bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence homology between two membrane transport ATPases, the Kdp-ATPase of Escherichia coli and the Ca2+-ATPase of sarcoplasmic reticulum.
Authors: Hesse J E , Wieczorek L , Altendorf K , Reicin A S , Dorus E , Epstein W
Published: May 2006

We have determined the DNA sequences of the genes encoding the three structural proteins of the Kdp-ATPase, an ATP-driven potassium transport system of Escherichia coli. Regions of the predicted amino acid sequence of KdpB, the phosphorylated protein of the system, are homologous to regions of the Ca2+-ATPase of rabbit sarcoplasmic reticulum. The phosphorylated aspartate residue of the latter is within a region of homology.
Molecular cloning and the total nucleotide sequence of the human c-Ha-ras-1 gene activated in a melanoma from a Japanese patient.
Authors: Sekiya T , Fushimi M , Hori H , Hirohashi S , Nishimura S , Sugimura T
Published: May 2006

The transforming gene of malignant melanoma tissue obtained from a Japanese patient and maintained in nude mice has been cloned in its biologically active form and identified as the c-Ha-ras-1 gene, a homologue of the viral Ha-ras gene. Nucleotide sequence analysis revealed that the genetic alteration responsible for the transforming activity of the melanoma oncogene was localized to a single point mutation in the second exon. The transversion of adenine to thymine results in the substitution of leucine for glutamine as amino acid residue 61 of the predicted p21 protein. Other nucleotide sequences spanning a 2.9-kilobase segment including the entire exons and introns were found to be exactly the same as those in a proto-oncogene from a normal Caucasian reported previously, except for base alterations explained as polymorphic differences.
Fluorescence quantum yield of visual pigments: evidence for subpicosecond isomerization rates.
Authors: Doukas A G , Junnarkar M R , Alfano R R , Callender R H , Kakitani T , Honig B
Published: May 2006

The fluorescence quantum yields (phi f) for bovine and squid rhodopsins are determined. Both pigments yield similar results, with an average value for phi f of 1.2 (+/- 0.5) X 10(-5). Since the estimated radiative lifetime of rhodopsin is 5 nsec, the rate constant of the process that competes with fluorescence must be on the order of 0.1 psec. Given the large quantum yield for isomerization of rhodopsin's retinal chromophore, this process is likely to correspond to the motion along retinal's C11-C12 torsional coordinate that leads to cis-trans isomerization. An empirical excited-state potential energy curve along this coordinate is derived. It is shown that subpicosecond torsional motion to highly twisted nonfluorescing regions of the potential is possible and, in fact, likely. Our results require the existence of a barrier-less excited-state potential energy curve and suggest that cis-trans isomerization occurs in less than 1 psec.
Maturation of catalase precursor proceeds to a different extent in glyoxysomes and leaf peroxisomes of pumpkin cotyledons
Authors: Yamaguchi Junji , Nishimura Mikio , Akazawa Takashi
Published: May 2006

As an approach to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in greening pumpkin cotyledons, catalase molecules were purified from the two different types of microbody and their structural properties were compared. The purified glyoxysomal catalase was found to consist of four identical subunits (55 kDa), whereas the leaf peroxisomal catalase contains two different forms of monomeric subunit (55 and 59 kDa). These different catalase species cross-reacted with the rabbit antibody raised against the glyoxysomal enzyme. During gel filtration on an Ultrogel AcA 34 column, the leaf peroxisomal 55-kDa polypeptide eluted slightly faster than the leaf peroxisomal 59-kDa polypeptide. The profile of catalase activities exactly paralleled the elution pattern of the 55-kDa molecules, which indicated that the 59-kDa polypeptide was enzymically inactive. Peptide mapping analysis using
Staphylococcus aureus
protease V8 showed that the glyoxysomal 55-kDa polypeptide was identical to the leaf peroxisomal 55-kDa species, whereas the leaf peroxisomal 59-kDa polypeptide had a different primary structure from the 55-kDa polypeptide. In an
in vitro
translation system directed by mRNA isolated from etiolated and green cotyledons, glyoxysomal and leaf peroxisomal catalases were synthesized as the identical 59-kDa polypeptide. From peptide mapping analysis, the
in vitro
-translated 59-kDa polypeptide was found to have a nearly identical primary structure to that of the leaf peroxisomal 59-kDa species.
In vivo
pulse-chase labeling experiments using etiolated cotyledons showed the conversion of the 59-kDa polypeptide to the 55-kDa molecular species. The overall results strongly indicate that the 59-kDa polypeptide is a precursor form of catalase in pumpkin cotyledons.
Characterization of a cloned DNA sequence that is present at centromeres of all human autosomes and the X chromosome and shows polymorphic variation.
Authors: Jabs E W , Wolf S F , Migeon B R
Published: May 2006

We have identified a human DNA recombinant (p308) with a 3.0-kilobase (kb) BamHI insert that hybridizes in situ exclusively to the centromeric region of all human autosomes and the X chromosome. This highly repetitive sequence is significantly enriched on several chromosomes, most prominently on chromosome 6. In all individuals, the majority of genomic repeats are organized as tandem 3.0-kb BamHI repeats, each containing one Taq I site; the others are organized into BamHI and Taq I repeats of variable size that have some chromosome specificity. Using mouse-human hybrids, we have defined the specific organization of this sequence on chromosomes 6, 3, and X. In some individuals, there are differences in the number and nature of the tandem repeats. These polymorphisms segregate in families as if chromosome specific. Although variable from one chromosome to another, 308 contains sequences homologous to DNA present in centric heterochromatin of essentially all human chromosomes and is evolutionarily conserved. Therefore, a significant component of pericentric DNA is similar for all human chromosomes.
Cryospectrokinetic characterization of intermediates in biochemical reactions: carboxypeptidase A.
Authors: Auld D S , Galdes A , Geoghegan K F , Holmquist B , Martinelli R A , Vallee B L
Published: May 2006

Cryospectrokinetic studies provide concurrent structural, kinetic, and chemical data on short-lived intermediates in the course of the interactions of enzymes with their substrates and of other, similar pairs of biomolecules. Subzero temperatures extend the lifetimes of these intermediates and, combined with rapid-mixing and rapid-scanning instrumentation, allow simultaneous measurement of both their physical-chemical and kinetic characteristics. For carboxypeptidase A, the spectra of a chromophoric, enzymatically functional cobalt atom at the active site signal the structure of the coordination complex during catalysis, while radiationless energy transfer between enzyme tryptophans and the fluorescent dansyl blocking group of rapidly hydrolyzed peptide and ester substrates provides the basis for measurement of the rates of formation and breakdown of intermediates. Subzero radiationless energy transfer kinetic studies of the zinc and cobalt enzymes disclose two intermediates in the hydrolysis of both peptides and esters and furnish all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2----E + P. The chemical and kinetic data indicate that neither of these is an acylenzyme intermediate. Both absorption and EPR spectra of the ES2 reaction intermediates consistently demonstrate the formation of transient metal complexes, differences between the effects induced by peptides and esters, and strong similarities between those induced by all peptides on the one hand and all esters on the other. The marked alterations of the cobalt spectra likely reflect the coordination of a substrate carboxyl and/or carbonyl group to the metal at a critical step in the course of catalysis. The cryospectrokinetic approach developed here in the mechanistic study of this metalloenzyme is applicable to the examination of transients of biochemical reactions in general. It will allow molecular characterization of previously elusive intermediates and greatly magnify the range of mechanistic questions that can be answered.
Distinct organization of methylcholanthrene- and phenobarbital-inducible cytochrome P-450 genes in the rat.
Authors: Sogawa K , Gotoh O , Kawajiri K , Fujii-Kuriyama Y
Published: May 2006

The complete nucleotide sequence of the methylcholanthrene-inducible cytochrome P-450c gene was determined by sequence analysis of cloned genomic DNA and the sequence, consisting of 524 amino acids, of the protein was deduced therefrom. The gene for the cytochrome was approximately 6.0 kilobases long and was split into seven exons. Comparison of the gene with that of the phenobarbital-inducible cytochrome P-450e showed that the gene structures for the two types of cytochrome P-450 differ greatly; the location, number, and size of intervening sequences are very dissimilar. However, the sequence homology between the two types of cytochrome suggests that the two genes have evolved from a common ancestor.
A Ca2+-activated channel from Xenopus laevis oocyte membranes reconstituted into planar bilayers.
Authors: Young G P , Young J D , Deshpande A K , Goldstein M , Koide S S , Cohn Z A
Published: May 2006

Plasma membrane fractions from Xenopus laevis oocytes were incorporated into planar lipid bilayers. We show the existence of numerous Ca2+-activated nonspecific channels that are more permeable to anions. These channels are activated by Ca2+ at micromolar concentration but not by Mg2+, Zn2+, or Mn2+, even at millimolar concentrations. Decreasing Ca2+ concentration to less than 1 microM decreases the time of channel opening until channels close completely in the absence of Ca2+ and in the presence of EGTA. I- and Br- are more permeable through this channel than Cl-. The time during which the channels remain open is also voltage-dependent, with the channels switching off at higher voltages in both polarities. Single-channel activity shows a conductance of 380 pS in 1 M NaCl and 1 mM CaCl2, with an average open lifetime of 1.5 s at 40 mV. Similar channels are found in different stages of oocyte maturation. These observations support the hypothesis that an increase in oocyte-free Ca2+ activates directly these channels, and the resultant Cl- efflux forms the ionic basis for the fertilization potential in X. laevis.
Cloning and sequence analysis of the human major histocompatibility complex gene DC-3 beta.
Authors: Boss J M , Strominger J L
Published: May 2006

The DC antigen is one of the class II major histocompatibility antigens involved in the regulation of the immune response. This molecule is a heterodimer composed of an alpha and a beta chain. Southern blot analysis of several homozygous cell lines shows that there are two DC beta genes. The DC-3 beta gene, corresponding to a polymorphic restriction fragment, was cloned and sequenced and found to exist in five exons spanning 8 kilobase pairs of DNA. These exons correspond to the functional domains of the DC beta protein. Comparison of the beta 1 domains of known DC beta chains shows that the polymorphism is clustered in four regions. A similar comparison of the mouse A beta sequences shows only two prominent diversity regions. The DC beta chain sequences are eight amino acids shorter than the A beta chain sequences due to the elimination of a small exon by an aberrant splice acceptor.
Use of monoclonal antibodies as sensitive and specific probes for biologically active human gamma-interferon.
Authors: Chang T W , McKinney S , Liu V , Kung P C , Vilcek J , Le J
Published: May 2006

Mouse monoclonal antibodies B1 and B3 are specific for natural and Escherichia coli-derived recombinant human gamma-interferon (IFN-gamma). The two antibodies recognize different epitopes of the IFN-gamma molecule and do not compete with each other's binding. We have used these two antibodies to construct a solid-phase, sandwich immunoradiometric assay for human IFN-gamma. Purified antibody B1 was coated on polystyrene beads (0.64 cm in diameter) and used as the solid-phase immunoadsorbent and antibody B3 was labeled with 125I and used as tracer. This assay can be completed in about 4 hr and is capable of detecting IFN-gamma levels in human serum or tissue culture fluids as low as 0.1 NIH reference unit/ml. Recombinant human IFN-gamma derived from E. coli was detectable at a concentration of 0.02 ng/ml. The assay appears to be specific for the biologically active forms of IFN-gamma, since after exposure to pH 2, 37 degrees C, or 56 degrees C, biological activity and reactivity in the immunoradiometric assay decreased in parallel. The immunoradiometric assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule.
Dose-related effect of acetylsalicylic acid on replication of varicella zoster virus in vitro.
Authors: Walz-Cicconi M A , Weller T H
Published: May 2006

Cultivation of human embryonic lung (HEL) cells in media containing acetylsalicylic acid (ASA) at 100 micrograms/ml and maintenance at this level after inoculation with either cell-free varicella zoster virus (VZV) or virus-infected cells resulted in a 2- to 4-fold increase in yields of cell-free virus released by sonication. The degree of enhancement was dependent upon multiplicity of infection and time of harvest. Enhanced viral yields were not consistently accompanied by an increase in the number of infected cells, nor was VZV plaque formation in HEL indicator cells significantly increased in the presence of ASA at 100 micrograms/ml. In the presence of ASA at 500-1000 micrograms/ml, VZV plaque formation was inhibited; this inhibition was partially reversible, depending on concentration and period of exposure to ASA. These findings may bear on the apparent association between ASA ingestion and the development of Reye syndrome after infection with varicella virus.
Racemization of aspartic acid and phenylalanine in the sweetener aspartame at 100 degrees C.
Authors: Boehm M F , Bada J L
Published: May 2006

The racemization half-lives (i.e., the time required to reach a D/L = 0.33) at pH 6.8 for aspartic acid and phenylalanine in the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester) were determined to be 13 and 23 hours, respectively, at 100 degrees C. Racemization at this pH does not occur in aspartame but rather in its diketopiperazine decomposition product. Our results indicate that the use of aspartame to sweeten neutral pH foods and beverages that are then heated at elevated temperature could generate D-aspartic acid and D-phenylalanine. The nutritive consequences of these D-amino acids in the human diet are not well established, and thus aspartame should probably not be used as a sweetener when the exposure of neutral pH foods and beverages to elevated temperatures is required. At pH 4, a typical pH of most foods and beverages that might be sweetened with aspartame, the half-lives are 47 hours for aspartic acid and 1200 hours for phenylalanine at 100 degrees C. Racemization at pH 4 takes place in aspartame itself. Although the racemization rates at pH 4 are slow and no appreciable racemization of aspartic acid and phenylalanine should occur during the normal use of aspartame, some food and beverage components could conceivably act as catalysts. Additional studies are required to evaluate whether the use of aspartame as a sugar substitute might not in turn result in an increased human consumption of D-aspartic acid and D-phenylalanine.
Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins.
Authors: Lacal J C , Santos E , Notario V , Barbacid M , Yamazaki S , Kung H , Seamans C , McAndrew S , Crowl R
Published: May 2006

The H-ras gene of the BALB murine sarcoma virus (BALB-MSV) was placed under the transcriptional control of the tightly regulated PL promoter of bacteriophage lambda in the expression vectors pEV-vrf-1 and pRC23. Upon derepression of the PL promoter, large amounts (10-20% of total cellular protein) of the H-ras gene product p21 are synthesized in Escherichia coli. We constructed three H-ras gene expression vectors, designated pJCL-H5, pJCL-E30, and pJCL-33. pJCL-H5 directs the synthesis of p21, a fusion protein whose four amino-terminal residues are replaced by eight amino acids coded for by plasmid sequences. The 13 5' coding nucleotides of the BALB-MSV H-ras gene missing in pJCL-H5 were regenerated in pJCL-E30 by inserting a pair of complementary synthetic oligodeoxynucleotides. As a result, pJCL-E30 encodes a p21 protein, p21T, of sequence identical to that of the transforming p21 protein of BALB-MSV. pJCL-33 is a derivative of pJCL-E30 in which the 12th codon, AAA, a lysine codon, was replaced by GGA, a glycine codon. Thus, pJCL-33 directs the synthesis of a p21 protein, p21N, whose sequence corresponds to that of a normal cellular p21 protein. We report the purification of H-ras p21 proteins to apparent homogeneity by a method involving solubilization with chaotropic agents followed by reverse-phase high-performance liquid chromatography.
Translational regulation of the L11 ribosomal protein operon of Escherichia coli: analysis of the mRNA target site using oligonucleotide-directed mutagenesis.
Authors: Baughman G , Nomura M
Published: May 2006

The L11 ribosomal protein operon in Escherichia coli consists of the genes for proteins L11 and L1 and is feedback regulated by the translational repressor L1. The mRNA target site for this repression is located close to the translation initiation site of the first L11 cistron. Several mutant plasmid molecules carrying altered nucleotide sequences in the L1 target site were constructed by site-directed in vitro mutagenesis using synthetic oligodeoxyribonucleotides. Specifically, we examined the importance of a presumptive double-stranded stem structure that is common among L1 binding sites on rRNA from a variety of organisms and in L11 mRNA. Mutational alterations that disrupt the stem structure were found to abolish translational regulation as analyzed both in vitro and in vivo. Two of the mutations were combined so that the stem structure was restored but with a different primary nucleotide sequence. This double mutant was shown to restore the original phenotype, the ability to be translationally regulated by L1. These experiments show the importance of the stem structure, but not its primary sequence, for the interaction of L1 with the mRNA and support the concept that mRNA target sites share some structural features with the corresponding ribosomal protein binding sites of rRNA.
Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract.
Authors: Hammond S L , Ham R G , Stampfer M R
Published: May 2006

A serum-free medium with bovine pituitary extract as the only undefined supplement has been developed for long-term culture of human mammary epithelial cells. This medium supports serial subculture of normal cells for 10-20 passages (1:10 splits) without conditioning or special substrates, and it supports rapid clonal growth with plating efficiencies up to 35%. It consists of an optimized basal nutrient medium, MCDB 170, supplemented with insulin, hydrocortisone, epidermal growth factor, ethanolamine, phosphoethanolamine, and bovine pituitary extract. Replacement of pituitary extract with prostaglandin E1 and ovine prolactin yields a defined medium that supports rapid clonal growth and serial subculture for three or four passages. Cultures initiated in these media from normal reduction mammoplasty tissue remain diploid and maintain normal epithelial morphology, distribution of cell-associated fibronectin, expression of keratin fibrils, and a low level of expression of milk fat globule antigen. Large cell populations can now be generated and stored frozen, permitting multiple experiments over a period of time with cells from a single donor. These media greatly extend the range of experiments that can be performed both conveniently and reproducibly with cultured normal and tumor-derived human mammary epithelial cells.
“Hopeful monsters,” transposons, and Metazoan radiation
Authors: Erwin Douglas H. , Valentine James W.
Published: May 2006

The appearance of many novel morphologies, frequently expressed taxonomically as new phyla, classes, or orders, occurs with such rapidity in evolutionary time that microevolutionary substitutions involving structural genes seem an implausible mechanism. It has been suggested that such novelties are produced by changes in developmental and regulatory structures and patterns rather than by an accumulation of single structural gene changes. The horizontal transmission of genetic material via RNA-based viruses between members of a population may rapidly create intrafertile sub-populations that differ markedly from their parents and form the basis of new morphological types, avoiding the usual fitness problems associated with “hopeful monsters.”
Cloning and characterization of cDNAs encoding human gastrin-releasing peptide.
Authors: Spindel E R , Chin W W , Price J , Rees L H , Besser G M , Habener J F
Published: May 2006

We have prepared and cloned cDNAs derived from poly(A)+ RNA from a human pulmonary carcinoid tumor rich in immunoreactivity to gastrin-releasing peptide, a peptide closely related in structure to amphibian bombesin. Mixtures of synthetic oligodeoxyribonucleotides corresponding to amphibian bombesin were used as hybridization probes to screen a cDNA library prepared from the tumor RNA. Sequencing of the recombinant plasmids shows that human gastrin-releasing peptide (hGRP) mRNA encodes a precursor of 148 amino acids containing a typical signal sequence, hGRP consisting of 27 or 28 amino acids, and a carboxyl-terminal extension peptide. hGRP is flanked at its carboxyl terminus by two basic amino acids, following a glycine used for amidation of the carboxyl-terminal methionine. RNA blot analyses of tumor RNA show a major mRNA of 900 bases and a minor mRNA of 850 bases. Blot hybridization analyses using human genomic DNA are consistent with a single hGRP-encoding gene. The presence of two mRNAs encoding the hGRP precursor protein in the face of a single hGRP gene raises the possibility of alternative processing of the single RNA transcript.
Oxidation of leukotrienes at the omega end: demonstration of a receptor for the 20-hydroxy derivative of leukotriene B4 on human neutrophils and implications for the analysis of leukotriene receptors.
Authors: Clancy R M , Dahinden C A , Hugli T E
Published: May 2006

Leukotriene B4 [LTB4; (5S,12R)-5,12-dihydroxy-6,14-cis-8,10-trans-icosatetraenoic acid] and its 20-hydroxy derivative [20-OH-LTB4; (5S,12R)-5,12,20-trihydroxy-6,14-cis-8,10-trans-icosatetraenoic acid] are principal metabolites produced when human neutrophils (hPMNs) are stimulated by the calcium ionophore A23187. These compounds were purified to homogeneity by Nucleosil C18 and silicic acid HPLC and identified by UV absorption and gas chromatographic/mass spectral analyses. 20-OH-LTB4 is considerably more polar than LTB4 and interacts weakly with the hydrophobic Nucleosil C18 resin, whereas LTB4 interacts strongly, reflecting the hydrophobic C13-C20 domain in LTB4. Specific binding of highly purified [3H]LTB4 and [3H]20-OH-LTB4 to hPMNs was assessed. Binding of [3H]20-OH-LTB4 could be largely displaced by an excess of nonlabeled LTB4 or 20-OH-LTB4 but not by 15-hydroxyicosatetraenoic acid (15-HETE), (5S,12S)-5,12-dihydroxy-6,10-trans-8,14-cis-icosatetraenoic acid [(5S,12S)-diHETE], or the 6-trans stereoisomer of LTB4 at 1 microM. In contrast, [3H]LTB4 displays a high level of nonspecific binding to human PMNs, which makes assessment of the Kd for LTB4 binding unobtainable. Binding measurements for [3H]LTB4 were performed in a buffer containing bovine serum albumin, and under these conditions significantly less nonspecific binding was observed. The apparent Kd for high-affinity binding sites on human PMNs at 0 degrees C was 31.3 X 10(-9) M for LTB4 and 14.3 X 10(-9) M for 20-OH-LTB4. In addition, we observed a saturable low-affinity receptor for 20-OH-LTB4 with a Kd of approximately 100 X 10(-9) M and 2 X 10(5) receptors per cell. The data from this study suggest that omega oxidation represents a major pathway for metabolism of LTB4 as well as other arachidonate metabolites. LTB4 and 20-OH-LTB4 express similar functional activities and share common binding properties to hPMNs but differ significantly in their physical properties. It is the unique physical characteristics of 20-OH-LTB4 that suggest that arachidonate metabolites oxidized at the omega position may be more important agents in inflammation than LTB4.
Differential expression of mRNAs for alpha- and beta-tubulin during differentiation of the parasitic protozoan Leishmania mexicana.
Authors: Fong D , Wallach M , Keithly J , Melera P W , Chang K P
Published: May 2006

The parasitic protozoan Leishmania mexicana amazonensis has two developmental stages: a motile flagellated promastigote stage and a sessile intracellular amastigote stage. In our previous work, cells of the promastigote stage were found to synthesize more tubulin protein than those of the amastigote stage. Here, tubulin mRNAs in these leishmanias were analyzed. Based on dot blot hybridization between total leishmanial RNA and tubulin-specific cDNA probes derived from chicken brain, amastigotes and promastigotes were found to have approximately equal amounts of alpha- and beta-tubulin mRNAs. RNA blotting of leishmanial RNA, using chicken tubulin cDNA probes, showed that amastigotes and promastigotes both gave a single mRNA species of 2100 nucleotides for alpha-tubulin in roughly similar quantities. However, such analysis for beta-tubulin revealed mainly a single mRNA species of 3600 nucleotides for amastigotes and three species of 2800, 3600, and 4400 nucleotides for promastigotes, the smallest mRNA being the most predominant. Thus, regulation of gene expression appears to be different only for beta-tubulin between the two developmental stages of this protozoan.
Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus.
Authors: Kroos L , Kaiser D
Published: May 2006

A promoterless trp-lac fusion fragment was inserted near one end of the bacterial transposon Tn5 in the correct orientation to fuse lacZ gene expression to promoters outside Tn5. The resulting transposon, Tn5 lac, retains the kanamycin-resistance gene of Tn5 and transposes in Escherichia coli at 6% the frequency of Tn5 to many different sites in a bacteriophage lambda target. Expression of beta-galactosidase, the product of the lacZ gene, from Tn5 lac insertions in phage lambda depends both on insertion into a transcription unit in the correct orientation and on the regulation of the promoter of the transcription unit, verifying that by transposition Tn5 lac can fuse lacZ expression to outside promoters. An insertion of Tn5 lac in bacteriophage P1 was isolated and used to introduce Tn5 lac into Myxococcus xanthus, a bacterium that undergoes multicellular development. Stable kanamycin-resistant transductants are obtained that contain no P1 DNA sequences but have Tn5 lac inserted at different sites in the Myxococcus chromosome. Individual transductants express different levels of beta-galactosidase. A chromogenic substrate of beta-galactosidase, 5-bromo-4-chloro-3-indolyl beta-D-galactoside, is toxic in Myxococcus when cleaved in large amounts. In principle, Tn5 lac could be used to assay transcription in any bacterium in which Tn5 can transpose and beta-galactosidase can be measured.
Rotational isomerism about the 17(20)-bond of steroids and euphoids as shown by the crystal structures of euphol and tirucallol.
Authors: Nes W D , Wong R Y , Benson M , Landrey J R , Nes W R
Published: May 2006

The influence of configuration at C-20 on rotation about the 17(20)-bond in steroids and euphoids was examined by x-ray crystallographic studies of the C-20 epimers euphol and tirucallol. The H atom on C-20 was in back next to C-18 in the crystal structures of both of the compounds, and C-22 was found to be cis-oriented ("left-handed") to C-13 in euphol and trans-oriented to it ("right-handed") in tirucallol. The results, which are consistent with the known left-handed crystal structure of 24(25)-dihydroeuphol and right-handed crystal structure of cholesterol and other natural sterols, lend further credence to the earlier suggestion that rotational isomerism at the 17(20)-bond can arise in C-20 epimers and that there is preference for an arrangement with the 20-H atom adjacent to C-18.
Intermediate filament cDNAs from BHK-21 cells: demonstration of distinct genes for desmin and vimentin in all vertebrate classes.
Authors: Quax W , van den Heuvel R , Egberts W V , Quax-Jeuken Y , Bloemendal H
Published: May 2006

Recombinant cDNA plasmids for the intermediate filament proteins desmin and vimentin were constructed from baby hamster kidney (BHK-21) mRNA. Analysis of four desmin clones gave a sequence of 1574 nucleotides, which is 75% of the total mRNA length. The derived amino acid sequence for hamster desmin shows 92% overall homology with chicken desmin; the homology with hamster vimentin is highest in the alpha-helical middle part (74%). The 3'-noncoding region of desmin mRNA is found to be 677 nucleotides long. With the aid of 5'- and 3'-specific probes, it has been established that there is a single gene for desmin in the hamster genome. This gene expresses a single mRNA species of 2.2 kilobases. Hybridization experiments of a number of DNAs with desmin and vimentin probes show that there are distinct restriction enzyme fragments carrying vimentin and desmin sequences in the genome of representatives of all vertebrate classes.
Homology of adenoviral E3 glycoprotein with HLA-DR heavy chain.
Authors: Chatterjee D , Maizel J V
Published: May 2006

The Mr 19,000 adenovirus glycoproteins coded by the E3 region that is expressed on the cell membrane and presumably binds to HLA class I antigen shows sequence homology to the alpha-chain of human HLA-DR and to the kappa-chain of human IgM. The homology extends to a conserved domain present in all of the major histocompatibility complex antigens, beta 2-microglobulins, and immunoglobulins. Predicted beta-sheet secondary structures are similar between the Mr 19,000 adenovirus protein and these immune system proteins. Evolutionary and functional implications of the homology are discussed.
Inhibition by restricted-calorie diet of lymphoproliferative disease and renal damage in MRL/lpr mice.
Authors: Fernandes G , Good R A
Published: May 2006

Restriction of calorie intake from the time of weaning greatly prolongs life, and it inhibits development and expression of the lymphoproliferative syndrome, renal disease, and decline of certain immunologic functions with age in MRL/lpr mice. This dramatic influence of diet on mice of this short-lived autoimmunity-prone strain, while associated with decreased rate of growth, is not associated with debilitation or apparent disease in the MRL/lpr mice. The massive lymphadenopathy and splenomegaly that developed in the putatively well-fed animals was prevented by dietary restriction, as were histopathologic abnormalities of thymus, spleen, lymph nodes, and kidneys.
Thyrotropin-releasing hormone stimulates GTP hydrolysis by membranes from GH4C1 rat pituitary tumor cells.
Authors: Hinkle P M , Phillips W J
Published: May 2006

Thyrotropin-releasing hormone (TRH) stimulates prolactin production by GH4C1 rat pituitary tumor cells, which possess high-affinity membrane receptors for the peptide. TRH caused up to a 50% increase in the activity of a low-Km GTPase in membranes from GH4C1 cells. The TRH stimulatory effect was maximal at GTP concentrations of 1 microM or lower. TRH caused an increase in GTPase activity of between 0.2 and 20 pmol of GTP hydrolyzed per mg of protein per min, depending on GTP concentration, while TRH binding was 0.3 pmol/mg of protein. TRH did not stimulate GTPase activity in membranes from GH12C1, or GH-Y cells, two pituitary lines lacking TRH receptors. Stimulation of GTPase depended on occupancy of the TRH receptor; half-maximal increases in GTPase activity required 46 nM TRH and 25 nM [N3-methyl-His]TRH, but the TRH free acid was inactive. The apparent Kds of these peptides for receptors were similar when measured under the same conditions. The fact that TRH binding to receptors is regulated by guanyl nucleotides, together with the demonstration of TRH stimulation of low-Km GTPase activity, suggests that the TRH receptor is associated with a guanyl nucleotide regulatory protein in the lactotroph membrane.
Nuclease protection analysis of ribonucleoprotein complexes: use of the cytotoxic ribonuclease alpha-sarcin to determine the binding sites for Escherichia coli ribosomal proteins L5, L18, and L25 on 5S rRNA.
Authors: Huber P W , Wool I G
Published: May 2006

A rapid and convenient method has been devised to determine the binding sites for proteins on RNA. The procedure is an adaptation of one used to map DNA-protein complexes by protection against nuclease digestion. The method uses the cytotoxic ribonuclease alpha-sarcin, which hydrolyzes purines in both single- and double-stranded regions of RNA. It has been authenticated by confirming the binding sites for the Escherichia coli ribosomal proteins L18 and L25 on 5S rRNA and its value has been established by identifying the attachment site for protein L5. The procedure should be useful for the analysis of other ribonucleoprotein complexes.
tRNAMetf2 gene in the leader region of the nusA operon in Escherichia coli.
Authors: Ishii S , Kuroki K , Imamoto F
Published: May 2006

The promoter-proximal portion of the operon containing the Escherichia coli nusA gene has been cloned. Its nucleotide sequence shows that genes for tRNAMetf2 and a 15-kilodalton protein of unknown function precede the nusA protein gene. The sequence suggests that the three genes form a single transcription unit. Consistent with this hypothesis, purified RNA polymerase formed full-length transcripts on the cloned DNA in vitro, although transcription was frequently arrested at the intercistronic site(s) between the gene for tRNAMetf2 and the 15-kilodalton protein.
Direct isolation of the functional human thymidine kinase gene with a cosmid shuttle vector.
Authors: Lau Y F , Kan Y W
Published: May 2006

We have developed a new recombinant DNA cloning system to isolate directly the functional unit of the human thymidine kinase (TK) gene. The system utilizes a cosmid vector that can shuttle cloned DNA sequences between bacteria and mammalian cells. A complete human cosmid library was constructed and DNA from the total library was transfected to mouse L cells deficient in TK (LTK-) by calcium phosphate precipitation. The transfected cells were then selected with hypoxanthine/aminopterin/thymidine (HAT) medium, and one HAT-resistant cell clone was isolated. This cell line became resistant to HAT selection by acquiring the TK gene derived from the human cosmid library. As the cosmid vector contains the cohesive ends of the bacteriophage, we could directly retrieve the human DNA sequences from the transformed mouse L cells. Total DNA from the transformed TK+ L cells was packaged in vitro with lysogenic bacterial extracts and used to infect Escherichia coli. One of the two recombinant cosmids isolated contained a 43.8-kilobase human DNA insert and was capable of converting TK- L cells to the TK+ phenotype in both acute and stable transformation assays. Thus, we have isolated the functional human TK gene in this recombinant cosmid. The gene was further localized on a 14.5-kilobase BamHI DNA fragment, and it transcribed a mature mRNA of about 1,500 nucleotides. This method of gene isolation has several special features: (i) an intact structural gene can be cloned directly based on its function without knowledge of its amino acid or nucleotide sequence; (ii) the functional gene sequences can be recovered faster and more efficiently than with the usual DNA transfection method; and (iii) in conjunction with cell-sorting techniques, this method can be used to clone genes encoding cell surface markers.
Uptake of lectins by pulmonary alveolar type II cells: subsequent deposition into lamellar bodies.
Authors: Williams M C
Published: May 2006

Type II cells of the alveolar epithelium of adult rats have been shown to internalize the ferritin-labeled lectin from Maclura pomifera (MPA-F). This alpha-galactose-binding molecule binds specifically to the apical plasma membrane of the cells. Once within the cell the lectin is cycled from pinocytic vesicles, to multivesicular bodies of two types, and finally to lamellar bodies, the storage granules of surfactant. Those multivesicular bodies that first contain MPA-F lack detectable lysosomal enzymes, while those labeled later are reactive. For 30-60 min, uptake of MPA-F is blocked by adding methyl alpha-D-galactoside to the instillate. Lectins that have no or limited binding to type II cells are taken up in amounts similar to fluid phase markers. These observations indicate that type II cells can take up substances from alveoli by the process of adsorptive endocytosis and deposit the ingested material into lamellar bodies.
Body size and food size in freshwater zooplankton
Authors: Bogdan Kenneth G. , Gilbert John J.
Published: May 2006

We used double-label liquid scintillation techniques to measure the efficiencies with which eight different-sized zooplankton species ingested four cell types relative to a standard cell type (
Chlamydomonas
). Efficiency ratios (ERs: clearance rate on cell type X ÷ clearance rate on
Chlamydomonas
) on the three ultraplankton (<5 μm in diameter) cells (a coccoid bacterium and the algae
Synechococcus
and
Nannochloris
) varied greatly among zooplankton species but were not correlated with zooplankton body length. Variation in ERs on a much larger (17 × 14 μm) algal cell (
Cryptomonas
) was only partly explained by zooplankton body length. The eight zooplankton species were classified into three functional groups: (
i
) species having moderate to high ERs on all ultraplankton (0.4 < ER < 1.6) and ERs on
Cryptomonas
proportional to their body lengths (
Conochilus, Diaphanosoma
, and probably
Keratella cochlearis
and
Ceriodaphnia
); (
ii
) species having extremely low ERs on bacteria (mean ER < 0.05), higher but still low ERs on ultraphytoplankton (ER generally < 0.4), and ERs on
Cryptomonas
proportional to their body lengths (
Bosmina, Diaptomus
copepodites and adults); (
iii
) species having extremely low ERs on all ultraplankton (mean ER < 0.05) and ERs on
Cryptomonas
much higher than expected given their body lengths (
Keratella crassa, Polyarthra
, and
Diaptomus
nauplii). These functional groups follow neither taxonomic nor body-length groupings. We conclude that zooplankton body length may influence the maximal particle size a species can ingest but has little influence on the ingestion of smaller particles. Two frequently used models relating zooplankton body size and food size are unrealistic.
Transcriptional activation of the rat liver tyrosine aminotransferase gene by cAMP.
Authors: Hashimoto S , Schmid W , Schütz G
Published: May 2006

The enzyme tyrosine aminotransferase (Tyr-ATase; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), which is synthesized in rat liver, is induced by glucocorticoids, insulin, and glucagon or its intracellular mediator cAMP. We have used cloned TyrATase genomic and cDNA sequences to study the mechanism of induction by cAMP. RNA blot analysis shows that cAMP causes a rapid 5-fold increase in TyrATase mRNA concentration in rat liver. Transcription in isolated rat liver nuclei was studied to determine the relative rate of transcription of the TyrATase gene after cAMP administration. We show that the accumulation of TyrATase mRNA after cAMP stimulation is a consequence of transcriptional activation of the TyrATase gene. Combined dexamethasone and cAMP treatment leads to higher TyrATase mRNA concentrations than each inducer alone, which implies that dexamethasone and cAMP act by distinct mechanisms.
A dual role for the Ca2+-requiring proteinase in the degradation of hemoglobin by erythrocyte membrane proteinases.
Authors: Pontremoli S , Melloni E , Sparatore B , Michetti M , Horecker B L
Published: May 2006

Binding of hemoglobin chains to erythrocyte membranes is an obligatory step in the conversion of hemoglobin to acid-soluble products by erythrocyte proteinases. This binding requires limited proteolysis of the hemoglobin chains and also modification of the inner surface of the erythrocyte membrane, both of which result from the action of a soluble Ca2+-requiring neutral proteinase. Final digestion of the bound hemoglobin chains in the membrane complex results from the action of intrinsic membrane endopeptidases. Regulation of the activity of the Ca2+-requiring proteinase by the substrate provides a mechanism for the initiation of selective protein turnover.
Inhibition of epidermal growth factor-induced mitogenesis by amiloride and an analog: evidence against a requirement for Na+/H+ exchange.
Authors: Besterman J M , Tyrey S J , Cragoe E J , Cuatrecasas P
Published: May 2006

We have tested the hypothesis that the rapid stimulation of Na+/H+ exchange by epidermal growth factor (EGF) is a requirement for induction of mitogenesis. BALB/c 3T3 cells exposed for 4 hr at 37 degrees C to both EGF at 1 ng/ml and either 0.2-1 mM amiloride (an inhibitor of Na+/H+ exchange) or 10 microM MK-685 (an amiloride analog and more potent inhibitor of Na+/H+ exchange) incorporated no less [methyl-3H]thymidine during a 1-hr pulse 20 hr later than did cells exposed for 4 hr to EGF alone. Control experiments utilizing low external pH (to dissociate EGF from its receptor) and anti-EGF antibodies indicated that the failure of amiloride to inhibit mitogenesis when copresent with EGF during the first 4 hr was not due to incomplete removal of EGF and complete removal of amiloride at t4. Cells incubated with 200 microM amiloride for 24 hr showed nearly complete inhibition of stimulation by EGF. In comparison, cells incubated with 10 microM MK-685 for 24 hr showed only a slight inhibition of stimulation by EGF. Incubations with amiloride or MK-685 for shorter periods of time indicated that only amiloride inhibited mitogenesis and that this inhibition happened between 4 (t4) and 10(t10) hr after EGF addition, during which time increases in RNA and protein synthesis (required for mitogenesis) occurred. Amiloride inhibited both RNA and protein syntheses in intact cells during this prereplicative period, while MK-685 was without effect. We conclude that (i) inhibition of EGF-induced mitogenesis by amiloride is due not to inhibition of EGF-stimulated Na+/H+ exchange but rather to inhibition of necessary events occurring during the hours immediately prior to the onset of DNA synthesis, these events probably being RNA and protein synthesis and (ii) in cell culture medium buffered with CO2/HCO3-, complete inhibition of EGF-stimulated Na+/H+ exchange does not inhibit EGF-induced mitogenesis and, thus, stimulation of Na+/H+ exchange is not necessary for induction of mitogenesis by EGF.
Nucleotide sequence of the spo0B gene of Bacillus subtilis and regulation of its expression.
Authors: Bouvier J , Stragier P , Bonamy C , Szulmajster J
Published: May 2006

The spo0B gene is one of the genes involved in initiation of sporulation of Bacillus subtilis. This gene, previously cloned into the pHV33 shuttle vector, is expressed in Escherichia coli and B. subtilis. We have determined the sequence of 1118 base pairs (bp) of the DNA insert carrying the spo0B gene. The promoter sequence of this gene shows the canonical T-A-T-A-A-T region at 10 bp from the transcriptional start (-10 region) but an unusual sequence, T-T-T-T-C-T-, in the -35 region. The nucleotide sequence shows an open reading frame encoding a 192-amino-acid polypeptide of Mr 22,542, which is close to the molecular weight of the spo0B product synthesized in E. coli minicells. To investigate the regulation of the spo0B gene under a variety of physiological conditions, we constructed an in-frame fusion between the spo0B promoter proximal region and the lacZ gene of E. coli. This hybrid gene was subsequently integrated into the B. subtilis chromosome, and the beta-galactosidase activity was measured. It was found that the spo0B gene is preferentially expressed during exponential growth; it is not induced by exhaustion of the growth medium nor repressed by glucose.
Novel B-cell maturation factor from spontaneously autoimmune viable motheaten mice.
Authors: Sidman C L , Marshall J D , Masiello N C , Roths J B , Shultz L D
Published: May 2006

Both in vivo and in vitro, mice homozygous for the viable motheaten mutation show severe immunodeficiency, polyclonal B-cell activation and Ig secretion, and spontaneous production of a lymphokine [B-cell maturation factor (BMF)] that directly drives the maturation of normal or tumor B cells to the state of active Ig secretion. BMF from motheaten mice is distinct from previously identified forms in its cells of origin (B cells) and biochemical characteristics (apparent Mr 15,000 by gel filtration and NaDodSO4/PAGE; pI 4.3 by chromatofocusing). Among the known murine single-gene models of autoimmunity, only motheaten mice show high levels of spontaneous BMF production, which therefore may be an important component in the development of this form of autoimmunity/immunodeficiency disease. The coincidence of spontaneous BMF production and uncontrolled Ig secretion within the same mutant mouse constitutes the strongest evidence to date for a significant physiological (in vivo) role for BMFs.
Nucleotide sequence of cDNA and derived amino acid sequence of human complement component C9.
Authors: DiScipio R G , Gehring M R , Podack E R , Kan C C , Hugli T E , Fey G H
Published: May 2006

The nucleotide sequence coding for the ninth component of human complement (C9) has been determined and the corresponding amino acid sequence has been derived. A human liver cDNA library was screened by the colony-hybridization technique using two radiolabeled oligonucleotide probes that correspond to known regions of the C9 amino acid sequence. Two recombinant plasmids were isolated and their cDNA inserts were sequenced. The derived protein sequence consists of 537 amino acids in a single polypeptide chain. A profile of the hydropathic index versus sequence number indicates that the amino-terminal half of C9 is predominantly hydrophilic in character whereas the carboxyl-terminal section of this protein is more hydrophobic. The amphipathic organization of the primary structure of C9 is consistent with the known potential of polymerized C9 to penetrate lipid bilayers, causing the formation of transmembrane channels.
Anti-mRNA: specific inhibition of translation of single mRNA molecules.
Authors: Pestka S , Daugherty B L , Jung V , Hotta K , Pestka R K
Published: May 2006

A plasmid was constructed to generate RNA complementary to the beta-galactosidase mRNA under control of the phage lambda PL promoter. When this anti-mRNA was produced, synthesis of beta-galactosidase was dramatically inhibited (98%). Syntheses of galactoside permease and transacetylase, whose coding sequences are downstream of the beta-galactosidase coding region, are inhibited to a lesser degree, 80% and 55%, respectively. The generation of anti-mRNA that can be targeted to inhibit a single species of mRNA molecule within cells provides a potent mechanism by which specific transcripts can be translationally inactivated. This can be used to determine the function of proteins as well as to select cloned genes in a single rapid and convenient step.