bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific modulation of sodium channels in mammalian nerve by monoclonal antibodies.
Authors: Meiri H , Goren E , Bergmann H , Zeitoun I , Rosenthal Y , Palti Y
Published: May 2006

Monoclonal antibodies (mAbs) were generated against the sodium channels in the intact membrane of the eel electroplax. These antibodies bind to nodes of Ranvier, as indicated by immunofluorescence. When externally applied to rat nerve fibers one of these mAbs blocks impulse conduction. In voltage-clamp experiments, this mAb was found to attenuate sodium current amplitude without affecting the time course. The dose-response curve was very steep and had an ED50 of 133 nM. About half of the mAb effect was shown to be due to a shift, in the hyperpolarizing direction, of the steady-state sodium inactivation versus membrane potential curve. The remaining effect was voltage- and time-independent. This mAb had no effect on the potassium or leakage currents. The results indicate that on the external surface of the sodium channel, there are a number of antigenically similar determinants, which are functionally linked to specific elements of the sodium conductance system. These functionally related determinants were preserved through the course of evolution.
Evidence for methoxatin (pyrroloquinolinequinone) as the cofactor in bovine plasma amine oxidase from resonance Raman spectroscopy.
Authors: Moog R S , McGuirl M A , Cote C E , Dooley D M
Published: May 2006

Resonance Raman spectra of the 2,4-dinitrophenylhydrazine derivatives of bovine plasma amine oxidase [amine:oxygen oxidoreductase (deaminating) (copper-containing), EC 1.4.3.6] have been measured. Detailed comparisons to the spectra of the corresponding derivatives of methoxatin (pyrroloquinolinequinone), pyridoxal, and other aldehydes and diones provide further evidence that covalently bound methoxatin or a closely similar derivative is the organic cofactor in copper-containing amine oxidases.
Absolute electronegativity and hardness correlated with molecular orbital theory
Authors: Pearson Ralph G.
Published: May 2006

The concepts of absolute electronegativity, χ, and absolute hardness, η, are incorporated into molecular orbital theory. A graphic and concise definition of hardness is given as twice the energy gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital. Useful correlations can now be made between chemical behavior, visible-UV absorption spectra, optical polarizability, ionization potentials, and electron affinities.
Identification of the parasite transferrin receptor of Plasmodium falciparum-infected erythrocytes and its acylation via 1,2-diacyl-sn-glycerol.
Authors: Haldar K , Henderson C L , Cross G A
Published: May 2006

The transferrin receptor of schizont-infected erythrocytes of Plasmodium falciparum (Gambian clone FCR-3/A2) is a parasite-encoded protein of Mr 102,000, which is present in purified erythrocyte membranes. Polyclonal antiserum to the purified Mr 102,000 protein was raised in rabbits. At physiological pH, immunoaffinity-purified protein bound human ferrotransferrin but not apotransferrin. Conversely, antibody to human transferrin was used to purify the ferrotransferrin-receptor complex from infected cells. The isolated receptor was specifically recognized by the polyclonal rabbit antiserum raised against the Mr 102,000 protein. Preliminary analysis indicated that, unlike the human receptor, the plasmodial transferrin receptor is not a disulfide linked dimer but a single polypeptide acylated via 1,2-diacyl-sn-glycerol.
On the molecular mechanisms of the Schiff base deprotonation during the bacteriorhodopsin photocycle
Authors: Chronister Eric L. , Corcoran Timothy C. , Song Li , El-Sayed M. A.
Published: May 2006

Using optical flash photolysis and time-resolved Raman methods, we examined intermediates formed during the photocycle of bacteriorhodopsin (bR), as well as the bR color change, as a function of pH (in the 7.0-1.5 region) and as a function of the number of bound Ca
2+
ions. It is found that at a pH just below 3 or with less than two bound Ca
2+
per bR, the deprotonation (the L
550
→ M
412
) step ceases, yet the K
610
and L
550
analogues are still formed as in native bR. The lack of deprotonation in the photocycle of both acid blue and deionized blue bR and the similarity of their Raman spectra as well as of their K
610
and L
550
analogues strongly suggest that both blue samples have nearly the same retinal active site. It is suggested that in both blue species, bound cations are removed via a proton-cation exchange equilibrium, either on the cation exchange column for the deionized sample or in solution for the acid blue sample. The proton-cation exchange equilibrium is found to quantitatively account for the pH dependence of the purple-to-blue color change. The different mechanisms responsible for the large reduction (≈11 units) of the pK
a
value of the protonated Schiff base (PSB) during the photocycle are discussed. The absence of the L
550
→ M
412
deprotonation process in both blue species is discussed in terms of the previously proposed cation model for the deprotonation of the PSB during the photocycle of native bR. The extent of the deprotonation and the blue-to-purple color change are found to follow the same dependence on either the pH or the amount of cations added to deionized blue bR. This observed correlation is briefly discussed.
Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments.
Authors: Saito I , Stark G R
Published: May 2006

Charomids are cosmid vectors up to 52 kilobases (kb) long, bearing 1-23 copies of a 2-kb spacer fragment linked in head-to-tail tandem arrays. Like cosmids and lambda phage, charomids can be packaged in vitro for efficient introduction into bacteria. Charomids contain a polylinker with nine unique restriction sites for cloning and can be used without preparing vector arms. Using a charomid of appropriate size, one can clone inserts of any size up to 45 kb. For example, charomid 9-36 (9 cloning sites, 36 kb long) is too small to be packaged efficiently without an insert and can be used to clone fragments of 2-16 kb. The structure of charomids facilitates restriction mapping of the insert DNA and, after cloning, all the spacer fragments can be removed easily. After enrichment by size fractionation in an agarose gel, a specific single-copy genomic sequence can be cloned rapidly from approximately 3 micrograms of DNA. Using charomid 9-36, we have cloned and mapped an amplified novel DNA fragment from a cell line resistant to N-(phosphonoacetyl)-L-aspartate and carrying about 100 copies of the CAD (carbamoyl-phosphate synthetase/aspartate carbamoyltransferase/dihydroorotase) gene. The fragment lies at the center of an inverted duplication of this gene.
Biochemical basis for cisplatin and 5-fluorouracil synergism in human ovarian carcinoma cells.
Authors: Scanlon K J , Newman E M , Lu Y , Priest D G
Published: May 2006

The human ovarian cell line A2780 was exposed to either cisplatin (10 microM) or 5-fluorouracil (5FUra) (5 microM) for 1 hr. Cytotoxicity was less than 14% with either agent alone. Cisplatin (10 microM) and 5FUra (5 microM) in combination for 1 hr caused a 76% reduction in cell growth. Thymidine (dThd, 10 microM), if given concomitantly with the combination of cisplatin and 5FUra, completely protected the tumor cells. A 30-min exposure to cisplatin increased the intracellular pools of 5,10-methylenetetrahydrofolate and tetrahydrofolate 2.5-fold. The capacity of intact cells to form 5-fluorodeoxyuridylate (FdUMP)-thymidylate (dTMP) synthase complex when incubated with fluorodeoxyuridine (FdUrd) was enhanced 2.5-fold when the cells were pretreated with cisplatin. These experiments demonstrate that cisplatin can increase the availability of the reduced folate necessary for tight binding of FdUMP to dTMP synthase, thus enhancing the cytotoxicity of the cisplatin and 5FUra combination.
Molecular cloning of cDNA for human prothymosin alpha.
Authors: Goodall G J , Dominguez F , Horecker B L
Published: May 2006

A cDNA library was constructed from human spleen mRNA and screened for clones containing cDNAs coding for prothymosin alpha. A clone containing a 503-base-pair insert including the entire coding sequence for the translated portion of the mRNA was isolated. The deduced amino acid sequence confirms and completes the partial sequence of human prothymosin alpha determined by protein sequencing methods. The presence of an initiator codon immediately preceding the codon for the NH2-terminal serine residue and of a terminator codon immediately following the codon for Asp-109, the COOH-terminal residue, suggests that prothymosin alpha is synthesized without formation of a larger precursor polypeptide. Analysis of the 5' sequence preceding the initiator methionine codon excluded the presence of a signal peptide in the translated sequence.
Tomographic three-dimensional reconstruction of cilia ultrastructure from thick sections.
Authors: McEwen B F , Radermacher M , Rieder C L , Frank J
Published: May 2006

We have applied a computer-based tomographic technique to reconstruct the three-dimensional ultrastructure of newt lung cilia. Epon-embedded samples were cut into 0.25-micron-thick sections that were imaged at 1 MV with a high-voltage electron microscope. For the reconstruction shown, a tilt series of 53 micrographs was taken at tilt angles between -54 degrees and +50 degrees. The reconstruction was accomplished from these projections using a weighted back-projection algorithm. The 12-nm resolution of the reconstruction was sufficient to resolve the outer doublet and central pair microtubules, dynein arms, radial spokes, and central sheath structures. The reconstruction can be viewed from various angles and with appropriate parts cut away to reveal structural features of interest. The sense of depth in these views can be enhanced by stereo viewing of shaded surface images. From this reconstruction, we determined that newt lung cilia contain the more common triplet grouping of radial spokes.
Polymorphism of HLA-DR beta chains in DR4, -7, and -9 haplotypes: implications for the mechanisms of allelic variation.
Authors: Gregersen P K , Moriuchi T , Karr R W , Obata F , Moriuchi J , Maccari J , Goldberg D , Winchester R J , Silver J
Published: May 2006

We have isolated and sequenced cDNA clones corresponding to the DR beta 1 and DR beta 2 loci from two homozygous B-cell lines typed as DR7 (Burkhart) and DR9 (ISK). These nucleotide sequences were compared to beta 1 and beta 2 chains of other DR haplotypes. The first-domain sequences of beta 2 chains are identical in DR4 and DR7 haplotypes. In addition, there is strong sequence homology within the 3' untranslated regions of beta 1 genes from DR4, -7, and -9 haplotypes, thus confirming the close evolutionary relationship among these three haplotypes. In contrast, the first-domain sequences of beta 1 molecules from these haplotypes are very different from each other and do not reflect the DR4, -7, -9 family relationship. Two explanations for the differences in degree of diversity between beta 1 and beta 2 chains are suggested. The differences may be a consequence of selection pressures; this implies functional differences for products of the beta 1 and beta 2 loci. Alternatively, closely linked segments of the human class II region may differ in their underlying rates of variation, independent of selection pressures, and this may in part account for the extraordinary diversity found in the beta 1 first domain.
Interleukin 2 mediates the inhibition of oligodendrocyte progenitor cell proliferation in vitro.
Authors: Saneto R P , Altman A , Knobler R L , Johnson H M , de Vellis J
Published: May 2006

In the immune system, T-lymphocyte proliferation depends on interleukin 2 [IL-2 (T-cell growth factor)] interaction with specific receptors. In this study we show that IL-2 can specifically inhibit the proliferation of neonatal rat oligodendrocyte progenitor cells cultured in a serumless, chemically defined medium (oligodendrocyte-defined medium; ODM). IL-2 inhibited both [3H]thymidine incorporation and increase in cell number. Specificity was shown by precipitating IL-2 activity with anti-IL-2 antiserum. Furthermore, growth inhibition depended on the expression of Tac (an anti-IL-2 receptor monoclonal antibody)-positive receptors (IL-2 receptor). When cells were cultured in the presence of IL-2, both Tac-positive staining and growth inhibition were no longer expressed. The addition of interleukin 1 had no effect on [3H]thymidine incorporation or changes in cell number. However, when IL-1 was subsequently added together with IL-2, Tac expression and IL-2-mediated inhibition of cell proliferation was induced. This inhibitory effect was not due to a sensitive subpopulation because greater than 90% of the culture was Tac positive. Taken together, these data show that IL-2 can specifically inhibit oligodendrocyte proliferation and acts via Tac-positive receptors.
Octapeptides deduced from the neuropeptide receptor-like pattern of antigen T4 in brain potently inhibit human immunodeficiency virus receptor binding and T-cell infectivity.
Authors: Pert C B , Hill J M , Ruff M R , Berman R M , Robey W G , Arthur L O , Ruscetti F W , Farrar W L
Published: May 2006

The differentiation antigen T4, present on the helper/inducer subset of T lymphocytes, is thought to serve as the receptor for the human immunodeficiency virus (HIV). We find that a 60-kDa protein, immunoprecipitable by monoclonal antibody (mAb) OKT4, is present on membranes from human brain as well as human T cells. Furthermore, the radioiodinated HIV envelope glycoprotein [125I-labeled gp120 (125I-gp120)] can be specifically covalently affixed to a molecule present on rat, monkey, and human brain membranes to yield a complex that is indistinguishable from that formed on human T cells. T4 antigen has been studied on unfixed squirrel monkey, rat, and human brain sections by autoradiography using the mAb OKT4. A highly conserved neuroanatomical pattern has been demonstrated, suggesting an analogous organization in these three mammalian brains. Furthermore, the localization of 125I-gp120 receptor binding appears similar to that of T4 and is highly reminiscent of patterns for many previously characterized neuropeptide receptors. A computer-assisted analysis of gp120 suggested that a previously unremarkable octapeptide sequence within the gp120 protein, which we have synthesized and termed "peptide T," may play an important role in HIV attachment. Thus, peptide T and three rationally designed peptide analogs, each with a systematic amino acid substitution, potently inhibit specific 125I-gp120 binding to brain membranes. Additionally, when tested in a viral infectivity assay, these peptides show the same rank order and similar absolute potency to block HIV infection of human T cells. Thus, peptide T may provide a useful pharmacological or immunological basis for the control and treatment of AIDS.
New scientific opportunities and old obstacles in vaccine development.
Authors: Warren K S
Published: May 2006

The present status of and priorities for vaccine development are described, and the historical conditions under which vaccines have been developed are contrasted with newer technologies for such development. Current programs, the opportunities they present, and the obstacles to their implementation are summarized.
The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA.
Authors: Eschenfeldt W H , Berger S L
Published: May 2006

Clones for human prothymosin alpha have been identified in cDNA libraries from staphylococcal enterotoxin A-stimulated normal human lymphocytes and from simian virus 40-transformed fibroblasts. The 1198-base-pair fibroblast clone has been sequenced. The encoded protein is highly acidic (54 residues out of 111) and shares greater than 90% sequence homology with rat prothymosin alpha. The peptide "hormone" thymosin alpha 1 appears at positions 2-29 of the prothymosin alpha amino acid sequence. There is no N-terminal signal peptide. Examination of mouse and human tissues revealed the presence of prothymosin alpha mRNA in kidney, liver, spleen, normal lymphocytes (predominantly T cells), human T-cell leukemia virus-infected T cells, and myeloma cells (B-cell lineage). Prothymosin alpha mRNA is inducible; upon mitogen stimulation it increased greater than 15-fold above the level found in resting lymphocytes. Similarly, serum-deprived NIH 3T3 cells responded to serum restitution with an increase in prothymosin alpha mRNA. Characterization of human genomic DNA by Southern blot analysis disclosed a complicated pattern consistent with genetic polymorphism. These data suggest that prothymosin alpha plays an intracellular role tied to cell proliferation. There is no evidence that it serves as a precursor for secreted thymic peptides. However, given the complexity at the genomic level, multiple functions, including a putative secretory capability, cannot be excluded.
Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.
Authors: Beck von Bodman S , Schuler M A , Jollie D R , Sligar S G
Published: May 2006

We have totally synthesized a gene that codes for rat hepatic cytochrome b5. The 5' flanking region was designed for efficient expression of this gene in Escherichia coli by incorporating an optimum ribosome binding site and spacer region. Both a soluble form, analogous to the protease-treated microsomal protein, as well as the complete cytochrome with hydrophobic membrane anchor, was constructed and expressed. Transformants with the gene for the soluble protein overproduce authentic cytochrome b5 to a level of 8% of the total cell protein. The complete cytochrome is expressed to a lesser extent with most of the protein found in the cell membrane fraction. This represents complete synthesis and bacterial expression of a mammalian metalloprotein gene. Cytochrome b5 is normally a six-coordinate low spin heme protein with histidine-39 and histidine-63 as axial ligands. We have replaced histidine-63 with a methionine residue by cassette mutagenesis, utilizing specific restriction enzyme sites engineered into the synthetic gene. The resultant protein has histidine-39 as sole axial ligand and is five-coordinate high spin in the ferric resting state, as indicated by optical and electron spin resonance spectroscopy. The ability to generate mutant cytochrome b5 in high yield is a crucial step in understanding heme protein folding, protein-protein recognition and binding, and biological electron transfer processes.
Sequential state generation by model neural networks.
Authors: Kleinfeld D
Published: May 2006

Sequential patterns of neural output activity form the basis of many biological processes, such as the cyclic pattern of outputs that control locomotion. I show how such sequences can be generated by a class of model neural networks that make defined sets of transitions between selected memory states. Sequence-generating networks depend upon the interplay between two sets of synaptic connections. One set acts to stabilize the network in its current memory state, while the second set, whose action is delayed in time, causes the network to make specified transitions between the memories. The dynamic properties of these networks are described in terms of motion along an energy surface. The performance of the networks, both with intact connections and with noisy or missing connections, is illustrated by numerical examples. In addition, I present a scheme for the recognition of externally generated sequences by these networks.
Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton.
Authors: Conboy J , Kan Y W , Shohet S B , Mohandas N
Published: May 2006

Protein 4.1 is an important structural protein that is expressed in erythroid and in a variety of non-erythroid tissues. In mammalian erythrocytes, it plays a key role in regulating membrane physical properties of mechanical stability and deformability by stabilizing spectrin-actin interaction. We report here the molecular cloning and characterization of human erythrocyte protein 4.1 cDNA and the complete amino acid sequence of the protein derived from the nucleotide sequence. Probes prepared from the cloned erythrocyte protein 4.1 cDNA hybridized with distinct mRNA species from a wide variety of non-erythroid tissues, including brain, liver, placenta, pancreas, and intestine, implying substantial homology between erythroid and non-erythroid protein 4.1. The availability of cloned erythrocyte protein 4.1 cDNA should facilitate the study of the functional characteristics of this protein in erythroid as well as non-erythroid cells.
Genetic polymorphism and exon changes of the constant regions of the human T-cell rearranging gene gamma.
Authors: Lefranc M P , Forster A , Rabbitts T H
Published: May 2006

The genomic nucleotide sequences of the constant-region (C) genes of the human T-cell rearranging gene gamma are given. These sequences show considerable allelic and nonallelic variation. Allelic variants exist at both C gamma 1 and C gamma 2 loci in coding regions (as well as in restriction enzyme sites). Both C gamma genes are in the same transcriptional orientation. Moreover, the organization of the nonallelic C gamma genes reveals some interesting features: the C gamma 1 gene, like the mouse C gamma gene, has three exons, whereas the C gamma 2 gene has four exons, including a duplicated second exon that would create a putative protein with an enlarged constant region. However, these two duplicated exons in C gamma 2 have lost the cysteine residue that is thought to be involved in the interchain disulfide bridge.
Molecular analysis of the maize anthocyanin regulatory locus C1.
Authors: Cone K C , Burr F A , Burr B
Published: May 2006

The C1 gene of maize plays a regulatory role in the production of anthocyanin pigments in the aleurone layer of the endosperm. As an initial step toward understanding the molecular details of how C1 controls pigment biosynthesis, we cloned the C1 gene. This was accomplished by first cloning a mutable allele of C1, c1-m5, which contains the transposable element Spm. A combination of molecular and genetic analysis was used to identify the Spm at the C1 locus. Individual genomic DNAs from a population in which the c1-mutable phenotype was segregating with the recessive c1 phenotype were digested with methyl-sensitive restriction enzymes and probed with a small DNA fragment derived from a defective Spm. One Sal I restriction fragment complementary to the Spm probe was shown to be present in the DNA of individuals with the c1-m5 phenotype but absent from DNA of individuals with a recessive c1 phenotype. Subsequent cloning and restriction analysis of this fragment revealed sequences flanking the Spm that proved to be C1-specific. A DNA fragment derived from the flanking sequences was then used as a probe to clone the wild-type C1 gene and several additional alleles of C1, including one stable recessive, two mutations caused by Ds insertions, one mutation induced by insertion of a defective Spm, and two dominant mutations, C1-S and C1-I. RNA blot hybridization analysis of three C1 alleles indicates that C1 regulation of the Bz1 and A1 structural genes in the anthocyanin biosynthetic pathway is at the transcriptional level.
Human c-myb protooncogene: nucleotide sequence of cDNA and organization of the genomic locus.
Authors: Majello B , Kenyon L C , Dalla-Favera R
Published: May 2006

We have isolated cDNA clones of the human c-myb mRNA that contain approximately 3.4 kilobases of the approximately 3.8-kilobase mRNA sequence. Nucleotide sequence analysis shows that the c-myb mRNA contains an open reading frame of 1920 nucleotides, which could encode a 72-kDa protein. The cDNA nucleotide sequence and the predicted amino acid sequence of the c-myb protein are highly homologous to the corresponding chicken and mouse proteins. In particular, a region toward the NH2 terminus of the protein containing a 3-fold tandem repeat of 51 residues is evolutionarily conserved and is the only region of homology with the Drosophila c-myb protein. This region may represent a functionally important structure, most likely the DNA-binding domain. cDNA clones have been used to isolate genomic clones and to define a preliminary intron/exon organization of the c-myb gene. Identification of 5' and 3' coding and noncoding exons indicates that the human c-myb locus spans a 40-kilobase region.
Intracytoplasmic phosphorylation sites of Tac antigen (p55) are not essential for the conformation, function, and regulation of the human interleukin 2 receptor.
Authors: Hatakeyama M , Minamoto S , Taniguchi T
Published: May 2006

Tac antigen, the receptor for human interleukin 2 (IL-2), contains in its intracytoplasmic region a serine residue (Ser-247) that is seemingly the predominant site of protein kinase C-mediated phosphorylation. A number of studies on growth factor receptors have suggested the importance of phosphorylation in receptor structure, function, and regulation. In this study, we generated site-directed mutations in the Tac antigen cDNA to generate mutant receptors in which Ser-247 or Thr-250, a probable site of minor phosphorylation, was replaced with another amino acid that is not accessible to phosphorylation. Study of the expression of these mutant genes in a T-lymphoid cell line has provided no evidence as to the essential role of the above-mentioned residues in determining the degree of receptor affinity, its ability for signal transduction, and phorbol ester-mediated regulation of the receptor. Our results strongly suggest the existence of an IL-2 receptor "complex" in which the Tac antigen is associated with another molecule(s) that is involved in receptor structure, function, and regulation.
Glucocorticoid receptor immunoreactivity in monoaminergic neurons of rat brain.
Authors: Härfstrand A , Fuxe K , Cintra A , Agnati L F , Zini I , Wikström A C , Okret S , Yu Z Y , Goldstein M , Steinbusch H
Published: May 2006

A monoclonal antibody against the rat liver glucocorticoid receptor was used in combination with rabbit antibodies against tyrosine hydroxylase, phenylethanolamine N-methyltransferase, and 5-hydroxytryptamine to demonstrate strong glucocorticoid receptor immunoreactivity in large numbers of central monoaminergic nerve cell bodies of the male rat. The receptor immunoreactivity was predominantly located in the nucleus, whereas the tyrosine hydroxylase, phenylethanolamine N-methyltransferase, and 5-hydroxytryptamine were detected mainly in the cytoplasm. The vast majority of the noradrenergic nerve cell bodies of groups A1-A7 and of the 5-hydroxytryptaminergic cell bodies of groups B1-B9 were found to contain strong glucocorticoid receptor immunoreactivity. The majority of the phenylethanolamine N-methyltransferase-immunoreactive nerve cells of the adrenergic cell groups C1-C3 and of the dorsal subnuclei of the nucleus tractus solitarius in the medulla oblongata were also strongly immunoreactive for glucocorticoid receptor. In the midbrain dopaminergic groups A8-A10, moderately (A8, A9) to strongly (A10) glucocorticoid receptor-immunoreactive cells were found, ranging from 40 to 75% of the total population. In the hypothalamic dopaminergic cell groups, all the cells of groups A12 and A14, as well as the majority of the dopaminergic cells of the zona incerta (A13), were found to contain moderate to strong glucocorticoid receptor immunoreactivity, but none of the large dopaminergic cells of the posterior hypothalamus (A11) showed such immunoreactivity.
Hormonal modulation of the quantity and in situ activity of tyrosine hydroxylase in neurites of the median eminence.
Authors: Wang P S , Porter J C
Published: May 2006

The role of ovarian hormones in the control of the quantity and activity of tyrosine hydroxylase (TyrOHase) in neurites of the median eminence of the rat was investigated. TyrOHase was quantified by an immunoblot assay using purified rat TyrOHase as the standard. Treatment of ovariectomized animals with progesterone, but not estradiol, resulted in a significant reduction in the amount of TyrOHase in the median eminence. The in situ activity of the enzyme was assayed by measuring the rate of synthesis of L-3,4-dihydroxyphenylalanine (dopa), and the results were expressed as mol of dopa per hr per mol of TyrOHase. In animals treated with both estradiol and progesterone for 3 days, the in situ activity of TyrOHase in the median eminence was 114 +/- 13.5 (mean +/- SEM) compared to 26 +/- 4.7 for the controls. Estradiol or progesterone alone was much less effective than was the combination of estradiol and progesterone. To ascertain whether the effect of estradiol and progesterone on TyrOHase activity was reflected in the secretion of dopamine into hypophyseal portal blood, ovariectomized rats were treated for 3 days with both estradiol and progesterone or with the solvent vehicle. The concentration of dopamine in portal plasma of the hormone-treated animals was 1.93 +/- 0.533 ng/ml compared to 0.34 +/- 0.094 ng/ml in vehicle-treated animals. We conclude that the quantity and in situ molar activity of TyrOHase in neurites of the median eminence as well as the secretion of dopamine from these neurites are modulated by the combined action of estradiol and progesterone.
The eigenfunction problem in higher dimensions: Exact results
Authors: Knight Bruce W. , Sirovich Lawrence
Published: May 2006

A hermitian integral kernel in
N
-space may be mapped to a corresponding Hamiltonian in 2
N
-space by the Wigner transformation. Linear simplectic transformation on the phase space of the Hamiltonian yields a new kernel whose spectrum is unchanged and whose eigenfunctions follow from an explicit unitary transformation. If an integral kernel has a Wigner transform whose surfaces of constant value are concentric ellipsoids, then the Wigner transform yields exact results to the eigenfunction problem. Such behavior is asymptotically generic near extrema of the Wigner transform, from which follow simple and robust asymptotic results for the ends of the eigenvalue spectrum and for the corresponding eigenfunctions.
Human HSP70 promoter contains at least two distinct regulatory domains.
Authors: Wu B J , Kingston R E , Morimoto R I
Published: May 2006

The expression of the human HSP70 gene is induced by a wide range of physiological stresses, including exposure to heat shock and heavy metals, or under nonstress conditions, such as in response to serum stimulation. We have previously demonstrated that in either case the regulated expression is at the primary level of transcription. To determine whether transcription is mediated through a single or multiple genetic elements, we have dissected the sequences upstream of the transcription start site of the human HSP70 gene by constructing chimeric genes retaining variable amounts of 5' flanking regions fused to the bacterial gene encoding chloramphenicol acetyltransferase. Transcription from the chimeric genes was determined by S1 nuclease analysis of separate stable transfectants. The sequences required for heat shock and cadmium induction lie between -107 and -68. Within this region is the sequence CTGGAATATTCCCG, which is identical in 12/14 positions with the heat shock element of Drosophila heat shock genes, and a separate sequence, CGNCCCGG, which is homologous to the core of the human metallothionein II metal-responsive element. The sequences required for serum-stimulated transcription are distinct from the heat shock element. The sequence CCAAT at -68 is required for high levels of correctly initiated transcripts, and a purine-rich sequence, GAAGGGAAAAG, at -58 is required for serum stimulation. The human HSP70 promoter contains at least two regulatory domains--a distal domain responsive to heat shock or cadmium and a proximal domain responsive to stimulation by serum.
Molecular cloning, germ-line transformation, and transcriptional analysis of the zeste locus of Drosophila melanogaster.
Authors: Gunaratne P H , Mansukhani A , Lipari S E , Liou H C , Martindale D W , Goldberg M L
Published: May 2006

Approximately 170 kilobase pairs (kb) of contiguous DNA sequences derived from bands 3A3,4 of the Drosophila melanogaster X chromosome have been isolated by molecular cloning. Sequences required for the wild-type expression of the zeste locus are located within a 6-kb fragment of this chromosomal region, as shown by phenotypic rescue of zeste mutants in P element-mediated germ-line transformation. Expression of zeste is correlated with a 2.2-kb poly(A)+ RNA species transcribed at all postzygotic stages of Drosophila development. Many zeste alleles, including several producing neomorphic phenotypes, are not associated with detectable rearrangements of DNA.
Human T-cell clones used to define functional epitopes on HLA class II molecules.
Authors: Weyand C M , Goronzy J , Fathman C G
Published: May 2006

Polyclonal reagents have been used to define HLA class II molecules in conventional serologic and cellular typing. We generated human alloreactive T-cell clones to analyze the functional fine specificities of HLA class II molecules that might be important for the phenomenon of HLA and disease association. We chose to examine HLA-Dw14, an HLA-D specificity that has been associated with juvenile rheumatoid arthritis. In this paper we have presented data that suggest that conventional cellular typing does not reflect the distribution of T-cell epitopes on major histocompatibility complex class II molecules. We describe three alloreactive T-cell clones that have defined three separate Dw14-associated T-cell epitopes. Two of these epitopes were on a DR-region molecule; the third was located on a DQ-region product. In a panel of unrelated DR4-positive donors, these three DW14-associated determinants were present in a high frequency but were not linked to each other. Within the tested panel of DR4-positive cells, all possible combinatorial arrangements of these three allodeterminants were seen. The concurrent expression of any two of the three allodeterminants was equivalent to a positive typing response for Dw14. Our finding that HLA-Dw14 is not characterized by a unique allodeterminant but by the combinatorial recognition of independently distributed T-cell interaction sites suggests that analysis of HLA and disease association may be more clearly demonstrated through the use of human T-cell clones.
Relationship of VH and VL genes encoding three idiotypic families of anti-p-azobenzenearsonate antibodies.
Authors: Robbins P F , Rosen E M , Haba S , Nisonoff A
Published: May 2006

We describe here an intrastrain, cross-reactive idiotype (CRI), CRID, associated with anti-p-azobenzenearsonate antibodies of the A/J strain of mouse and distinguishable, by some but not by all of its idiotopes, from the major anti-p-azobenzenearsonate idiotype (CRIA). Molecules carrying the CRID idiotype have heavy chain variable-segment sequences that are identical or nearly identical to that of the germ-line-encoded heavy chain variable sequence of CRIA. Their light chain variable sequences are very similar to those present in a third idiotypic family, CRIC, that is a minor CRI in the A/J strain but a major CRI in BALB/c. This appears to represent a form of combinational diversity, in which the heavy and light chain variable region genes of two unrelated idiotypic families interact to form a third family, all involving antibodies of the same antigen-binding (anti-p-azobenzenearsonate) specificity. The D region of CRID, in the six monoclonal representatives studied (three IgMs, three IgGs), is unusual in that it comprises a single amino acid--arginine or serine; there are eight amino acids in the D region of CRI+A antibodies. Three different heavy chain joining regions, JH1, JH2, and JH3, are utilized. A serological reagent was developed that identifies CRID; it was used to show that the idiotype is present in relatively high concentration, comparable to that of CRIA, in anti-p-azobenzenearsonate antibodies taken soon after primary immunization. In serum taken later CRIA greatly predominates, but CRID persists at low levels.
Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects.
Authors: Sigal I S , Gibbs J B , D'Alonzo J S , Temeles G L , Wolanski B S , Socher S H , Scolnick E M
Published: May 2006

We report that residues Lys-16 and Asp-119 play critical roles in the guanine nucleotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala119]Ha to induce changes characteristic of cellular transformation was much greater than that of normal Ha and similar to that of the oncogenic [Val12, Thr59]Ha. In this assay, [Asn16]Ha and [Val12, Asn16, Thr59]Ha were similar in potency to normal Ha. In yeast cells, Ha proteins with reduced nucleotide affinity exert a dominant temperature-dependent lethality that is avoided by the coexpression of the activated yeast ras gene [Ala18, Val19]RAS2. We interpret the biological consequences of reducing the nucleotide affinity of ras proteins in terms of two opposing factors: a growth-promoting effect, resulting from an increase in the GDP-GTP exchange rate, and a growth-limiting effect, resulting from an increase in the nucleotide-free ras protein species.
Inhibition of tumor-cell-mediated extracellular matrix destruction by a fibroblast proteinase inhibitor, protease nexin I.
Authors: Bergman B L , Scott R W , Bajpai A , Watts S , Baker J B
Published: May 2006

Human fibrosarcoma (HT-1080) cells, in contrast to normal fibroblasts, rapidly hydrolyze the glycoprotein, collagen, and elastin extracellular matrix (ECM) synthesized by cultured rat aortic smooth muscle cells. This degradation occurs at a rapid rate in the presence of serum, indicating that the cellular proteases responsible are relatively insensitive to serum proteinase inhibitors. Here it is shown that protease nexin I (PNI), a fibroblast-secreted inhibitor of urokinase, plasmin, and certain other serine proteinases, effectively inhibited the HT-1080 cell-mediated degradation of this ECM. PNI at 2.0 nM significantly inhibited matrix destruction for 1-2 days and at 0.2 microM caused a virtually complete inhibition that persisted for the entire 10-day period of observation. Inhibition of ECM destruction was accompanied by a transient arrest of HT-1080 cell proliferation that took place during the first 3 days after PNI addition. PNI did not inhibit the growth of normal fibroblasts and also did not inhibit the growth of HT-1080 cells that were seeded onto plastic dishes rather than onto ECM. Like many types of malignant cells, HT-1080 cells release large amounts of urokinase. Antibody against this plasminogen activator partially protected ECM from HT-1080 cell-mediated hydrolysis, indicating that it may have been a target of PNI. One potential physiological function of PNI could be to help maintain the integrity of connective tissue matrices, protection that malignant cells could overcome by secreting proteinases in excessive amounts.
Intermediate filaments in alpha-keratins.
Authors: Fraser R D , MacRae T P , Parry D A , Suzuki E
Published: May 2006

Previous x-ray diffraction studies on the alpha-keratins of hair and wool have revealed that the intermediate filaments (IF) have a helical structure rendered imperfect by a precisely defined dislocation. It has also been possible to deduce a surface lattice for the IF and to determine the number of IF molecules associated with each lattice point. In this work this information is combined with data on the ionic interactions between the coiled-coil rope segments of the IF molecules to provide a plausible model for the pattern of interactions that stabilize the framework of the IF in the "hard" alpha-keratins. Similar interaction studies of the proteins from the IF in the so-called "soft" alpha-keratin from the stratum corneum layer of the skin suggest that they are likely to have an essentially similar pattern.
Two acyl-coenzyme A oxidases in peroxisomes of the yeast Candida tropicalis: primary structures deduced from genomic DNA sequence.
Authors: Okazaki K , Takechi T , Kambara N , Fukui S , Kubota I , Kamiryo T
Published: May 2006

We report the complete nucleotide sequence of two genes encoding major peroxisomal polypeptides (PXPs) of Candida tropicalis. One, POX4, encodes PXP-4, which is the most abundant polypeptide in cells grown on oleic acid, and the other, POX5, is the gene for PXP-5. Each of the two polypeptides was found to be the subunit of a distinct long-chain acyl-coenzyme A oxidase: acyl-CoA oxidase II (PXP-4) or acyl-CoA oxidase I (PXP-5). Both the genes had no intron and gave a single open reading frame. The NH2-terminal sequences, except the initiator methionine, and the calculated molecular weights of the deduced polypeptides were consistent with those of the respective PXPs. Well-conserved sequences of 12 and 16 hydrophobic amino acids were present in the middle of the polypeptide, instead of at the NH2 terminus, and may be internal signal sequences for the peroxisomal location of PXPs. Although the two polypeptides were significantly homologous throughout their sequences, the local homologies in two regions out of five were markedly diverged from the average (63%); the homology in the second region was 93%, whereas that in the fourth one was only 24%. The implications of this finding are discussed in respect to the multiplicity of peroxisomal enzymes and the presence of multifunctional proteins in peroxisomes.
Primary structure of the human melanoma-associated antigen p97 (melanotransferrin) deduced from the mRNA sequence.
Authors: Rose T M , Plowman G D , Teplow D B , Dreyer W J , Hellström K E , Brown J P
Published: May 2006

p97 is a cell-surface glycoprotein that is present in most human melanomas but only in trace amounts in normal adult tissues. To determine the structure of this tumor-associated antigen and to identify its functional domains, we have purified and cloned p97 mRNA and determined its nucleotide sequence. The mRNA encodes a 738-residue precursor, which contains the previously determined N-terminal amino acid sequence of p97. After removal of a 19-residue signal peptide, the mature p97 molecule comprises extracellular domains of 342 and 352 residues and a C-terminal 25-residue stretch of predominantly uncharged and hydrophobic amino acids, which we believe acts as a membrane anchor. Each extracellular domain contains 14 cysteine residues, which form seven intradomain disulfide bridges, and one or two potential N-glycosylation sites. Protease digestion studies show that the three major antigenic determinants of p97 are present on the N-terminal domain. The domains are strikingly homologous to each other (46% amino acid sequence homology) and to the corresponding domains of human serum transferrin (39% homology). Conservation of disulfide bridges and of amino acids thought to compose the iron binding pockets suggests that p97 is also related to transferrin in tertiary structure and function. We propose that p97 be renamed melanotransferrin to denote its original identification in melanoma cells and its evolutionary relationship to serotransferrin and lactotransferrin, the other members of the transferrin superfamily.
Erythrocyte-neutrophil interactions: formation of leukotriene B4 by transcellular biosynthesis.
Authors: McGee J E , Fitzpatrick F A
Published: May 2006

Studies on the mechanism of leukotriene B4 biosynthesis in suspensions composed of neutrophils plus erythrocytes indicate that human erythrocytes convert neutrophil-derived leukotriene A4 into leukotriene B4. Leukotriene B4 formation by neutrophils in the presence of erythrocytes exceeded that from corresponding suspensions of neutrophils alone. The increase was proportional to the erythrocyte content of the suspension. The erythrocyte-dependent increase in leukotriene B4 biosynthesis did not equal the arithmetic sum of calcium ionophore-dependent biosynthesis by neutrophils plus calcium ionophore-dependent biosynthesis by erythrocytes, since erythrocytes produced no leukotriene B4 upon incubation with ionophore A23187. Erythrocytes did not stimulate 5-lipoxygenase activity within neutrophils, since the erythrocyte effect was confined to enzymatic hydration: leukotriene B4 increased coincident with decreased formation of 5,12-dihydroxyicosatetraenoic acids derived from nonenzymatic hydration. Biosynthesis of leukotriene B4 within the erythrocyte, from neutrophil-derived leukotriene A4, was established by comparing the effect of normal erythrocytes with erythrocytes containing a leukotriene A4 hydrolase that was inactivated by the substrate. In the latter case, leukotriene B4 formation increased by only 30-40%; in the former case, it increased by 100-200%. Transcellular biosynthesis of leukotriene B4 from erythrocyte-neutrophil interactions explains the paradoxical presence of leukotriene A4 hydrolase within erythrocytes, a cell incapable of synthesizing leukotriene A4; affords a mechanism to overcome rate limitations or "suicide inactivation" of leukotriene A4 hydrolase in neutrophils; exploits a cryptic capacity within erythrocytes, provisionally dormant cells in terms of icosanoid biosynthesis; indicates that the biosynthetic capacity of cell combinations is not necessarily equivalent to the sum of their separate capacities.
Molecular cloning of the mouse cell adhesion molecule uvomorulin: cDNA contains a B1-related sequence.
Authors: Schuh R , Vestweber D , Riede I , Ringwald M , Rosenberg U B , Jäckle H , Kemler R
Published: May 2006

A clone (F20) containing coding sequences for the cell adhesion molecule uvomorulin was isolated by immunological techniques from cDNA library in the expression vector lambda gt11. The beta-galactosidase-uvomorulin fusion protein was used to affinity purify anti-uvomorulin antibodies. Affinity-purified antibodies recognized uvomorulin from cell lysates of embryonal carcinoma cells and reacted with the cell surface of embryonal carcinoma cells. The 1.8-kilobase cDNA insert hybridized to a single 4.3-kilobase poly(A)+ RNA species found only in cells expressing uvomorulin. Part of the nontranslated 3' sequences of the cloned uvomorulin cDNA is homologous to the interspersed B1 repeat of the mouse genome.
The orangutan adult alpha-globin gene locus: duplicated functional genes and a newly detected member of the primate alpha-globin gene family.
Authors: Marks J , Shaw J P , Shen C K
Published: May 2006

We have cloned and sequenced the complete alpha 1- and alpha 2-globin genes of the orangutan, and here we compare them to the homologous genes of the human. The pattern of similarity apparent among the genes is most consistent with a model of gene correction operating on the primate alpha-globin cluster. This correction breaks down in both human and orangutan in the 3'-untranslated region at 14 base pairs downstream from the termination codon. The unit evolutionary period values calculated for either the replacement substitution or the silent substitutions are only slightly higher than the previously established molecular clock predicts. The 7-base-pair insertion in intron 2 of the human alpha 1-globin gene is not present in either orangutan gene, suggesting that this insertion is not the cause of the sequence divergence in the 3'-untranslated regions of primate alpha 2- and alpha 1-globin genes. Finally, blotting hybridization and partial DNA sequencing reveal a newly detected member of the primate alpha-globin gene family, which is located downstream from the duplicated adult alpha-globin genes.