bluuebunny/crossref_metadata_2025 huggingface dataset:
Lignin-degrading enzyme from
Phanerochaete chrysosporium
: Purification, characterization, and catalytic properties of a unique H
2
O
2
-requiring oxygenase
Authors: Tien Ming , Kirk T. Kent
Published: May 2006

An extracellular lignin-degrading enzyme from the basidiomycete
Phanerochaete chrysosporium
Burdsall was purified to homogeneity by ion-exchange chromatography. The 42,000-dalton ligninase contains one protoheme IX per molecule. It catalyzes, nonstereospecifically, several oxidations in the alkyl side chains of lignin-related compounds: C
α
—C
β
cleavage in lignin-related compounds of the type aryl—C
α
HOH—C
β
HR—C
γ
H
2
OH (R = -aryl or -
O
-aryl), oxidation of benzyl alcohols to aldehydes or ketones, intradiol cleavage in phenylglycol structures, and hydroxylation of benzylic methylene groups. It also catalyzes oxidative coupling of phenols, perhaps explaining the long-recognized association between phenol oxidation and lignin degradation. All reactions require H
2
O
2
. The C
α
—C
β
cleavage and methylene hydroxylation reactions involve substrate oxygenation; the oxygen atom is from O
2
and not H
2
O
2
. Thus the enzyme is an oxygenase, unique in its requirement for H
2
O
2
.
Complex carbohydrate diets are not capable of maintaining normal plasma calcium and phosphorus levels in vitamin D-deficient rats.
Authors: Underwood J L , Phelps M E , DeLuca H F
Published: May 2006

Substitution of Cerelose (glucose monohydrate) for complex carbohydrate (whole wheat flour) does not alter plasma calcium levels in vitamin D-deficient rats, contrary to a previous report. It is suspected that whole wheat flour may contain traces of vitamin D that result in a slower rate of depletion than found with Cerelose diets. Vitamin D-deficient rats showing low plasma 1,25-dihydroxyvitamin D3 levels and no detectable 25-hydroxyvitamin D3 levels in their blood show a hypocalcemia (5.6 mg/dl) and normal phosphatemia whether fed whole wheat or Cerelose diets.
Rotational barrier of a molybdenum-molybdenum quadruple bond
Authors: Collman James P. , Woo L. Keith
Published: May 2006

The synthesis and characterization of molybdenum(II) porphyrin dimers containing unbridged metal-metal quadruple bonds are presented. Variable temperature 300 MHz
1
H NMR studies of
meso
-substituted derivatives provide novel solution evidence for both the existence of quadruple bonds and for a barrier to rotation about the metal-metal bonds in these complexes. The activation energy for this rotational process is 10.1 ± 0.5 kcal·mol
-1
.
Cardiac alpha- and beta-myosin heavy chain genes are organized in tandem.
Authors: Mahdavi V , Chambers A P , Nadal-Ginard B
Published: May 2006

Two ventricular myosin heavy chains (MHCs), alpha and beta, which exhibit different levels of ATPase activity, are differentially expressed during development, in response to thyroid hormone and in several pathological conditions. We have isolated and analyzed the structure of the genes coding for alpha- and beta-MHC mRNAs in the rat. Detailed analysis of eight overlapping MHC genomic clones shows that the alpha- and beta-MHC genes are organized in tandem and span 50 kilobases of the chromosome. The beta-MHC gene, predominantly expressed in late fetal life, is located 4 kilobases upstream from the alpha-MHC gene, predominantly expressed in the adult. These two genes are very closely related at the nucleotide sequence level, suggesting that they have arisen by duplication of a common ancestor, yet their expression in the ventricular myocardium has been shown to be regulated in an antithetic fashion by thyroid hormone.
Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12.
Authors: Gibbs J B , Ellis R W , Scolnick E M
Published: May 2006

The 21,000-dalton protein (p21) encoded by the ras oncogene of Harvey murine sarcoma virus (v-Ha-ras) becomes phosphorylated (pp21) in vivo and in vitro on threonine residue 59. p21 molecules encoded by cellular ras genes (c-Ha-ras-1) contain an alanine at position 59, and thus these p21 molecules are not phosphorylated. In this investigation, recombinant ras genes have been constructed between the 5' p21 coding region of normal (EC) or oncogenically activated (EJ) human c-Ha-ras-1 and the 3' p21 coding region of v-Ha-ras to generate p21 molecules containing a threonine phosphoacceptor site at position 59 and a glycine (EC/v-Ha) or valine (EJ/v-Ha) at residue 12. In transformed NIH 3T3 mouse fibroblast cells labeled with [35S]methionine, the ratio of pp21 to p21 was strikingly modulated by the amino acid at residue 12. v-Ha-ras p21 has an arginine at position 12, and 24% of total p21 was in the phosphorylated form. A glycine at residue 12 decreased the amount of pp21 to 14% of total p21, and a valine at residue 12 dramatically increased this value to 50%. In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM). These results indicate that a biochemical activity of p21 distinguishes between previously observed biological differences of normal and activated human ras genes.
Aging of proteins: isolation and identification of a fluorescent chromophore from the reaction of polypeptides with glucose.
Authors: Pongor S , Ulrich P C , Bencsath F A , Cerami A
Published: May 2006

Proteins exposed to glucose over long periods are known to undergo physicochemical changes including crosslinking and formation of brown fluorescent pigments of poorly characterized structure. Acid hydrolysis of both browned poly(L-lysine) and browned bovine serum albumin is found to release a major fluorescent chromophore, which after alkalinization is extractable into organic solvents and which can be purified by silica gel chromatography. The fluorescence properties of this compound very closely resemble those of the bulk browned polypeptides. By NMR, mass spectroscopy, and chemical derivatization, this compound is assigned the structure 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI). Confirmation was obtained by independent chemical synthesis from furylglyoxal and ammonia. The incorporation of two peptide-derived amine nitrogens and two glucose residues in FFI strongly suggests that peptide-bound FFI precursors are implicated in the crosslinking of proteins by glucose in vivo. This reaction has potential implications in the understanding of glucose-mediated protein modifications and their role in the complications of diabetes and aging.
Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis.
Authors: Macara I G , Marinetti G V , Balduzzi P C
Published: May 2006

The transforming protein of avian sarcoma virus UR2, p68v-ros, has an associated tyrosine-specific protein kinase activity similar to that of p60v-src and several other oncogene products. However, this activity has not been linked unequivocally to transformation, and the physiological action of these proteins remains in doubt. We now have found that immunoprecipitated p68v-ros also is associated with phosphatidylinositol (PtdIns) kinase (ATP:PtdIns 4-phosphotransferase, EC 2.7.1.67) activity. PtdIns 4,5-bisphosphate [PtdIns(4,5)P2] specifically inhibits both this activity and the autophosphorylation of p68v-ros. Moreover, cells transformed by UR2 showed significant increases in 32P-labeling of PtdIns 4-phosphate (PtdIns4P) and PtdIns(4,5)P2 and in the formation of their catabolites, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, as compared to uninfected cells. These results suggest that a physiologically relevant function of oncogene kinases might be the phosphorylation of PtdIns and that increased turnover of PtdIns4P and PtdIns(4,5)P2 might play a role in transformation by increasing the formation of diacylglycerol, a catabolite of polyphosphoinositides that activates kinase C. This protein copurifies with the phorbol ester receptor, and its activation is likely to be intimately linked with mitogenesis. This hypothesis suggests a mechanism whereby certain oncogene proteins might cause the unrestricted growth typical of transformed cells and could explain why tumor promoters mimic many of the effects of transformation.
Assignment of the human gene for the low density lipoprotein receptor to chromosome 19: synteny of a receptor, a ligand, and a genetic disease.
Authors: Francke U , Brown M S , Goldstein J L
Published: May 2006

The availability of a species-specific monoclonal antibody that recognizes the low density lipoprotein (LDL) receptor of human but not hamster origin permitted assignment of the structural gene for the human receptor to chromosome 19. The antibody was used to detect the human LDL receptor in a series of hamster-human somatic cell hybrids by two assays: (i) a structural assay that measured cellular incorporation of [35S]methionine into immunoprecipitable receptor and (ii) a functional assay that measured the rate of receptor-dependent uptake and degradation of the 125I-labeled anti-receptor monoclonal antibody. Both assays showed that the human LDL receptor was expressed in 15 out of 20 hybrid cell lines. Expression of the human LDL receptor was 100% concordant with the presence of human chromosome 19; all other human chromosomes showed at least 25% discordance. As expected, the gene for the LDL receptor (LDLR) is located on the same chromosome as the gene for the disease familial hypercholesterolemia, which has been previously mapped to chromosome 19 by pedigree studies and is caused by allelic mutations at the LDL receptor locus. The gene for apolipoprotein E, a ligand for the LDL receptor, is also known to be located on chromosome 19, raising the possibility of an evolutionary link between a protein ligand and its receptor.
Disappearance and reappearance of B cells after in vivo treatment with monoclonal anti-I-A antibodies.
Authors: Waldor M K , Hardy R R , Hayakawa K , Steinman L , Herzenberg L A , Herzenberg L A
Published: May 2006

Previous studies have shown that treatment with antibodies to the murine I-A antigen encoded in the major histocompatibility complex attenuates experimental allergic encephalitis and experimental autoimmune myasthenia gravis. These studies were conducted with SJL mice, an inbred strain that is highly susceptible to the induction of these diseases. Here we show that injection of monoclonal anti-I-A antibody in the amounts used for the above studies rapidly depletes B cells. Fluorescence-activated cell sorter (FACS) multiparameter analysis of the B-cell subpopulations in treated animals shows that maximum depletion occurs around 5 days after treatment and that recovery of some subpopulations i still incomplete 1 month later. SJL mice are more sensitive to this B-cell depletion and recover more slowly than putatively normal C3H.Ighb (CKB) mice. Some components of the primary, secondary and tertiary IgG antibody responses are reduced in anti-I-A-treated SJL animals immunized after the first and second anti-I-A injections. The persistence of some antibody response impairment well beyond the time when anti-I-A disappears raises a note of caution concerning human therapy protocols based on the injection of anti-Ia antibodies.
Type beta transforming growth factor: a bifunctional regulator of cellular growth.
Authors: Roberts A B , Anzano M A , Wakefield L M , Roche N S , Stern D F , Sporn M B
Published: May 2006

Type beta transforming growth factor (TGF-beta) is a two-chain polypeptide of 25,000 daltons isolated from many tissues, including bovine kidney, human placenta, and human platelets. It has been characterized by its ability to stimulate reversible transformation of nonneoplastic murine fibroblasts, as measured by the formation of colonies of these cells in soft agar (ED50 = 4 pM TGF-beta for NRK fibroblasts). We now show that the response of cells to TGF-beta is bifunctional, in that TGF-beta inhibits the anchorage-dependent growth of NRK fibroblasts and of human tumor cells by increasing cell cycle time. Moreover, the anchorage-independent growth of many human melanoma, lung carcinoma, and breast carcinoma cell lines is inhibited by TGF-beta at concentrations in the same range as those that stimulate colony formation of NRK fibroblasts (average ED50 = 10-30 pM TGF-beta for inhibition). Whereas epidermal growth factor and TGF-beta synergize to induce anchorage-independent growth of NRK fibroblasts, their effects on the growth of A-549 human lung carcinoma cells are antagonistic. The bifunctional response of cells to TGF-beta is further demonstrated in Fischer rat 3T3 fibroblasts transfected with a cellular myc gene. In these cells TGF-beta synergizes with platelet-derived growth factor to stimulate colony formation but inhibits the colony formation induced by epidermal growth factor. The data indicate that the effects of TGF-beta on cells are not a function of the peptide itself, but rather of the total set of growth factors and their receptors that is operant in the cell at a given time.
Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo.
Authors: Zoumbos N C , Gascon P , Djeu J Y , Young N S
Published: May 2006

We have investigated interferon as a mediator of hematopoietic suppression in bone marrow failure. Interferon production by stimulated peripheral blood mononuclear cells from patients with aplastic anemia was significantly higher than that observed in controls; spontaneous interferon production by these cells was also high for more than half of aplastic anemia patients. Circulating interferon, not detectable in normal individuals, was detected in 10 of 24 patients. Interferon is a potent inhibitor of hematopoietic cell proliferation and, therefore, may be the mediator of suppression in many in vitro models employing patients' cells and sera. The possible pathogenic importance of interferon in aplastic anemia was suggested by an increase in hematopoietic colony formation in vitro after exposure of bone marrow cells to antiinterferon antisera (277 +/- 71% increase for patients compared to 1.6 +/- 1.6% for normal individuals). Interferon levels in the bone marrow sera of aplastic anemia patients were high (mean = 203 international units (IU)/ml, n = 8), even in comparison to circulating levels in the same patients. Normal bone marrow sera also contained measurable interferon but at lower levels (41 IU/ml, n = 16), indicating that interferon may be a normal bone marrow product. High concentration of bone marrow interferon, possibly due to abnormal immunologic activity or a reaction to virus infection of the bone marrow, may mediate hematopoietic suppression in aplastic anemia patients.
Vasoactive intestinal peptide-containing neurons in the paraventricular nucleus may participate in regulating prolactin secretion.
Authors: Mezey E , Kiss J Z
Published: May 2006

Vasoactive intestinal polypeptide (VIP) immunoreactivity is present in varicosities and fibers in the hypothalamic paraventricular nucleus (PVN) in normal control animals. Adrenalectomy and lactation combined with colchicine treatment results in the appearance of a large population of VIP-immunopositive cell bodies in the parvocellular part of the PVN. Adrenalectomy, as well as lactation, significantly increases the number of VIP-positive fibers in the external zone of the median eminence. These observations suggest that the VIP-immunopositive neurons in the PVN may participate in regulating prolactin and corticotropin secretion.
Isolation and partial characterization of a fatty acid binding protein in rat liver plasma membranes.
Authors: Stremmel W , Strohmeyer G , Borchard F , Kochwa S , Berk P D
Published: May 2006

When [14C]oleate-bovine serum albumin complexes were incubated in vitro with rat liver plasma membranes (LPM), specific, saturable binding of oleate to the membranes was observed. Maximal heat-sensitive (i.e., specific) binding was 3.2 nmol/mg of membrane protein. Oleate-agarose affinity chromatography of Triton X-100-solubilized LPM was used to isolate a single 40-kDa protein with high affinity for oleate. On gel filtration, the protein comigrated with various fatty acids but not with [14C]bilirubin, [35S]sulfobromophthalein, [14C]taurocholate, [14C]phosphatidylcholine, or [14C]cholesteryloleate. A rabbit antibody to this membrane fatty acid-binding protein gave a single precipitin line with the antigen but no reactivity with concentrated cytosolic proteins, LPM bilirubin/sulfobromophthalein-binding protein, or rat albumin or other rat plasma proteins. The antibody selectively inhibited heat-sensitive binding of [14C]oleate to LPM. Immunofluorescence studies localized the antigen in liver-cell plasma membranes as well as in other major sites of fatty acid transport. These data are compatible with the hypothesis that this protein may act as a receptor in a hepatocellular uptake mechanism for fatty acids.
Complete amino acid sequence of human hemopexin, the heme-binding protein of serum.
Authors: Takahashi N , Takahashi Y , Putnam F W
Published: May 2006

We have determined the complete primary structure of human hemopexin, a plasma beta-glycoprotein that specifically binds one heme with high affinity and transports it to hepatocytes for salvage of the iron. Human hemopexin (Mr approximately equal to 63,000) consists of a single polypeptide chain containing 439 amino acid residues with six intrachain disulfide bridges. The amino-terminal threonine residue is blocked by an O-linked galactosamine oligosaccharide, and the protein has five glucosamine oligosaccharides N-linked to the acceptor sequence Asn-X-Ser/Thr. The 18 tryptophan residues are arranged in four clusters, and 12 of the tryptophans are conserved in homologous positions. Computer-assisted analysis of the internal homology in amino acid sequence indicates that hemopexin consists of two similar halves, thus suggesting duplication of an ancestral gene. Limited tryptic digestion cleaves apohemopexin after arginine-216 into two half-molecules, whereas heme-saturated hemopexin is cleaved after lysine-101. The half-molecules are connected by a histidine-rich hinge-like region that contains two glucosamine oligosaccharides. A structural model for human hemopexin is proposed that is based on these properties and on computer-assisted predictions of the secondary structure and the hydrophilic/hydrophobic character. In this model alpha-helices and beta-turns predominate, and the two halves are connected by an exposed connecting region in apohemopexin that becomes inaccessible to trypsin in hemesaturated hemopexin. Many segments of hemopexin are similar to sequences of other heme proteins, but no overall structural relationship of hemopexin to any other heme protein was identified.
Simultaneous comparison of three protein sequences.
Authors: Murata M , Richardson J S , Sussman J L
Published: May 2006

Currently there are several computer algorithms available for aligning two biological sequences. When more than two sequences are to be aligned, however, pairwise comparisons using these methods rarely lead to a consistent alignment of the sequences. One obvious solution to this problem is to compare all the sequences simultaneously. Here we present an algorithm for the simultaneous comparison of three biological sequences. The algorithm is an extension of the method developed by S. B. Needleman and C. D. Wunsch, but it decreases the almost prohibitively long computing time required by a direct naive extension to a practical level: it takes time proportional to the cube of the mean sequence length, in comparison to the fifth power time taken by the direct extension. Simultaneous comparison not only gives a consistent alignment of the three sequences, but it could also reveal homologous residues in the sequences that might be overlooked by the pairwise comparisons. As an example of the application of the algorithm, three copper-containing proteins, plastocyanin, stellacyanin, and cucumber basic blue protein, are compared.
Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.
Authors: Povirk L F , Goldberg I H
Published: May 2006

When defined-sequence DNA from the lacl region of plasmid pMC1 was treated with the nonprotein chromophore of neocarzinostatin in the presence of various thiols, the predominant lesions were direct strand breaks, occurring primarily at thymine and adenine residues. In the presence of glutathione, however, alkali-dependent strand breaks, occurring at certain cytosine residues, were also detected but were virtually absent when other thiols were used. Chromophore-induced release of free cytosine base from [3H]cytosine-labeled DNA was 2- to 3-fold greater with glutathione than with the other thiols. These results suggest that the alkali-dependent strand break is some form of apyrimidinic site. These sites were substrates for endonuclease IV of Escherichia coli, although a 5-fold greater concentration of enzyme was required for their cleavage than was required for cleavage of apurinic sites in depurinated DNA. These sites were also less sensitive to E. coli endonuclease VI (exonuclease III) by a factor of at least 5 and less sensitive to E. coli endonuclease III by a factor of at least 10. These and other results suggest that these sites are chemically different from normal apurinic/apyrimidine sites. When chromophore-induced apyrimidinic sites were quantitated as alkali-dependent breaks at 11 specific sites in the lacl gene, a correlation was found between occurrences of these lesions and the reported frequencies of G-C to A X T transitions at the same sites. All occurrences of the trinucleotide sequence A-G-C, including the ochre 21 mutational hot spot, were particularly prominent sites. The selective formation of endonuclease-resistant apyrimidinic sites at specific cytosine residues may explain the high frequency of G X C to A X T transitions in the mutational spectrum of neocarzinostatin.
Maintenance of differentiated rat hepatocytes in primary culture.
Authors: Isom H C , Secott T , Georgoff I , Woodworth C , Mummaw J
Published: May 2006

Normal adult rat hepatocytes remained viable and functional for at least 43 days when plated on collagen-coated dishes and fed chemically defined medium supplemented with dimethyl sulfoxide (Me2SO). Hepatocytes isolated by collagenase perfusion and cultured in the presence or absence of Me2SO were (i) examined by light and electron microscopy for morphological changes; (ii) analyzed for the production of albumin and other plasma proteins; and (iii) tested by autoradiography for DNA synthesis. Me2SO-treated cells continued to produce specific plasma proteins during the entire culture period; albumin production was consistently high (11-19 micrograms/ml of culture medium per 24 hr) from day 2 to at least day 43 after plating. Ultrastructural analyses demonstrated that Me2SO-treated hepatocytes resembled those from intact liver in organization of cytoplasmic organelles and cellular junctions. The optimal concentration for observing the morphological and biochemical effects of Me2SO was 2% (vol/vol). We conclude that supplementation of chemically defined medium with Me2SO enables maintenance of differentiated hepatocytes in culture for extended periods of time.
Isolation of a gene enhancer within an amplified inverted duplication after "expression selection".
Authors: Ford M , Davies B , Griffiths M , Wilson J , Fried M
Published: May 2006

We have attempted to isolate and identify cellular expression sequences from F9 teratocarcinoma DNA by utilizing their ability to reactivate a selectable gene devoid of its own expression sequences (expression selection). Restriction nuclease-digested F9 cellular DNA was ligated to a polyoma virus (Py) DNA fragment which contains an intact transforming region but is incapable of inducing transformation because it lacks the viral 5' enhancer sequence. The ligation mixture was used to transfect Rat-1 cells and a transformed cell line, 3B, was isolated. The 3B cell line contained a single type of Py DNA insert, which was molecularly cloned as an 18-kilobase BglII fragment. A weak cellular enhancer was identified in a 4.7-kilobase BamHI fragment upstream from the Py sequences. Both the Py DNA and the enhancer sequences were found to be present in an inverted duplication in the 3B clone. The presence of this structure in 3B genomic DNA was confirmed by the analysis of selectively isolated inverted duplicated sequences, and the structure was found to be at least 22 kilobases long. In the 3B cell line, the inverted duplicated sequences containing the Py and enhancer sequences are quite stable and are amplified 20- to 40-fold. The strongly transformed phenotype of the 3B cells may be a result of this amplification. The formation of inverted duplications as a part of the amplification mechanism as well as a general strategy for the cloning of inverted duplicated (amplified) sequences is discussed.
Gene organization of DC and DX subregions of the human major histocompatibility complex.
Authors: Okada K , Boss J M , Prentice H , Spies T , Mengler R , Auffray C , Lillie J , Grossberger D , Strominger J L
Published: May 2006

The DC and DX subregions of the human major histocompatibility complex (MHC) have been cloned from a cosmid library made from a human B-cell line, Priess. The DC subregion, 48 kilobases, includes the DC alpha and DC beta genes. A second DC-like region, the DX subregion, 35 kilobases, contains the DX alpha gene and a newly found beta gene termed DX beta. Since the DC and DX genes are highly homologous in nucleotide sequence, gene size, exon-intron organization, and direction of transcription, the DC and DX subregions were presumably generated by duplication of an ancestral alpha-beta gene pair. Nucleotide sequencing indicates that all four genes have intact coding sequences and promoter regions. Homology between the upstream promoter sequences of these four genes and seven other class II genes at nucleotides -69 to -78 and -98 to -110 highlights these previously described conserved elements. Moreover, a striking conservation of flanking alpha-gene-specific and beta-gene-specific sequences has been observed. Comparison of Southern blots of Priess DNA with DC alpha and DC beta cDNA probes with isolated cosmid clones showed that (i) the human chromosome encodes only two DC alpha-related and two DC beta-related genes, namely, DC alpha, DX alpha, DC beta, and DX beta, and (ii) the DC and DX subregions are homozygous in Priess cells.
Human lambda light-chain constant region gene CMor lambda: the primary structure of lambda VI Bence Jones protein Mor.
Authors: Frangione B , Moloshok T , Prelli F , Solomon A
Published: May 2006

Serologic, structural, and genetic analyses have shown that the constant (C) region of human kappa light chains is encoded by a single gene, whereas that of lambda chains is encoded by multiple genes. We have determined the complete C region amino acid sequence of two monoclonal lambda VI light chains, Bence Jones proteins Sut and Mor. The C region of lambda chains Sut and Mor consists of 105 residues, as is characteristic for human lambda light chains, of which 102 are identical in sequence. Protein Sut has the C region sequence associated with the C lambda isotype Mcg-, Kern-, Oz+ and represents a product of the C lambda 3 (Kern-, Oz+) gene. Protein Mor has a C region sequence associated with Mcg-, Kern-, and Oz- proteins but differs from protein Sut by the presence of three amino acid interchanges at positions 168, 176, and 194. These substitutions distinguish protein Mor from lambda chains encoded by the C lambda 1 (Mcg+), C lambda 2 (Kern-, Oz-), and C lambda 3 (Kern-, Oz+) genes and provide further evidence for polymorphism of the human C lambda genome. The gene encoding the C region sequence of lambda chain Mor is designated CMor lambda.
Nucleotide sequence and structure of the human apolipoprotein E gene.
Authors: Paik Y K , Chang D J , Reardon C A , Davies G E , Mahley R W , Taylor J M
Published: May 2006

The gene for human apolipoprotein E (apo-E) was selected from a library of cloned genomic DNA by screening with a specific cDNA hybridization probe, and its structure was characterized. The complete nucleotide sequence of the gene as well as 856 nucleotides of the 5' flanking region and 629 nucleotides of the 3' flanking region were determined. Analysis of the sequence showed that the mRNA-encoding region of the apo-E gene consists of four exons separated by three introns. In comparison to the structure of the mRNA, the introns are located in the 5' noncoding region, in the codon for glycine at position -4 of the signal peptide region, and in the codon for arginine at position +61 of the mature protein. The overall lengths of the apo-E gene and its corresponding mRNA are 3597 and 1163 nucleotides, respectively; a mature plasma protein of 299 amino acids is produced by this gene. Examination of the 5' terminus of the gene by S1 nuclease mapping shows apparent multiple transcription initiation sites. The proximal 5' flanking region contains a "TATA box" element as well as two nearby inverted repeat elements. In addition, there are four Alu family sequences associated with the apo-E gene: an Alu sequence located near each end of the gene and two Alu sequences located in the second intron. This knowledge of the structure permits a molecular approach to characterizing the regulation of the apo-E gene.
Ultrashort-loop positive feedback of corticotropin (ACTH)-releasing factor to enhance ACTH release in stress.
Authors: Ono N , Bedran de Castro J C , McCann S M
Published: May 2006

Previous experiments have shown that intraventricular injection of ovine corticotropin (ACTH)-releasing factor (oCRF) in doses too low to elevate plasma ACTH by direct action on the pituitary does not lower plasma ACTH, suggesting that the peptide lacks a negative ultrashort-loop feedback action to suppress its own release under resting conditions. The present study was performed to determine whether oCRF has any action to alter CRF release in stress. The peptide was injected into the third ventricle or external jugular vein of freely moving ovariectomized female rats 5 min prior to application of ether stress. When oCRF was injected into the third ventricle in doses of 500 pg (0.1 pmol) or less, there was no significant alteration in plasma ACTH prior to ether stress; however, there was a significantly enhanced increase in plasma ACTH 2 and 5 min after ether stress applied 5 min after intraventricular injection of oCRF at doses of 50 (0.01 pmol) or 150 pg (0.03 pmol). These results suggest that the peptide acts on structures adjacent to the third ventricle to augment stress-induced CRF release. To rule out the possibility that the sensitivity of the pituitary itself to CRF increases dramatically following stress, 10 or 100 ng of oCRF was injected i.v. These doses produced a significant dose-related increase in plasma ACTH at 2, 5, or 15 min. In other groups receiving the same doses of oCRF and ether stressed 5 min later, plasma ACTH was significantly higher 2 or 5 min after ether stress when compared with plasma ACTH in ether-stressed saline-injected animals. However, in contrast with the results of intraventricular injection of oCRF, the release of ACTH was no greater than that obtained by summing the independent effects of exogenous oCRF and the CRF released by stress. We conclude that CRF may have a positive ultrashort-loop feedback action to enhance stress-induced ACTH release and that this enhancement is not due to increased sensitivity of anterior pituitary corticotrophs to CRF.
Thyroid hormones increase Na+-H+ exchange activity in renal brush border membranes.
Authors: Kinsella J , Sacktor B
Published: May 2006

Na+-H+ exchange activity, i.e., amiloride-sensitive Na+ and H+ flux, in renal proximal tubule brush border (luminal) membrane vesicles was increased in the hyperthyroid rat and decreased in the hypothyroid rat, relative to the euthyroid animal. A positive correlation was found between Na+-H+ exchange activity and serum concentrations of thyroxine (T4) and triiodothyronine (T3). The thyroid status of the animal did not alter amiloride-insensitive Na+ uptake. The rate of passive pH gradient dissipation was higher in membrane vesicles from hyperthyroid rats compared to the rate in vesicles from hypothyroid animals, a result which would tend to limit the increase in Na+ uptake in vesicles from hyperthyroid animals. Na+-dependent phosphate uptake was increased in membrane vesicles from hyperthyroid rats; Na+-dependent D-glucose and L-proline uptakes were not changed by the thyroid status of the animal. The effect of thyroid hormones in increasing the uptake of Na+ in the brush border membrane vesicle is consistent with the action of the hormones in enhancing renal Na+ reabsorption. Further, the regulation of transtubular Na+ flux has now been shown to be concomitant with modulation of the entry of Na+ into the tubular cell across its luminal membrane, mediated by the exchange reaction, and with the previously reported control of the pumping of Na+ out of the cell across its basolateral membrane, mediated by the Na+,K+-ATPase.
Synthesis of small nuclear ribonucleoprotein particles by the malarial parasite Plasmodium falciparum.
Authors: Francoeur A M , Gritzmacher C A , Peebles C L , Reese R T , Tan E M
Published: May 2006

Sera from patients with autoimmune diseases have been used to identify small nuclear ribonucleoprotein particles (snRNPs) present in higher eukaryotic cells and also in dinoflagellates. Previously these sera have not detected crossreactive snRNP protein antigens of other lower eukaryotes such as yeast, Tetrahymena, or Dictyostelium. We report that anti-Sm, anti-U1-RNP, and anti-La/SS-B human antisera react with specific snRNP protein antigens synthesized by the protozoan Plasmodium falciparum, the human malarial parasite. These results suggest that the structure and antigenicity (and thus probably the function) of snRNPs have been widely conserved in eukaryote evolution.
Betaine synthesis in chenopods: Localization in chloroplasts
Authors: Hanson Andrew D. , May Ann Marie , Grumet Rebecca , Bode Janet , Jamieson Gene C. , Rhodes David
Published: May 2006

Plants from several families (Chenopodiaceae, Gramineae, Compositae) accumulate betaine (glycine betaine) in response to salt or water stress via the pathway: choline → betainal (betaine aldehyde) → betaine. Betaine accumulation is probably a metabolic adaptation to stress. Intact protoplasts from leaves of spinach (
Spinacia oleracea
) oxidized [
14
C]choline to betainal and betaine, as did protoplast lysates. Upon differential centrifugation, the [
14
C]choline-oxidizing activity of lysates sedimented with chloroplasts. Chloroplasts purified from protoplast lysates by a Percoll cushion procedure retained strong [
14
C]choline-oxidizing activity (1-3 nmol/mg of chlorophyll per hr), although the proportion of the intermediate, [
14
C]betainal, in the reaction products was usually higher than for protoplasts. Isolated chloroplasts also readily oxidized [
14
C]betainal to betaine (20-100 nmol/mg of chlorophyll per hr). Light increased the oxidation of both [
14
C]choline and [
14
C]betainal by isolated chloroplasts ≈3-fold; this light-stimulation was abolished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Similar results were obtained with another chenopod (
Beta vulgaris
) but not with pea (
Pisum sativum
), a species that accumulates no betaine. The chloroplast site for betaine synthesis in chenopods contrasts with the mitochondrial site in mammals.
Tumorigenic and metastatic properties of "normal" and ras-transfected NIH/3T3 cells.
Authors: Greig R G , Koestler T P , Trainer D L , Corwin S P , Miles L , Kline T , Sweet R , Yokoyama S , Poste G
Published: May 2006

To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Ha-ras-1 gene derived from the human T24 bladder carcinoma cell line and compared them with untransfected NIH/3T3 cells. Whereas subcutaneous implantation of NIH/3T3 cells in the supraclavicular region produced palpable tumors that failed to metastasize, NIH/3T3 cells inoculated in the footpad gave rise to malignant tumors that metastasized to the lung. Under identical conditions and irrespective of the site of implantation, A51 cells formed rapidly growing primary tumors that produced pulmonary metastases. In an assay for experimental metastasis, intravenously injected NIH/3T3 cells gave rise to pulmonary nodules only at high cell inocula and in long-term survivors (90 days after injection). In contrast, A51 cells formed multiple lung tumor colonies detectable 14 days after injection. These results indicate that "normal" untransfected NIH/3T3 cultures contain subpopulations of cells that express malignant properties and that transfection of NIH/3T3 cells with activated c-Ha-ras-1 accelerates formation of metastases.
Deletion mapping of HLA and chromosome 6p genes.
Authors: Levine F , Erlich H , Mach B , Leach R , White R , Pious D
Published: May 2006

We have analyzed a set of heterozygous mutants with deletions that encompass parts of HLA and surrounding regions of chromosome 6p. By a combination of Southern blotting, serologic, enzymatic, and cytogenetic analyses, we have ordered eight independent deletion break points into a sequence that divides chromosome 6p into six regions. The deletion mutants have been used in conjunction with the Southern blot technique to map HLA and other 6p gene probes into those regions. On the basis of these and other data we propose a genetic and physical map of HLA and surrounding regions of chromosome 6p. We find that for HLA probes, most of which hybridize with more than one gene, the multiple copies recognized by single probes map to single regions. Any chromosome 6p gene can now be regionally mapped by using these mutants.
Inhibition of platelet aggregation by a monoclonal antibody against human fibronectin.
Authors: Dixit V M , Haverstick D M , O'Rourke K , Hennessy S W , Broekelmann T J , McDonald J A , Grant G A , Santoro S A , Frazier W A
Published: May 2006

A monoclonal antibody (A3.3) has been generated against human platelet fibronectin (FN). A3.3 reacts with human plasma FN but with no other plasma proteins. A3.3 was found to inhibit thrombin- or ionophore A23187-stimulated aggregation of gel-filtered platelets in a concentration-dependent manner in both an aggregometer assay and a sensitive well plate aggregation assay. The antibody does not block secretion of serotonin. Four other anti-FN monoclonal antibodies that recognize different epitopes on FN than A3.3 does have no effect on platelet aggregation. A3.3 does not block the adhesion of CHO cells to FN-coated surfaces, indicating that it does not bind to the identified cell-binding domain of FN. A3.3 reacts with a 160/140-kDa doublet, known to contain the cell-binding domain, that is produced by digestion of FN with elastase or thermolysin. However, the antibody does not react with lower molecular weight species that also contain the cell-binding domain or with any of the other identified domains of FN. The A3.3 epitope is extremely protease sensitive and the smallest fragment found in any digest that retains reactivity with A3.3 is a 70-kDa peptide produced in low yield by mild thermolytic cleavage of FN. These data suggest that A3.3 defines a functional site present on both the platelet and plasma FN molecule that has a direct role in platelet aggregation.
Substituted benzamides as ligands for visualization of dopamine receptor binding in the human brain by positron emission tomography.
Authors: Farde L , Ehrin E , Eriksson L , Greitz T , Hall H , Hedström C G , Litton J E , Sedvall G
Published: May 2006

Two substituted benzamides, FLB 524 and raclopride, were labeled with 11C and examined for their possible use as ligands for positron emission tomography (PET)-scan studies on dopamine-2 (D-2) receptors in the brains of monkeys and healthy human subjects. Both ligands allowed the in vivo visualization of D-2 receptor binding in the corpus striatum caudate nucleus/putamen complex in PET-scan images. [11C]Raclopride showed a high ratio of specific striatal to nonspecific cerebellar binding, and the kinetics of binding of this ligand made it optimal for PET studies. The in vivo binding of [11C]raclopride in the striatum of cynomolgus monkeys was markedly reduced by displacement with haloperidol. This and previous in vitro data indicate that [11C]raclopride binds selectively to striatal D-2 dopamine receptors. In healthy human subjects, [11C]raclopride binding in the caudate nucleus/putamen was 4- to 5-fold greater than nonspecific binding in the cerebellum. In comparison with previously available ligands for PET-scan studies on central dopamine receptors in man, [11C]raclopride appears to be advantageous with regard to (i) specificity of binding to D-2 receptors, (ii) the high ratio between binding in dopamine-rich (caudate, putamen) and dopamine-poor (cerebellum) human brain regions, and (iii) rapid association and reversibility of specific binding. [11C]Raclopride should be a valuable tool for characterizing D-2 receptors in the brains of patients with neuropsychiatric disorders.
Unidirectional circulation in a prebiotic photochemical cycle
Authors: Careri Giorgio , Wyman Jeffries
Published: May 2006

In this brief note, we suggest the possibility that a soliton-assisted unidirectional photochemical cycle has played a role in prebiotic evolution. This suggestion is based on a model calculation which is itself based on the detection of Davydov-type soliton overtones in acetanilide.
Mutagenic repair in Escherichia coli: products of the recA gene and of the umuD and umuC genes act at different steps in UV-induced mutagenesis.
Authors: Bridges B A , Woodgate R
Published: May 2006

When excision-deficient Escherichia coli carrying umuC or umuD alleles were exposed to visible light several hours after ultraviolet irradiation, base-pair-substitution mutations were induced in these normally non-UV-mutable bacteria. It is argued that delayed photoreversal of pyrimidine dimers removes blocks to DNA replication and allows the "survival" and expression of misincorporated bases. A model for UV mutagenesis is proposed with two steps: (i) misincorporation opposite a photoproduct, which can be mediated directly by RecA protein, and (ii) bypass, only the latter process requiring umuD+ and umuC+ alleles. Basal levels of gene products are sufficient for at least some misincorporation events, although induced levels of umuD and umuC gene products are necessary for the bypass step. umuC bacteria containing the recA441 allele showed a greater yield of mutants, and those containing recA430 a reduced yield, following delayed photoreversal. The lexA51 allele (which results in constitutive derepression of RecA protein production) did not significantly alter the yield of mutants but caused them to appear marginally sooner in a recA441 umuC strain. These results emphasize that the nature of the RecA protein and not its concentration is paramount in determining the level of misincorporation. Experiments with recA441 umuC bacteria at 43 degrees C and 30 degrees C suggest that the misincorporation effect is unlikely to be attributable to cleavage of a DNA binding protein such as a repressor or a component of the polymerase complex. Moreover, misincorporation seems to occur without the need for induced synthesis of any other protein under recA control.
The c-myc oncogene is translocated to the involved chromosome 12 in mouse plasmacytoma.
Authors: Erikson J , Miller D A , Miller O J , Abcarian P W , Skurla R M , Mushinski J F , Croce C M
Published: May 2006

Although it is known that the c-myc oncogene is rearranged in a head-to-head fashion with the immunoglobulin heavy chain locus in mouse plasmacytomas, it has not been clear whether the c-myc oncogene is translocated to the heavy chain locus on mouse chromosome 12 or whether the heavy chain locus is translocated to the c-myc locus on mouse chromosome 15. To determine which of these two possibilities is correct, we hybridized Chinese hamster fibroblasts with J558 mouse plasmacytoma cells that carry a reciprocal chromosome translocation between chromosomes 12 and 15, and we examined the segregating hybrids for the presence of the normal and rearranged mouse c-myc genes, for the presence of different regions of the mouse heavy chain locus, and for the presence of genes located on mouse chromosomes 12 and 15. The results of this analysis indicate that, as in human Burkitt lymphomas with the 8;14 chromosome translocation, the c-myc gene is translocated to the heavy chain locus in mouse plasmacytomas. Thus the orientation of the heavy chain locus on mouse chromosome 12 and of the c-myc gene on mouse chromosome 15 is the same as the orientation of the homologous loci in man.
Three-dimensional structure of beta 2-microglobulin.
Authors: Becker J W , Reeke G N
Published: May 2006

The three-dimensional structure of beta 2-microglobulin, the light chain of the major histocompatibility complex class I antigens, has been determined by x-ray crystallography. An electron density map of the bovine protein was calculated at a nominal resolution of 2.9 A by using the methods of multiple isomorphous replacement and electron density modification refinement. The molecule is approximately 45 X 25 X 20 A in size. Almost half of the amino acid residues participate in two large beta structures, one of four strands and the other of three, linked by a central disulfide bond. The molecule thus strongly resembles Ig constant domains in polypeptide chain folding and overall tertiary structure. Amino acid residues that are the same in the sequences of beta 2-microglobulin and Ig constant domains are predominantly in the interior of the molecule, whereas residues conserved among beta 2-microglobulins from different species are both in the interior and on the molecular surface. In the crystals studied, the molecule is clearly monomeric, consistent with the observation that beta 2-microglobulin, unlike Ig constant domains, apparently does not form dimers in vivo but associates with the heavy chains of major histocompatibility complex antigens. Our results demonstrate that, at the level of detailed three-dimensional structure, the light chain of the major histocompatibility class I antigens belongs to a superfamily of structures related to the Ig constant domains.
Interleukin 2 (IL-2) augments transcription of the IL-2 receptor gene.
Authors: Depper J M , Leonard W J , Drogula C , Krönke M , Waldmann T A , Greene W C
Published: May 2006

We demonstrate that purified interleukin 2 (IL-2) can directly upregulate IL-2 receptor expression on phytohemagglutinin-activated T lymphocytes maintained in culture until IL-2 receptor expression had markedly declined. The IL-2-induced increase in IL-2 receptor number is maximal within 12 hr, requires new RNA and protein synthesis, and is mediated by an interaction of ligand with the high-affinity receptors for IL-2. IL-2 stimulation results in increased accumulation of IL-2 receptor mRNA within 4 hr, while an increase in IL-2 receptor gene transcription is detected within 30 min in isolated nuclei. In addition, IL-2 incubation results in increased amounts of c-myc and transferrin receptor mRNA, but it does not augment levels of mRNA encoding the beta chain of the T-cell receptor for antigen. These results demonstrate that IL-2 can directly upregulate transcription and expression of its own receptor and, therefore, indicate that IL-2 may regulate IL-2-dependent immune responses, in part, by influencing the expression of IL-2 receptors.