal and nitroprusside-stimulated cGMP levels were significantly increased (550 +/- 250 and 13,240 +/- 9950 fmol/mg of total cell protein, respectively). The calcium ionophore A23187 (10 microM) caused no increase in cGMP concentration in either cell type cultured alone but produced a 6-fold increase in cocultures. Neither aspirin nor 5,8,11,14-icosatetraynoic acid influenced these results. No changes in cAMP levels were detected. Using cocultures in which one cell type was grown on microcarrier beads, we have shown that cGMP increased only in vascular smooth muscle cells and was not dependent upon the formation of junctions between endothelium and smooth muscle cells. In long-term (48-hr) mixed-cell cocultures, but not in short-term microcarrier cocultures, amplification of the nitroprusside-induced increase in cGMP was observed. These results show that responses associated with endothelium-dependent relaxation can be reconstituted in cultured endothelial and vascular smooth muscle cells and that endothelium generates a humoral factor(s) that stimulates accumulation of smooth muscle cGMP and has a longer-term effect that amplifies guanylate cyclase stimulation by nitroprusside, a drug acting directly upon smooth muscle to stimulate formation of the cyclic nucleotide. Cultured cells provide a valuable model system for the study of endothelium-vascular smooth muscle interactions.
The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites.
Authors: Martin K , Huo L , Schleif R F
Published: May 2006

Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the araI site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when araI and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are araI, araO2, and perhaps the araBAD operon RNA polymerase binding site.
Walking along the rabies genome: is the large G-L intergenic region a remnant gene?
Authors: Tordo N , Poch O , Ermine A , Keith G , Rougeon F
Published: May 2006

Rabies cDNA clones, obtained by "walking along the genome" using two successive DNA primers, have allowed the sequence determination of the genes encoding the N, M1, M2, G, and the beginning of the L protein as well as the rabies intergenic regions. Start and stop transcription signals located at the border of each gene encoding a protein have been identified and are similar to the corresponding signals from vesicular stomatitis virus (VSV) and Sendai virus. Except for limited stretches of the nucleoprotein, there is no homology between corresponding structural proteins of these three viruses. Rabies intergenic regions are variable both in length and sequence. Evidence for the existence of a remnant protein gene in the 423 nucleotide long G-L intergenic region is presented. This finding is discussed in terms of the evolution of unsegmented negative-strand RNA viruses.
Murine protein H is comprised of 20 repeating units, 61 amino acids in length.
Authors: Kristensen T , Tack B F
Published: May 2006

A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized with this cDNA probe. Ten positives were colony-purified, and the largest plasmid cDNA insert, MH8 (4.4 kb), was sequenced by the dideoxy chain termination method. MH8 contained the complete coding sequence for the precursor of murine complement protein factor H (3702 bp), 100 bp of 5'-untranslated sequence, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length. Similar repetitive units are present in the C4b binding protein, the C3b-receptor (CR1), complement factor B and C2, and in beta 2-glycoprotein I and the interleukin 2 receptor. This finding suggests a common evolutionary origin for regions of these proteins.
Nucleoside kinases in T and B lymphoblasts distinguished by autoradiography.
Authors: Osborne W R
Published: May 2006

Nucleoside kinases catalyze the initial step leading to the accumulation of deoxypurine nucleotides that occurs in patients with inherited deficiencies of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This accumulation is thought to interfere with DNA synthesis in lymphocytes and, thus, to cause the immune defects associated with these enzymopathies. However, there is controversy about the identity of the nucleoside kinases that are responsible for intracellular phosphorylation of deoxyadenosine in adenosine deaminase deficiency and deoxyguanosine in purine nucleoside phosphorylase deficiency. To distinguish the nucleoside kinases present in T and B lymphoblastoid cells, we have coupled discontinuous PAGE with autoradiography. This procedure showed that deoxycytidine kinase (NTP:deoxycytidine 5'-phototransferase, EC 2.7.1.74), deoxyadenosine kinase (ATP:deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76), and adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) are all present in both T and B lymphoblasts. While adenosine kinase is expressed at nearly equal levels in B and T cells, the deoxynucleoside kinases are expressed at much lower levels in B cells than in T cells. The autoradiographic data agreed with assays of the nucleoside kinase activities. Molecular weights were determined by using 5-10% polyacrylamide gels. Mr values were 29,000 for adenosine kinase, 41,000 for deoxyadenosine kinase, and 53,000 for deoxycytidine kinase and its isozyme. The reduced expression of deoxycytidine and deoxyadenosine kinases in B lymphoblasts may account for the lower accumulation of deoxypurine nucleotides in B cells as compared with T cells.
Mapping second messenger systems in the brain: differential localizations of adenylate cyclase and protein kinase C.
Authors: Worley P F , Baraban J M , De Souza E B , Snyder S H
Published: May 2006

[3H]Forskolin and [3H]phorbol 12,13-dibutyrate have been used to map the adenylate cyclase and phosphatidylinositol systems respectively in brain slices by light-microscopic autoradiography. [3H]Forskolin binding to brain sections is displaced potently by forskolin (KD approximately equal to 15 nM) and is enhanced by fluoride and GTP analogs, agents which activate the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Highest [3H]forskolin binding occurs in the corpus striatum, substantia nigra, hippocampus, and molecular layer of the cerebellum. Lesion studies demonstrate that binding sites in the substantia nigra are associated with striatal afferents, while hippocampal sites are localized to granule cell dendrites and mossy fiber terminals, and the intense binding in the cerebellar molecular layer is largely associated with granule cell axons and terminals. Protein kinase C mediates the activity of hormones and neurotransmitters, which act through the phosphatidylinositol cycle, and is labeled with high affinity by [3H]phorbol 12,13-dibutyrate. At many synapses, maps of adenylate cyclase and protein kinase C reveal reciprocal distributions, which may have implications for second messenger regulation of synaptic transmission.
Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes.
Authors: Suzdak P D , Schwartz R D , Skolnick P , Paul S M
Published: May 2006

The effects of ethanol on Cl- uptake were studied using a cell-free subcellular preparation from brain that contains a gamma-aminobutyric acid (GABA)/barbiturate receptor-sensitive Cl- transport system. In isolated vesicles prepared from rat cerebral cortex, ethanol, at concentrations that are present during acute intoxication (20-50 mM), stimulated 36Cl- uptake in a concentration-dependent and biphasic manner. The ethanol-stimulated uptake of 36Cl- was markedly inhibited by the GABA antagonists picrotoxin and bicuculline but not by a variety of other neurotransmitter receptor antagonists. The effects of ethanol in stimulating 36Cl- uptake in isolated brain vesicles were qualitatively and quantitatively similar to that of pentobarbital. Ethanol also markedly potentiated both muscimol- and pentobarbital-stimulated 36Cl- uptake at concentrations below those that directly stimulate 36Cl- uptake. Under our incubation conditions, ethanol did not release GABA, suggesting that it interacts with the postsynaptic GABA/barbiturate receptor complex. The ability of ethanol to stimulate GABA/barbiturate receptor-mediated Cl- transport may explain many of its pharmacological properties and provides a mechanism for the common psychopharmacological actions of ethanol, barbiturates, and benzodiazepines.
Potentiation of muscarinic and alpha-adrenergic responses by an analogue of guanosine 5'-triphosphate.
Authors: Evans M G , Marty A
Published: May 2006

Ca2+-dependent K+ and Cl- currents were recorded in isolated and dialyzed rat lacrimal gland cells by use of the tight-seal whole-cell recording technique. Under control conditions, application of acetylcholine (0.5-1.0 microM) resulted in the full activation of both types of current. When 50-200 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S], a nonhydrolyzable GTP analogue) was added to the intracellular solution, activation of both currents was seen with 1 nM acetylcholine, a dose 1/100th that needed under control conditions. Dialysis with solutions containing 200 microM GTP or cAMP had no, or only slight, potentiation effects. The effects of GTP[S] were obtained only when ATP was included in the intracellular solution. The potentiated responses to acetylcholine were blocked by increasing 10-fold the intracellular Ca2+-buffering capacity and were not dependent on external Ca2+. Thus, the potentiated responses appeared to result from a release of Ca2+ from internal stores. GTP[S] also greatly potentiated the Ca2+-dependent adrenergic (norepinephrine) response of this preparation. In addition, GTP[S] elicited in some cells transient responses without application of acetylcholine or norepinephrine. Finally, rapid and sustained responses were seen as soon as the cells were dialyzed with inositol trisphosphate (20 microM). These findings are discussed in terms of a possible role of a GTP-binding protein as a link between activation of muscarinic or adrenergic receptors and initiation of Ca2+ release by inositol trisphosphate.
Human glucose-6-phosphate dehydrogenase: primary structure and cDNA cloning.
Authors: Takizawa T , Huang I Y , Ikuta T , Yoshida A
Published: May 2006

The X-chromosome-linked glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) of humans and other mammals consists of a subunit with a molecular weight of about 58,000. The enzyme plays a key role in the generation of NADPH, particularly in matured erythrocytes, and the genetic deficiency of the enzyme is associated with chronic and drug- or food-induced hemolytic anemia in humans. The enzyme was purified to homogeneity from human erythrocytes. The complete amino acid sequence of the subunit, consisting of 531 amino acid residues, was determined by automated and manual Edman degradation of tryptic, chymotryptic, thermolytic, and cyanogen bromide peptides obtained from the enzyme. Based on the amino acid sequence data thus obtained, a 41-mer oligonucleotide with unique sequence was prepared. Two cDNA libraries constructed in phage lambda gt11--i.e., a human liver cDNA library and a human hepatoma Li-7 cDNA library--were screened with the synthetic nucleotide probe. Two positive clones, lambda G6PD-19 and lambda G6PD-25, were obtained from the hepatoma library. lambda G6PD-19 contained an insertion of 2.0 kilobase pairs (kbp), and encoded 204 amino acid residues that were completely compatible with the COOH-terminal portion of the enzyme. The insertion of the clone had a 3' noncoding region of 1.36 kbp. The other clone, lambda G6PD-25, had an insertion of 1.8 kbp and encoded 362 amino acid residues of G6PD. Southern blot analysis of DNA samples obtained from cells with and without the human X chromosome indicated that the cDNA hybridizes with a sequence in the X chromosome.
Homology of lipoprotein lipase to pancreatic lipase.
Authors: Ben-Avram C M , Ben-Zeev O , Lee T D , Haaga K , Shively J E , Goers J , Pedersen M E , Reeve J R , Schotz M C
Published: May 2006

Bovine milk lipoprotein lipase was subjected to amino acid sequence analysis. The first 19 amino-terminal residues were Asp-Arg-Ile-Thr-Gly-Gly-Lys-Asp-Phe-Arg-Asp-Ile-Glu-Ser-Lys-Phe-Ala-Leu- Arg. In addition, reversed-phase high-performance liquid chromatography of a tryptic digest of reduced and alkylated lipase resolved a number of peptides, five of which contained cysteine. Sequence analysis of the tryptic peptides revealed in most instances a close homology to porcine pancreatic lipase. Based on this homology, the relative alignment of the sequenced lipoprotein lipase peptides can be made. In addition, a potential binding site for the triacylglycerol substrate and a carbohydrate-binding domain for lipoprotein lipase are postulated.
Deprotonation of the Schiff base of rhodopsin is obligate in the activation of the G protein.
Authors: Longstaff C , Calhoon R D , Rando R R
Published: May 2006

Photolysis of rhodopsin leads to the formation of an activated intermediate that activates a G protein, thus beginning the visual cascade. This activated form of rhodopsin appears coincident in time with the spectroscopically defined intermediate, metarhodopsin II. Metarhodopsin I, the precursor of metarhodopsin II, contains a protonated Schiff base, whereas metarhodopsin II does not. The question of whether the deprotonation of the protonated Schiff base is obligate in the formation of activated rhodopsin was addressed by monomethylating the active-site lysine of permethylated rhodopsin and determining whether this pigment can activate the G protein upon photolysis. The photolysis of the new pigment, which absorbs at 520 nm, led to the formation of a relatively stable metarhodopsin I-like intermediate with a lambda max of approximately equal to 485 nm, with no apparent formation of either metarhodopsin II- or metarhodopsin III-like intermediates. The only probe available to detect formation of the active form of rhodopsin is G protein activation. Photolysis of the pigment in the presence of the G protein did not lead to measurable activation of the GTPase activity of the latter. These studies establish a functional link between Schiff base deprotonation and activation of the G protein. It is concluded that proton transfer from the protonated Schiff base of rhodopsin is obligate for the initiation of visual transduction.
Functional role of HLA class I cell-surface molecules in human T-lymphocyte activation and proliferation.
Authors: Taylor D S , Nowell P C , Kornbluth J
Published: May 2006

This investigation addressed the role of major histocompatibility complex-encoded class I molecules in the activation and proliferation of human lymphocytes. We studied the effect of antibodies specific for HLA-A and HLA-B locus gene products on mitogen-stimulated peripheral blood mononuclear cell (PBMC) subpopulations. Three individually derived, well-characterized anti-HLA class I monoclonal antibodies were demonstrated to inhibit the proliferation of human PBMC stimulated by either OKT3 or the calcium ionophore ionomycin. The antibody directed against HLA-A, -B, and -C locus gene products (W6/32) and the antibody directed against HLA-B locus gene products (4E) inhibited proliferation induced by either mitogen by 70-90%. The HLA-A locus-specific antibody (131), though inhibiting ionomycin-induced proliferation by 80-90%, was much less effective when OKT3 was the stimulus. The inhibition affected T4+ and T8+ cells and was not mediated by DR+ accessory cells. The inhibitory effect of these antibodies was associated with a decrease in the level of interleukin 2 activity present in culture supernatants, decreased interleukin 2 receptor expression, and decreased transferrin receptor expression and was not overcome by the addition of exogenous interleukin 2. Our results suggest that HLA class I molecules are directly involved in the early critical events of human lymphocyte activation and proliferation.
Promotion of the Whitten effect in female mice by synthetic analogs of male urinary constituents.
Authors: Jemiolo B , Harvey S , Novotny M
Published: May 2006

Two volatile constituents of male mouse urine, 2-(sec-butyl)-4,5-dihydrothiazole and dehydro-exo-brevicomin, were synthesized and tested for their ability to induce estrous cycle in female mice (the Whitten effect). The suppression of ovarian cycling activity that resulted from grouping the females was abolished by exposure to normal male urine. The synthetic compounds, when added together in appropriate concentrations to the (previously inactive) urine of castrated males, or even to water, were found to be as effective as normal male urine. The action of the synthetic compounds appears attenuated for singly caged females.
Conductive polymers derived from iron, ruthenium, and osmium metalloporphyrins: The shish-kebab approach
Authors: Collman James P. , McDevitt John T. , Yee Gordon T. , Leidner Charles R. , McCullough Laughlin G. , Little William A. , Torrance Jerry B.
Published: May 2006

The synthesis and characterization of pyrazine-bridged polymers of iron(II/III), ruthenium(II/III), and osmium(II/III) octaethylporphyrin (dubbed “shish-kebab” polymers) are presented. Optical and dc conductivity measurements reveal that the ruthenium and osmium polymers, when partially oxidized, are highly conductive. Electrochemical and ESR results are presented that indicate the existence of an interesting metal-centered conduction pathway. Unlike most of the previously reported porphyrinic molecular metals in which the conduction electrons are macrocyclic-based, electron transport in these materials proceeds exclusively along the metal-pyrazine backbone.
Replication of a plasmid bearing a human Alu-family repeat in monkey COS-7 cells.
Authors: Johnson E M , Jelinek W R
Published: May 2006

Monkey COS-7 cells were transformed with BLUR8 DNA, a pBR322 plasmid containing a human Alu-family sequence at the BamHI site. Within 24 hr of transformation 2-5% of the BLUR8 molecules recovered resisted cleavage by Dpn I, indicating they had replicated. Electron microscopy revealed appropriately sized circular molecules with replication bubbles whose centers were mapped to the Alu insert. A 16-base-pair deletion within the Alu sequence prevented replication. The results indicate that certain Alu sequences can serve as origins of replication in COS-7 cells.
How enzymes can capture and transmit free energy from an oscillating electric field.
Authors: Westerhoff H V , Tsong T Y , Chock P B , Chen Y D , Astumian R D
Published: May 2006

Recently, it has been demonstrated that free energy from an alternating electric field can drive the active transport of Rb+ by way of the Na+, K+-ATPase. In the present work, it is shown why many transmembrane enzymes can be expected to absorb free energy from an oscillating electric field and transduce that to chemical or transport work. In the theoretical analysis it turned out to be sufficient that (i) the catalytic process be accompanied by either net or cyclic charge translocation across the membrane and (ii) the stability of the enzyme states involved be asymmetric. Calculations based on a four-state model reveal that free-energy transduction occurs with sinusoidal, square-wave, and positive-only oscillating electric fields and for cases that exhibit either linear or exponential field-dependent rate constants. The results suggest that in addition to oscillating electric field-driven transport, the proposed mechanism can also be used to explain, in part, the "missing" free energy term in the cases in which ATP synthesis has been observed with insufficient transmembrane proton electrochemical potential difference.
Induction of distinct types of spontaneous electrical activities in mammary epithelial cells by epidermal growth factor and insulin.
Authors: Enomoto K , Cossu M F , Edwards C , Oka T
Published: May 2006

Electrophysiological measurements of the membrane potentials of mouse mammary epithelial cells in primary culture revealed the presence of spontaneous-oscillating-hyperpolarizing potentials in cells incubated with epidermal growth factor. The hyperpolarizing potentials were 5-20 mV in amplitude and about 10 sec in duration. The peak height of the response was reduced by hyperpolarization, and the input membrane resistance decreased during the response. The response was probably due to activation of K+ channels. The latency period for the epidermal growth factor induction of the hyperpolarizing potential was approximately 3 hr. In contrast, insulin induced spontaneous-depolarizing potentials that were about 5 mV in amplitude and 1 sec in duration. The depolarizing potentials were attributed to activity of ion channels, since the peak height was dependent on the membrane potential and the depolarizing potential was accompanied by a decrease of input membrane resistance. The time lag for the induction of the depolarizing potential was 6-12 hr. Other hormones involved in mammary cell differentiation, such as cortisol and prolactin, neither induced the depolarizing potentials nor changed the induction of depolarizing potential by insulin. In addition, other growth factors, such as nerve growth factor and fibroblast growth factor, elicited no electrical activity.
An evolutionary reduction principle for genetic modifiers.
Authors: Feldman M W , Liberman U
Published: May 2006

The joint evolution of major genes under viability selection and a modifier locus that controls recombination between the major genes, mutation at the major gene, or migration between two demes is studied. The modifying locus is selectively neutral and may have an arbitrary number of alleles. For each case a class of polymorphic equilibria exists in which the frequencies of the modifying alleles are those computed by assuming that the recombination, mutation, or migration rates were viabilities and in which the major and modifier loci are not statistically associated. These are called viability-analogous Hardy-Weinberg (VAHW) equilibria. A new allele introduced near these equilibria will enter the population if its marginal average rate of recombination, mutation, or migration (whichever applies) is less than the population average prior to its introduction. Stability properties of these VAHW equilibria are also reported.
Extracellular localization of pokeweed antiviral protein.
Authors: Ready M P , Brown D T , Robertus J D
Published: May 2006

Pokeweed antiviral protein is an enzyme of Mr 29,000 known to inactivate a wide variety of eukaryotic ribosomes. We have used electron microscopy to show that the antibody specific for the protein is bound within the cell wall matrix of leaf mesophyll cells from Phytolacca americana. Any penetration or breakage of the cell wall and membrane could allow the enzyme to enter the cytoplasm, where it is likely to inhibit protein synthesis in the damaged cell. We speculate that pokeweed antiviral protein is a defensive agent whose principal function is probably antiviral.