bluuebunny/crossref_metadata_2025 huggingface dataset:
Glial cells develop a laminar pattern before neuronal cells in the lateral geniculate nucleus.
Authors: Hutchins J B , Casagrande V A
Published: May 2006

The lateral geniculate nucleus, which lies between the retina and the striate cortex in the visual pathway of mammals, is often made up of several distinctive cell layers, or laminae. We have used immunohistochemical methods to localize two glial cell intermediate filament proteins, glial fibrillary acidic protein and vimentin, and have found that layering of glial cells is evident before neuronal cell layers develop in the lateral geniculate nucleus. The correlation between glial cell lamination and neuronal lamination is consistent with the suggestion that glia are guiding neurons not only during the early postmitotic migratory phase of development but also during the later formation of functional divisions such as layers and nuclei.
Bipolar cells in the turtle retina are strongly immunoreactive for glutamate.
Authors: Ehinger B , Ottersen O P , Storm-Mathisen J , Dowling J E
Published: May 2006

Strong glutamate immunoreactivity was observed by both light and electron microscopy in bipolar cells of the turtle (Pseudemys scripta elegans) retina after postembedding immunohistochemistry. Virtually all bipolar cells showed strong labeling, on average 18 times that of the Müller (glial) cells. The data suggest that both on- and off-center bipolar cells are glutamatergic. Photoreceptors were also labeled, but with a labeling intensity about half that of the bipolar cells. Other types of retinal neurons showed less immunoreactivity, except for a small population of strongly labeled amacrine cells.
Repair of 4,5',8-trimethylpsoralen monoadducts and cross-links by the Escherichia coli UvrABC endonuclease.
Authors: Jones B K , Yeung A T
Published: May 2006

Using an oligonucleotide model substrate, we observed two unusual mechanisms of UvrABC endonuclease in the repair of 4,5',8-trimethylpsoralen monoadducts and crosslinks. (i) UvrABC endonuclease usually incises a psoralen monoadduct only on the damaged strand. However, for one of the monoadducts we studied, incision on the complementary undamaged strand was also observed at a very low frequency, as though the adduct were on the thymine across from the damaged strand. Although the details of the erroneous incision are not yet known, such erroneous incision is potentially mutagenic. (ii) In cross-link repair, we observed that the UvrABC endonuclease incises the cross-linked DNA on either the furan side strand or the pyrone side strand. The incisions are not equally efficient. These data suggest that the structure of a psoralen cross-link, as seen by a repair enzyme, varies with the DNA sequence.
A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily.
Authors: Hazel T G , Nathans D , Lau L F
Published: May 2006

We previously identified, by cDNA cloning, a set of genes that are expressed during the G0/G1 transition (cell cycle reentry) in mouse fibroblasts. These immediate early genes are transcriptionally activated within minutes of addition of serum or purified growth factors, and their mRNAs are superinduced in the presence of protein-synthesis inhibitors. We report here that one of these genes, represented by nur/77 cDNA (originally called 3CH77), encodes a member of the superfamily of ligand-binding transcription factors that includes the steroid and thyroid hormone receptors. The nur/77 cDNA sequence encodes a protein of 601 amino acids containing two regions of sequence similarity to members of this nuclear receptor superfamily, corresponding to their DNA-binding and ligand-binding domains. These results suggest that the growth factor-inducible immediate early gene nur/77 encodes a ligand-binding protein that regulates the genomic response to growth factors.
Two interferon-induced nuclear factors bind a single promoter element in interferon-stimulated genes.
Authors: Kessler D S , Levy D E , Darnell J E
Published: May 2006

Nuclear proteins induced by interferon (IFN) treatment of human cells are capable of forming two specific complexes with DNA fragments containing the IFN-stimulated response element (ISRE). These two complexes, designated B2 and B3, are distinguished by differential migration in gel retardation assays. The factor that forms the B3 complex, termed IFN-stimulated gene factor 3 (ISGF-3), preexists in cells, is activated upon IFN treatment, and appears with kinetics paralleling those for transcriptional activation of IFN-stimulated genes. The factor that forms the B2 complex (ISGF-2) appears following a time lag after IFN treatment during which protein synthesis must occur. By extensive point mutagenesis of the ISREs from two IFN-stimulated promoters (ISG54 and ISG15), we demonstrate that the B2 and B3 complexes are formed by factors binding to the same DNA sequence. Mutations at this site decrease or eliminate transcriptional activation and impair binding of both ISGF-2 and ISGF-3. This analysis has shown that the ISGF-3 binding site is slightly broader than the ISGF-2 binding site, which is completely contained within the sequence necessary both for ISGF-3 binding and for transcriptional activation. The evidence strongly implicates ISGF-3 as the positive transcriptional regulator of IFN-stimulated genes.
Butyrate infusions in the ovine fetus delay the biologic clock for globin gene switching.
Authors: Perrine S P , Rudolph A , Faller D V , Roman C , Cohen R A , Chen S J , Kan Y W
Published: May 2006

The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal beta-globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switching.
Role of the overdrive sequence in T-DNA border cleavage in Agrobacterium.
Authors: Toro N , Datta A , Yanofsky M , Nester E
Published: May 2006

The T-DNA of the Ti plasmid of Agrobacterium is flanked by 25-base-pair imperfect direct repeats that are required in cis for transfer to the genome of the plant host. Another sequence, designated overdrive, is located adjacent to the right-border repeats and functions in cis to enhance tumor formation. We have examined the effect of the overdrive sequence on the early steps in T-DNA processing. We report here that overdrive greatly enhances cleavage by the site-specific endonuclease in Agrobacterium, perhaps by directing the endonuclease to the adjacent border sequences. We also show by a gel mobility-shift assay that overdrive affinity-purified proteins from acetosyringone-induced Agrobacterium cells interact with T-DNA border and overdrive sequences. Further, we show that in vivo the virC operon enhances cleavage at the T-DNA borders, most likely by interaction between the VirC1 protein and the overdrive sequence.
B-cell-specific and interferon-gamma-inducible regulation of the HLA-DR alpha gene.
Authors: Tsang S Y , Nakanishi M , Peterlin B M
Published: May 2006

We investigated the cis-acting sequences that function in the B-cell-specific and interferon-gamma-inducible expression of the HLA-DR alpha gene, a human class II major histocompatibility complex gene. The effects of 5' deletions on the activity of the DR alpha promoter and the influence of upstream DR alpha promoter elements on the activity of the herpes simplex virus thymidine kinase promoter were examined by a transient transfection assay in human B-, T-, and fibroblast cell lines. We show that the DR alpha gene is regulated by positive and negative cis-acting sequences between positions -1300 and +31 from the site of initiation of transcription. We also demonstrate that the DR alpha promoter sequences from positions -116 to -92 and from -136 to -80 are the minimal sequences required for conferring B-cell specificity and interferon-gamma inducibility upon the Herpes simplex virus thymidine kinase promoter, respectively.
Detailed delineation of an interferon-gamma-responsive element important in human HLA-DRA gene expression in a glioblastoma multiform line.
Authors: Basta P V , Sherman P A , Ting J P
Published: May 2006

In this report, we determined that induction of the DR alpha-chain by recombinant human interferon-gamma (IFN-gamma) in a human glioblastoma multiform cell line is transcriptionally regulated and showed that protein synthesis is not necessary for this to occur. The regions of the DR alpha-chain gene that are responsible for basal and recombinant IFN-gamma-induced gene transcription have been determined by gene transfer of a series of 5' deletion mutants in which the upstream region of the DR alpha chain was linked to a reporter gene, chloramphenicol acetyltransferase. Chloramphenicol acetyltransferase transcript and protein levels were determined by S1 nuclease protection and chloramphenicol acetyltransferase enzyme assays, respectively. By using these deletion mutants, we were able to draw the following conclusions. (i) One hundred and nine base pairs of upstream sequence contains the basic DR alpha-chain gene promoter and represents the minimal amount of sequence necessary for basal gene expression. (ii) An additional 9 base pairs of upstream sequence can mediate recombinant IFN-gamma induction. (iii) Maximal recombinant IFN-gamma induction requires at most an additional 23 base pairs of upstream sequence. (iv) The sequence between positions -267 and -141 does not appear to contain any additional positive or negative regulatory elements. These results suggest that the region between positions -141 and -109 contains a critical IFN-gamma-responsive element. Substitution mutagenesis was performed to confirm this suggestion.
M2 delta, a candidate for the structure lining the ionic channel of the nicotinic cholinergic receptor.
Authors: Oiki S , Danho W , Madison V , Montal M
Published: May 2006

A synthetic 23-mer peptide that mimics the sequence of the putative transmembrane M2 segment of the Torpedo californica acetylcholine receptor (AcChoR) delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe-Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms discrete ionic channels in phosphatidylcholine bilayers. In contrast, a synthetic peptide that mimics the sequence of the putative M1 transmembrane segment of the Torpedo AcChoR delta subunit--Leu-Phe-Tyr-Val-Ile-Asn-Phe-Ile-Thr-Pro-Cys-Val-Leu-Ile-Ser-Phe- Leu-Ala-Ser-Leu-Ala-Phe-Tyr--does not form channels. The synthetic M2 delta channel peptide exhibits features that are characteristic of the authentic AcChoR channel, such as single channel conductances, discrimination of cations over anions, and channel lifetimes for open and closed states in the millisecond time range. Energetic considerations suggest that an aggregate of five amphipathic alpha-helices conforms the channel. Thus, the M2 segment may be a component of the AcChoR channel structure.
Cadmium accelerates bone loss in ovariectomized mice and fetal rat limb bones in culture.
Authors: Bhattacharyya M H , Whelton B D , Stern P H , Peterson D P
Published: May 2006

Loss of bone mineral after ovariectomy was studied in mice exposed to dietary cadmium at 0.25, 5, or 50 ppm. Results show that dietary cadmium at 50 ppm increased bone mineral loss to a significantly greater extent in ovariectomized mice than in sham-operated controls. These results were obtained from two studies, one in which skeletal calcium content was determined 6 months after ovariectomy and a second in which 45Ca release from 45Ca-prelabeled bones was measured immediately after the start of dietary cadmium exposure. Furthermore, experiments with 45Ca-prelabeled fetal rat limb bones in culture demonstrated that Cd at 10 nM in the medium, a concentration estimated to be in the plasma of mice exposed to 50 ppm dietary Cd, strikingly increased bone resorption, from 27 +/- 2% (mean +/- SEM) 45Ca release in cultures with no added cadmium to 68 +/- 6% release in cultures containing cadmium (n = 4). These in vitro results indicate that cadmium may enhance bone mineral loss by a direct action on bone. Results of the in vivo studies are consistent with a significant role of cadmium in the etiology of Itai-Itai disease among postmenopausal women in Japan and may in part explain the increased risk of postmenopausal osteoporosis among women who smoke.
Identification and purification of a Drosophila protein that binds to the terminal 31-base-pair inverted repeats of the P transposable element.
Authors: Rio D C , Rubin G M
Published: May 2006

We have used DNase I footprinting and partially fractionated nuclear extracts from Drosophila Kc tissue culture cells to identify DNA-binding proteins that interact with the terminal repeats of P transposable elements. We have identified a binding activity that interacts specifically with a region of the 31-base-pair terminal inverted repeats that is directly adjacent to the duplication of target site DNA. Binding occurs to both the 5' and 3' inverted terminal repeats irrespective of the sequence of the duplicated target DNA. UV photochemical crosslinking studies suggest that the binding activity resides in a polypeptide of 65-70 kDa. Biochemical fractionation and oligonucleotide affinity chromatography have been used to purify the binding activity to near homogeneity and identify a polypeptide of 66 kDa in the highly purified preparations. The site to which binding occurs is included in a region absolutely required for P element transposition, suggesting that this binding protein may be a cellular factor involved in P element transposition.
Promoters largely determine the efficiency of repressor action.
Authors: Lanzer M , Bujard H
Published: May 2006

Operator sequence and repressor protein regulate the activity of the lac promoter over a greater than 1000-fold range. Combinations of the lac operator with other promoter sequences, however, differ vastly in the level of repression. The data presented show that the extent of repression is determined largely by the rates of complex formation of the competing systems operator-repressor and promoter-RNA polymerase and by the rate at which RNA polymerase clears the promoter. Moreover, up to 70-fold differences in the level of repression were found when the operator was placed in different positions within the promoter sequence. A kinetic model is proposed that explains the observed effects and that allows predictions on promoters controlled by negatively acting elements.
Admixture as a tool for finding linked genes and detecting that difference from allelic association between loci.
Authors: Chakraborty R , Weiss K M
Published: May 2006

Admixture between genetically different populations may produce gametic association between gene loci as a function of the genetic difference between parental populations and the admixture rate. This association decays as a function of time since admixture and the recombination rate between the loci. Admixture between genetically long-separated human populations has been frequent in the centuries since the age of exploration and colonization, resulting in numerous hybrid descendant populations today, as in the Americas. This represents a natural experiment for genetic epidemiology and anthropology, in which to use polymorphic marker loci (e.g., restriction fragment length polymorphisms) and disequilibrium to infer a genetic basis for traits of interest. In this paper we show that substantial disequilibrium remains today under widely applicable situations, which can be detected without requiring inordinately close linkage between trait and marker loci. Very disparate parental allele frequencies produce large disequilibrium, but the sample size needed to detect such levels of disequilibrium can be large due to the skewed haplotype frequency distribution in the admixed population. Such situations, however, provide power to differentiate between disequilibrium due just to population mixing from that due to physical linkage of loci--i.e., to help map the genetic locus of the trait. A gradient of admixture levels between the same parental populations may be used to test genetic models by relating admixture to disequilibrium levels.
Hypoxia induces DNA overreplication and enhances metastatic potential of murine tumor cells.
Authors: Young S D , Marshall R S , Hill R P
Published: May 2006

Cultured cells subjected to oxygen deprivation have been shown to undergo anomalous DNA synthesis, which can result in DNA overreplication and the generation of cellular variants [Rice, G. C., Hoy, C. & Schimke, R. T. (1986) Proc. Natl. Acad. Sci. USA 83, 5978-5982]. In the present study, murine tumor cells were exposed to severe hypoxia and then tested for their ability to form experimental metastases. Upon reoxygenation, cells transiently, yet dramatically, increased their metastatic potential. Flow cytometric analysis confirmed that hypoxia and reoxygenation induced cell cycle perturbations and DNA overreplication in these tumor cell lines. Fibrosarcoma cells with overreplicated DNA isolated by fluorescence-activated cell sorting proved to be highly metastatic, although cells with 2-4 times the haploid DNA content in populations treated with hypoxia were also markedly more metastatic than oxic populations. These results support the hypothesis that hypoxic conditions existing in regions of solid tumors promote cellular heterogeneity and tumor progression.
Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors.
Authors: Kato Y , Iwamoto M , Koike T , Suzuki F , Takano Y
Published: May 2006

Rabbit chondrocyte cultures on plastic dishes are capable of depositing a cartilaginous matrix, although the matrix does not calcify unless high levels of phosphate are added to the medium. In the present study, we cultivated a pelleted mass of rabbit growth-plate chondrocytes in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 micrograms of ascorbic acid per ml in a plastic centrifuge tube. These cells proliferated for several generations and then reorganized into a cartilage-like tissue that calcified without additional phosphate. The deposition of minerals was observed only after synthesis of a short-chain collagen and alkaline phosphatase. Serum factors were required for the increases in alkaline phosphatase and calcium contents. 5-Bromo-2'-deoxyuridine abolished the increases in uronic acid, alkaline phosphatase, and calcium contents. Transforming growth factor beta, at very low concentrations, suppressed the expression of the mineralization-related phenotype by chondrocytes. These results suggest that cartilage-matrix calcification can be controlled by growth factor(s) and that chondrocytes induce the mineralization of extracellular matrix when terminal differentiation is permitted in the absence of an artificial substrate.
Four exons encode a 93-base-pair insert in three neural cell adhesion molecule mRNAs specific for chicken heart and skeletal muscle.
Authors: Prediger E A , Hoffman S , Edelman G M , Cunningham B A
Published: May 2006

The neural cell adhesion molecule (N-CAM) is detected in chicken brain as three polypeptides of 180 kDa, 140 kDa, and 120 kDa that arise from a single gene by alternative splicing. Heart tissue, however, contains components of 150 kDa, 140 kDa, and 130 kDa; neither the differences in molecular mass among these components nor the difference between neural and cardiac N-CAM could be accounted for by variations in glycosylation alone. A cDNA clone isolated from an embryonic chicken heart library, [lambda N101B, 1.8 kilobases (kb)] contained a 93-base-pair (bp) insert not found in neural N-CAM cDNAs. In the N-CAM gene this sequence mapped within a large region between exons 12 and 13 and was derived from four exons (12A-D) of 15, 33, 42, and 3 bp. Exons 12C and 12D together coded for 15 amino acids very similar to the second half of the muscle-specific insert (MSD1) found in N-CAM cDNA from human muscle cell cultures [Dickson, G., Gower, H. J., Barton, C. H., Prentice, H. M., Elsom, V. L., Moore, S. E., Cox, R. D., Quinn, C., Putt, W. & Walsh, F. S. (1987) Cell 50, 1119-1130]; the sequences of 12A and 12B, however, were much less similar to the corresponding region of the MSD1 sequence. Two oligonucleotides, one specific to exons 12A plus 12B and one specific to exon 12C both recognized mRNA species of 6.4 kb, 4.3 kb, and 3.0 kb in chicken cardiac and skeletal muscle and no mRNA species in smooth muscle or brain. The 3' end of clone lambda N101B contained a sequence coding for a potential phosphatidylinositol linkage signal as does the smallest form of brain N-CAM. In heart cell membranes only the 130-kDa N-CAM polypeptide was released by phospholipase C, suggesting that this form of N-CAM is encoded by clone lambda N101B. The other heart N-CAM species (150 kDa and 140 kDa) may be transmembrane forms that include the 12A-D (and possibly other) inserts. Tissue-specific forms of N-CAM can thus be formed by alternative use of multiple small exons that may alter the conformation of the extracellular region of the molecule. Differential use or switching of these small exons in conjunction with the differential expression of larger exons specifying regions associated with the cell membrane and cytoplasmic domains may signal key events in embryogenesis and histogenesis.
Construction of a clonable, infectious, and tumorigenic mouse mammary tumor virus provirus and a derivative genetic vector.
Authors: Shackleford G M , Varmus H E
Published: May 2006

Molecular genetic studies of mouse mammary tumor virus (MMTV) have been hampered by the difficulty of cloning proviruses of milk-borne strains because of inhibitory sequences located in the gag gene. To surmount this barrier we have constructed a hybrid MMTV provirus composed of clonable 5' sequences (encompassing gag) from an endogenous MMTV provirus of C3H mice (Mtv-1) and 3' sequences from the milk-borne strain of MMTV in C3H mice, MMTV(C3H). Virions produced from XC cells transfected with this hybrid provirus are infectious in cell culture and tumorigenic in BALB/cJ mice. A vector derived from this provirus, containing the neomycin phosphotransferase gene (neo) and origins of replication from simian virus 40 and pBR322, is capable of transferring G418 resistance by virus infection in cell culture when supplied with viral proteins from either MMTV(C3H) or the hybrid MMTV. Expression of both hybrid and vector proviruses is inducible by dexamethasone in infected cells.
Rapid detection of human chromosome 21 aberrations by in situ hybridization.
Authors: Lichter P , Cremer T , Tang C J , Watkins P C , Manuelidis L , Ward D C
Published: May 2006

Plasmid clones containing up to 94 kilobases of single-copy DNA from band q22.3 of chromosome 21 and a complete pool of insert DNA from a chromosome 21 recombinant library have been used to rapidly detect numerical and structural aberrations of chromosome 21 by in situ hybridization in both metaphase and interphase cells. A trisomic karyotype, diagnostic of Down syndrome, is readily detected in nonmitotic cells because the majority of their nuclei exhibit three discrete foci of hybridization, in contrast to normal diploid cells, which show two foci. Chromosomal translocations involving chromosome 21 sequences were also detected with these probes, and the intranuclear location of 21q22.3 DNA sequences in "normal" human brain neurons was established with the plasmid DNA probe set. These results suggest that chromosome 21-specific probes may have utility in clinical diagnostics, especially by facilitating the direct analysis of interphase cells.
Early expression of a T-cell receptor beta-chain transgene suppresses rearrangement of the V gamma 4 gene segment.
Authors: von Boehmer H , Bonneville M , Ishida I , Ryser S , Lincoln G , Smith R T , Kishi H , Scott B , Kisielow P , Tonegawa S
Published: May 2006

beta transgenic mice have a T-cell receptor beta-chain gene that is prematurely expressed on the surface of CD4- CD8- thymocytes and paired with an uncharacterized non-T-cell receptor alpha-chain polypeptide. The rearrangement of the T-cell receptor variable region gamma chain gene segment V gamma 4, a component of the gamma-chain gene that is rearranged and expressed preferentially on thymocytes of normal adult mice, is severely repressed in beta transgenic mice. Consequently no gamma delta T-cell receptor heterodimers are detectable on the surface of adult thymocytes or splenic T cells. These results indicate that cells expressing alpha beta or gamma (V gamma 4)-delta TCRs originate from a common precursor in which the first productive rearrangement of either the beta or gamma locus determines the further differentiation pathway into either alpha beta or gamma delta T cells. The repression of V gamma 4 rearrangement by a preexisting beta-chain gene may be indicative of one of several mechanisms which ensure that gamma delta and alpha beta receptors do not as a rule appear on the surface of the same cell.
Stable expression of the tax gene of type I human T-cell leukemia virus in human T cells activates specific cellular genes involved in growth.
Authors: Wano Y , Feinberg M , Hosking J B , Bogerd H , Greene W C
Published: May 2006

Stable expression of the 40-kDa transactivator protein (Tax) from the type I human T-cell leukemia virus (HTLV-I) in Jurkat T cells leads to the activation and sustained expression of certain cellular genes that are transiently induced during normal T-cell growth. Cellular genes induced by Tax include those encoding the alpha subunit of the high-affinity interleukin 2 receptor (Tac), interleukin 2, and granulocyte/macrophage colony-stimulating factor. Tax induction of the interleukin 2 gene is synergistically amplified by mitogens that augment cytoplasmic levels of calcium. These changes in the pattern of cellular gene expression reflect a specific action of Tax, as they are undetectable in isogenically matched control cell lines expressing antisense tax cDNA. The spectrum of cellular genes regulated by Tax appears to be restricted: several other T-cell genes, either inducibly or constitutively expressed, are unaffected by this viral protein. These cell lines constitutively expressing Tax provide valuable reagents to explore the molecular basis for Tax action and to delineate the full spectrum of cellular genes regulated by this retroviral gene product.
Expression of the T-cell surface molecule CD2 and an epitope-loss CD2 mutant to define the role of lymphocyte function-associated antigen 3 (LFA-3) in T-cell activation.
Authors: Bierer B E , Peterson A , Barbosa J , Seed B , Burakoff S J
Published: May 2006

To define the role of the CD2-lymphocyte function-associated antigen 3 (LFA-3) interaction in T-cell activation, we have expressed a cDNA encoding the human CD2 molecule in a murine antigen-specific T-cell hybridoma. Expression of the CD2 molecule greatly enhances T-cell responsiveness to antigen; this enhancement is inhibited by anti-CD2 and anti-LFA-3 monoclonal antibodies (mAbs). CD2+ hybridomas produce interleukin 2 in response to combinations of anti-CD2 mAbs 9.6 and 9-1 and, in the presence of mAb 9-1, to sheep erythrocytes or to the LFA-3 antigen. Furthermore, hybridomas expressing a mutant CD2 molecule that has lost mAb 9.6 binding do not exhibit the enhanced response to antigen or the ability to respond to LFA-3 plus mAb 9-1, but these hybridomas retain the ability to respond to combinations of anti-CD2 mAbs. The role of the CD2-LFA-3 interaction in T-cell activation and the potential for other physiologic ligands for CD2 are discussed.
In situ hybridization of nucleus basalis neurons shows increased beta-amyloid mRNA in Alzheimer disease.
Authors: Cohen M L , Golde T E , Usiak M F , Younkin L H , Younkin S G
Published: May 2006

To determine which cells within the brain produce beta-amyloid mRNA and to assess expression of the beta-amyloid gene in Alzheimer disease, we analyzed brain tissue from Alzheimer and control patients by in situ hybridization. Our results demonstrate that beta-amyloid mRNA is produced by neurons in the nucleus basalis of Meynert and cerebral cortex and that nucleus basalis perikarya from Alzheimer patients consistently hybridize more beta-amyloid probe than those from controls. These observations support the hypothesis that increased expression of the beta-amyloid gene plays an important role in the deposition of amyloid in the brains of patients with Alzheimer disease.
Transformation of thebaine to oripavine, codeine, and morphine by rat liver, kidney, and brain microsomes.
Authors: Kodaira H , Spector S
Published: May 2006

Thebaine, an intermediate of morphine biosynthesis in the poppy plant, Papaver somniferum, was transformed to oripavine, codeine, and morphine by rat liver, kidney, and brain microsomes in the presence of an NADPH-generating system. The formation of morphine, codeine, and oripavine was identified by a specific RIA, HPLC, and GCMS. Thebaine also gave rise to four other compounds, which for the moment are unidentified. NADH dramatically increased the formation of both codeine and morphine when used together with an NADPH-generating system, especially in liver microsomes. NADPH is essential in the formation of oripavine from thebaine and morphine from codeine, while NADH is critical in the conversion of thebaine to codeine and from oripavine to morphine. Carbon monoxide or SKF 525A inhibited the conversion, indicating a role of cytochrome P-450. These results provide evidence for the enzymatic in vitro conversion by mammalian tissues of thebaine to morphine. The pathway is similar to that which exists in plants.
Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.
Authors: Antalis T M , Clark M A , Barnes T , Lehrbach P R , Devine P L , Schevzov G , Goss N H , Stephens R W , Tolstoshev P
Published: May 2006

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.
A Candida albicans dispersed, repeated gene family and its epidemiologic applications.
Authors: Scherer S , Stevens D A
Published: May 2006

Candida albicans causes a wide variety of infections but can readily be isolated from the skin and mucosa of healthy individuals. To enable high-resolution epidemiologic studies on this common pathogen, a species-specific DNA probe has been isolated from its genome. There are approximately equal to 10 copies of the sequence dispersed among the chromosome-sized DNA molecules resolved by pulsed-field electrophoresis. New DNA polymorphisms in this gene family arise at high rates. As a consequence, this probe will readily distinguish strains from different patients in the same hospital and from various sites in individual patients. The DNA polymorphisms detected by using this probe are largely due to internal changes in members of the family rather than movement to new genomic locations. This suggests recombination or gene conversion rather than transposition as the mechanism producing the observed variation.
lac permease of Escherichia coli containing a single histidine residue is fully functional.
Authors: Püttner I B , Kaback H R
Published: May 2006

Arg-302, His-322, and Glu-325, neighboring residues in putative helices IX and X of the lac permease (lacY gene product) of Escherichia coli, play an important role in lactose/H+ symport, possibly as components of a catalytic triad similar to that postulated for the serine proteases [Kaback, H. R. (1987) Biochemistry 26, 2071-2076]. By using restriction fragments of lacY genes harboring specific site-directed mutations, a fusion gene has been constructed that encodes a permease in which His-35 and His-39 are replaced with arginine, and His-205 with glutamine (RQHE permease). The resultant molecule contains a single histidine residue at position 322 and exhibits all of the properties of the wild-type permease. In addition, an analogous single-histidine permease was engineered with alanine at position 325 in place of glutamic acid (RQHA permease). This construct is defective in active transport but catalyzes exchange and counterflow normally. RQHA permease, like the single-histidine permease with Glu-325, also shows normal behavior with respect to N-ethylmaleimide inactivation, substrate protection, and binding. In addition to providing strong support for previous experiments, the engineered permease molecules should be useful for determining the apparent pK of His-322 under various conditions.
Oxygen and carbon isotopic compositions of gases respired by humans.
Authors: Epstein S , Zeiri L
Published: May 2006

Oxygen-isotope fractionation associated with respiration in human individuals at rest is linearly related to the fraction of the O2 utilized in the respiration process. The slope of this relationship is affected by a history of smoking, by vigorous exercise, and by the N2/O2 ratio of the inhaled gas. For patients who suffer anemia-related diseases, the slope of this relationship is directly proportional to their level of hemoglobin. These results introduce a new approach for studying the mechanisms of O2 consumption in human respiration and how they are affected by related diseases.
Okadaic acid: an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter.
Authors: Suganuma M , Fujiki H , Suguri H , Yoshizawa S , Hirota M , Nakayasu M , Ojika M , Wakamatsu K , Yamada K , Sugimura T
Published: May 2006

Okadaic acid is a polyether compound of a C38 fatty acid, isolated from a black sponge, Halichondria okadai. Previous studies showed that okadaic acid is a skin irritant and induces ornithine decarboxylase (OrnDCase; 3-hydroxyl-L-glutamate 1-carboxy-lyase, EC 4.1.1.17) in mouse skin 4 hr after its application to the skin. This induction was strongly inhibited by pretreatment of the skin with 13-cis-retinoic acid. A two-stage carcinogenesis experiment in mouse skin initiated by a single application of 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) and followed by application of 10 micrograms of okadaic acid twice a week revealed that okadaic acid is a potent additional tumor promoter: tumors developed in 93% of the mice treated with DMBA and okadaic acid by week 16. In contrast, tumors were found in only one mouse each in the groups treated with DMBA alone or okadaic acid alone. An average of 2.6 tumors per mouse was found in week 30 in the group treated with DMBA and okadaic acid. Unlike phorbol 12-tetradecanoate 13-acetate (TPA), teleocidin, and aplysiatoxin, okadaic acid did not inhibit the specific binding of [3H]TPA to a mouse skin particulate fraction when added up to 100 microM or activate calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in vitro when added up to 1.2 microM. Therefore, the actions of okadaic acid and phorbol ester may be mediated in different ways. These results show that okadaic acid is a non-TPA-type tumor promoter in mouse skin carcinogenesis.
Human liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells.
Authors: Tani K , Fujii H , Nagata S , Miwa S
Published: May 2006

Pyruvate kinase (PK) has four isozymes (L, R, M1, M2) that are encoded by two different genes. Among these isozymes, abnormalities of liver (L)-type PK is considered to be associated with hereditary nonspherocytic hemolytic anemia in humans. We isolated and determined the full-length sequence of human L-type PK cDNA. The cDNA contains 1629 base pairs encoding 543 amino acids, 68 base pairs of 5'-noncoding sequence, and 734 base pairs of 3'-noncoding sequence. The similarity between human and rat L-type PK was 86.9% at the nucleotide sequence level and 92.4% at the amino acid sequence level. The full-length L-type PK cDNA was placed under the promoter of simian virus 40 and introduced into monkey COS cells. Human L-type PK activity was detected in the extract of COS cells by the classical PK electrophoresis method.
Rapid regulation of c-myc protooncogene expression by progesterone in the avian oviduct.
Authors: Fink K L , Wieben E D , Woloschak G E , Spelsberg T C
Published: May 2006

The mRNA levels of genes known to be regulated by sex steroids are not altered until 1 hr or longer after steroid treatment, although the steroid receptor complexes are bound to nuclear acceptor sites within 5 min. In a search for early regulation of gene transcription, total chick oviduct RNA was isolated at various times after injection (i.p.) of progesterone and analyzed for c-myc expression. Levels of c-myc mRNA began to decrease in response to progesterone by 10 min after injection. The mRNA levels continued to decrease, reached a 70% reduction at 30 min, and returned to control values by 8 hr after steroid injection. Changes in alpha-tubulin mRNA levels were markedly less in these same RNA preparations. The effect was dependent on the dose of the steroid and was target-tissue specific. These changes occurred much more rapidly than changes in egg-white protein mRNA levels. Vehicle alone did not alter c-myc mRNA levels. Early regulated genes such as c-myc may represent the initial site of action of steroid receptors in the genome.
Transforming genes in chronic myelogenous leukemia.
Authors: Liu E , Hjelle B , Bishop J M
Published: May 2006

Chronic myelogenous leukemia (CML) is a hematopoietic malignancy characterized by an indolent chronic phase that invariably leads to a "blast crisis" indistinguishable from acute leukemia. Using a sensitive assay based on gene transfer and tumorigenesis, we sought evidence that damage to protooncogenes might figure in the progression from the chronic to the blast phase of CML. Seven of the 12 patients with CML examined in this manner harbored transforming genes. Mutations in RAS protooncogenes were detected in the leukemic cells from 1 of 6 chronic-phase patients, and 3 of 6 blast-crisis patients. In addition, a presently unidentified transforming gene (neither RAS nor RAF) was detected in 1 patient with chronic phase and 1 with blast crisis. Our data indicate that mutations in RAS genes may play diverse roles in the pathogenesis of CML.
Mean-field theory of a neural network.
Authors: Cooper L N , Scofield C L
Published: May 2006

A single-cell theory for the development of selectivity and ocular dominance in visual cortex has been generalized to incorporate more realistic neural networks that approximate the actual anatomy of small regions of cortex. In particular, we have analyzed a network consisting of excitatory and inhibitory cells, both of which may receive information from the lateral geniculate nucleus (LGN) and then interact through cortico-cortical synapses in a mean-field approximation. Our investigation of the evolution of a cell in this mean-field network indicates that many of the results on existence and stability of fixed points that have been obtained previously in the single-cell theory can be successfully generalized here. We can, in addition, make explicit further statements concerning the independent effects of excitatory and inhibitory neurons on selectivity and ocular dominance. For example, shutting off inhibitory cells lessens selectivity and alters ocular dominance (masked synapses). These inhibitory cells may be selective, but there is no theoretical necessity that they be so. Further, the intercortical inhibitory synapses do not have to be very responsive to visual experience. Most of the learning process can occur among the excitatory LGN-cortical synapses.
Identification of the major multiphosphorylation site in mammalian neurofilaments.
Authors: Lee V M , Otvos L , Carden M J , Hollosi M , Dietzschold B , Lazzarini R A
Published: May 2006

The sequence Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly repeats six times serially in the human midsized neurofilament (NF) protein (NF-M). To establish whether Lys-Ser-Pro-Val(Ala) is the major site for in vivo NF phosphorylation, peptides based on the human NF-M repeat were synthesized and chemically phosphorylated. These synthetic peptides were probed with 515 monoclonal antibodies (mAbs) that were raised to, and distinguished, several differentially phosphorylated forms of NF proteins. Studies with 95 of those mAbs that recognized the peptides before and after chemical phosphorylation demonstrated that a highly immunogenic epitope shared by the peptides is present in NFs from all species tested, including invertebrates. This suggests the phylogenetic conservation of a major NF phosphorylation site. Lastly, a cross-reactive antigenic determinant shared by the peptides and the major NF phosphorylation site was shown to exist in neurofibrillary tangles of patients with Alzheimer disease as well as in two neuron-specific microtubule-associated proteins (MAPs)--i.e., MAP2 and tau.
Diffusion in a rough potential.
Authors: Zwanzig R
Published: May 2006

Diffusion in a spatially rough one-dimensional potential is treated by analysis of the mean first passage time. A general expression is found for the effective diffusion coefficient, which can become very small at low temperatures.
The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.
Authors: Lopez J A , Chung D W , Fujikawa K , Hagen F S , Davie E W , Roth G J
Published: May 2006

The primary structure of the beta chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A lambda phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the beta chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5' untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding a signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3' noncoding region of 307 nucleotides. The 3' noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact beta chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the beta chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the alpha chain of GPIb and nine tandem repeats in leucine-rich alpha 2-glycoprotein. The leucine-rich sequence in the beta chain of GPIb is flanked on both sides by amino acid sequences that are similar to those flanking the leucine-rich tandem repeats of the alpha chain of GPIb and leucine-rich alpha 2-glycoprotein. The amino-terminal region of the beta chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein. The intracellular segment contains an unpaired cysteine and two potential sites for phosphorylation by cAMP-dependent protein kinase.
Activation of the protein-tyrosine kinase associated with the bombesin receptor complex in small cell lung carcinomas.
Authors: Gaudino G , Cirillo D , Naldini L , Rossino P , Comoglio P M
Published: May 2006

It has been hypothesized that bombesin-like peptides produced by small cell lung carcinomas may sustain deregulated proliferation through an autocrine mechanism. We have shown that the neuropeptide bombesin leads to the activation of a protein-tyrosine kinase that phosphorylates a 115-kDa protein (p115) associated with the bombesin receptor complex in mouse Swiss 3T3 fibroblasts. We now report that phosphotyrosine antibodies recognize a 115-kDa protein, phosphorylated on tyrosine, in four human small cell lung carcinoma cell lines producing bombesin but not in a nonproducer "variant" line. p115 from detergent-treated small cell lung carcinoma cells binds to bombesin-Sepharose and can be phosphorylated on tyrosine in the presence of radiolabeled ATP and Mn2+. As for the p115 immunoprecipitated from mouse fibroblast, the small cell lung carcinoma p115 can be phosphorylated in an immunocomplex kinase assay. However, the latter does not require the presence of exogenous bombesin for activity. Binding data, obtained by using radiolabeled ligand, suggest receptor occupancy in the cell lines producing bombesin. These observations are consistent with the hypothesis that proliferation in some human small cell lung carcinoma lines is under autocrine control, regulated through activation of bombesin receptors.
Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells.
Authors: Stevens R L , Fox C C , Lichtenstein L M , Austen K F
Published: May 2006

The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of 35S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although [35S]heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. We here demonstrate that human lung mast cells of 96% purity incorporate [35S] sulfate into separate heparin and chondroitin sulfate proteoglycans in an approximately equal to 2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin [35S]sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin [35S]sulfate E proteoglycans and the [35S]heparin proteoglycans were exocytosed from the [35S]sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of 35S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.