bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential expression of the mouse homeobox-containing gene Hox-1.4 during male germ cell differentiation and embryonic development.
Authors: Wolgemuth D J , Viviano C M , Gizang-Ginsberg E , Frohman M A , Joyner A L , Martin G R
Published: May 2006

Hox-1.4 is a mouse homeobox-containing gene (initially identified as HBT-1), whose expression appears to be testis-specific in the adult animal. Examination of Hox-1.4 transcripts in RNA from testes of mutant mice deficient in germ cells confirms that Hox-1.4 expression within the testis is germ cell-specific. Enriched populations of spermatogenic cells were used to localize the expression of Hox-1.4 specifically to germ cells that have entered into and progressed beyond the meiotic prophase stage of differentiation and to demonstrate the presence of two different size Hox-1.4 transcripts. Examination of RNA from teratocarcinoma cell cultures and mouse embryos at 10.5-16.5 days of gestation demonstrated the presence of several Hox-1.4 transcripts, which are larger than those present in germ cells. In the midgestation fetus, Hox-1.4 expression is most abundant in the spinal cord.
Characterization of the alpha-tubulin gene family of Arabidopsis thaliana.
Authors: Ludwig S R , Oppenheimer D G , Silflow C D , Snustad D P
Published: May 2006

The genome of Arabidopsis thaliana (Linnaeus) Heynhold was shown to contain an alpha-tubulin gene family consisting of at least four genes and/or pseudogenes. The primary structure of a transcribed alpha-tubulin gene was determined. A comparison of the predicted amino acid sequence of the A. thaliana alpha-tubulin with the predicted amino acid sequences of alpha-tubulins of Chlamydomonas reinhardtii, Stylonychia lemnae, and Homo spaiens reveals a high degree of homology; 90%, 87%, and 83% identity, respectively. Thus, a plant alpha-tubulin exhibits a high degree of homology to the alpha-tubulins of protists and animals. The coding sequence of the A. thaliana alpha-tubulin gene is interrupted by four introns, which occur at positions different from those of the less numerous introns of C. reinhardtii and rat alpha-tubulin genes. S1 nuclease mapping data showed that transcription is initiated 99 +/- 1 base pairs upstream from the translation initiation codon. Both 5' and 3' noncoding gene-specific probes were used to examine the expression of the alpha-tubulin gene in leaves, roots, and flowers by hybridization to total RNA isolated from these tissues. The results showed that the alpha-tubulin gene was transcribed in all three tissues.
Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia.
Authors: Kita T , Nagano Y , Yokode M , Ishii K , Kume N , Ooshima A , Yoshida H , Kawai C
Published: May 2006

In this study, we questioned whether in vivo probucol could prevent the progression of atherosclerosis in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia. At 2 months of age, eight WHHL rabbits were divided into two groups. Group A (n = 4) was fed standard rabbit chow for 6 months. Group B (n = 4) was fed standard rabbit chow containing 1% probucol for 6 months. At the end of the experiments, average plasma concentrations of cholesterol were 704 +/- 121 mg/dl in group A and 584 +/- 61 mg/dl in group B, respectively. The percentage of surface area of total thoracic aorta with visible plaques in group A versus group B was 54.2% +/- 18.8% versus 7.0% +/- 6.3%, respectively. What was noteworthy was that the percentage of plaque in the descending thoracic aorta was almost negligible (0.2% +/- 0.2%) in group B rabbits compared to that in group A rabbits (41.1% +/- 20.2%). Low density lipoproteins (LDL) isolated from WHHL rabbits under treatment with probucol (group B) were shown to be highly resistant to oxidative modification by cupric ion and to be minimally recognized by macrophages. On the contrary, LDL from group A rabbits incubated with cupric ion showed a 7.4-fold increase in peroxides (thiobarbituric acid-reactive substances) and a 4.3-fold increase in the synthesis of cholesteryl ester in macrophages compared to those of LDL from group B rabbits. Thus, probucol could definitely prevent the progression of atherosclerosis in homozygous WHHL rabbits in vivo by limiting oxidative LDL modification and foam cell transformation of macrophages.
Astrocyte gene expression in Creutzfeldt-Jakob disease.
Authors: Manuelidis L , Tesin D M , Sklaviadis T , Manuelidis E E
Published: May 2006

Gliosis (hyperplasia and hypertrophy of astrocytes), the fundamental response of the central nervous system to tissue destruction, typically becomes apparent only several weeks after injury. The biochemical hallmark of this response is a marked accumulation of the specific astrocyte intermediate filament glial fibrillary acidic protein (GFAP). To date despite its importance, the mechanisms of GFAP gene regulation have not been studied in any developmental or pathological system to our knowledge, and the molecular signals for GFAP mRNA and protein accumulation are not defined. In Creutzfeldt-Jakob disease, a progressive dementing illness caused by an "unconventional agent," we find steadily increasing elevations of GFAP mRNA throughout the later stages of disease, using two independent GFAP cDNA clones, representing the entire insert or the 3'-noncoding region (pScr-1). The accumulation of GFAP, assessed immunocytochemically, follows GFAP mRNA elevation. A 5-fold stimulation of GFAP gene expression precedes the development of florid histologic lesions in the cerebrum, and in the cerebellum 5- to 6-fold increases occurred with no detectable spongiform changes at any time during disease. Therefore, these GFAP changes cannot be simply a response to neuronal damage. These effects are directly or indirectly caused by high local concentrations of agent and possibly involve a humoral factor.
Human fetal cerebellar and cortical tissue transplanted to the anterior eye chamber of athymic rats: electrophysiological and structural studies.
Authors: Bickford-Wimer P , Granholm A C , Bygdeman M , Hoffer B , Olson L , Seiger A , Strömberg I
Published: May 2006

Human fetal tissue fragments from cortex cerebri and cerebellum were grafted to the anterior chamber of the eye of adult athymic nude rats. The grafts were obtained from tissue fragments recovered after elective routine abortions, performed in weeks 8-11 of gestation. Both cerebellar and cortex cerebri grafts survived and developed in the anterior chamber of the eye for 1-4 months. The transplants slowly became vascularized from the host iris. The grafts developed blood vessels with laminin-immunoreactive walls and contained relatively high amounts of glial fibrillary acidic protein- and neurofilament-immunoreactivity in the neuropil after 4 months in oculo. Recordings of extracellular action potentials from the grafts revealed spontaneously active neurons with action-potential waveforms similar to those observed in immature rodents. Morphologically, the grafts showed no signs of rejection. Clusters and bands of large neurons resembling Purkinje cells and dense aggregates of smaller granule-like cells could be found in the cerebellar grafts. Large neurons were also seen in the cortex grafts. Taken together, these data suggest that the athymic rat may serve as a useful tool for studies of central nervous system tissue from otherwise immunologically incompatible species.
Local cerebral metabolic effects of L-dopa therapy in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in monkeys.
Authors: Porrino L J , Burns R S , Crane A M , Palombo E , Kopin I J , Sokoloff L
Published: May 2006

The quantitative 2-deoxy[14C]glucose autoradiographic method was used to map the distribution of alterations in local cerebral glucose utilization that accompanies clinically effective chronic L-dopa therapy of rhesus monkeys made parkinsonian by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This pattern of changes was compared to the effects of a similar treatment regimen in normal monkeys. L-Dopa (100 mg with 10 mg carbidopa) was administered orally to normal and parkinsonian monkeys 3 times daily for 60-120 days prior to measurement of local cerebral glucose utilization. In parkinsonian monkeys treated with L-dopa, signs and symptoms of parkinsonism were controlled or suppressed, and widespread increases in glucose utilization were seen throughout the brain. Cerebral metabolic activity was increased both in areas rich in dopaminergic receptors, such as the caudate and putamen, and in nondopaminergic areas involved in motor functions. In many structures the rates of glucose utilization in L-dopa-treated parkinsonian monkeys were increased to levels that far exceeded rates measured in normal monkeys. In sharp contrast, similar treatment with L-dopa in normal monkeys had little if any effect on local cerebral glucose utilization. L-Dopa, then, appears to have an action in animals with selective lesions of the substantia nigra pars compacta produced by MPTP that is distinctly different from its effects in the normal monkey.
In vitro digestibility and amino acid composition of pearl millet (Pennisetum typhoides) and other cereals.
Authors: Ejeta G , Hassen M M , Mertz E T
Published: May 2006

The purpose of this study was to compare in vitro digestibility, protein distribution patterns, and amino acid composition of pearl millet with other major cereals. Digestibility values for the pearl millet varieties were higher than that of sorghum and comparable to that of maize. In contrast to sorghum, digestibility of pearl millet and maize did not decrease significantly upon cooking. Protein distribution patterns of uncooked pearl millet and shifts in the different fractions as a result of cooking also resembled that of maize and not sorghum. The amino acid profile of pearl millet is more favorable than that of normal sorghum and normal maize and is comparable to the small grains, wheat, barley, and rice. On the basis of these findings, it appears that pearl millet is a nutritious and well-digested source of calories and proteins for humans.
Cloning of partial cDNA encoding differentiation and tumor-associated mucin glycoproteins expressed by human mammary epithelium.
Authors: Gendler S J , Burchell J M , Duhig T , Lamport D , White R , Parker M , Taylor-Papadimitriou J
Published: May 2006

Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins (Mr greater than 250,000) that are developmentally regulated, tumor-associated, and highly immunogenic. Studies using monoclonal antibodies directed to these glycoproteins suggest that their molecular structures can vary with differentiation stages in the normal gland and in malignancy. To analyze the molecular nature of these glycoproteins, milk mucin was affinity-purified and deglycosylated with hydrogen fluoride, yielding bands at 68 and 72 kDa on silver-stained gels. Polyclonal and monoclonal antibodies to the stripped core protein were developed and used to screen a lambda gt11 expression library of cDNA made from mRNA of the mammary tumor cell line MCF-7. Seven cross-reacting clones were isolated, with inserts 0.1-1.8 kilobases long. RNA blot analysis, using as a probe the 1.8-kilobase insert subcloned in plasmid pUC8 (pMUC10), revealed transcripts of 4.7 and 6.4 kilobases in MCF-7 and T47D mammary tumor cells, whereas normal mammary epithelial cells from pooled milks have additional transcripts. The expression of mRNA correlates with antigen expression as determined by binding of two previously characterized anti-mucin monoclonal antibodies (HMFG-1 and HMFG-2) to seven cell lines. Restriction enzyme analysis detected a restriction fragment length polymorphism when human genomic DNA was digested with EcoRI or HinfI.
A second form of the beta subunit of signal-transducing G proteins.
Authors: Gao B , Gilman A G , Robishaw J D
Published: May 2006

A complementary DNA (cDNA) has been isolated that encodes a second form of the beta subunit of signal-transducing guanine nucleotide-binding regulatory proteins (G proteins). The cDNA corresponds to a 1.8-kilobase mRNA, and nucleotide sequence analysis indicates that the encoded polypeptide consists of 340-amino acid residues with a Mr of 37,335. Although the deduced polypeptide is the same size as that reported previously for the beta subunit, 10% of the amino acid residues are different. Furthermore, the 5' and 3' non-translated regions of this cDNA show no significant homology with those reported previously for cDNAs that encode the beta subunit. These data refute prior conclusions that there is only one form of the G protein beta subunit.
Augmentation of bursting pacemaker activity by egg-laying hormone in Aplysia neuron R15 is mediated by a cyclic AMP-dependent increase in Ca2+ and K+ currents.
Authors: Levitan E S , Kramer R H , Levitan I B
Published: May 2006

Release of the neuropeptide egg-laying hormone (ELH) from Aplysia bag cell neurons augments the endogenous bursting pacemaker activity of neuron R15. We have studied the ionic mechanisms underlying the effect of ELH in voltage-clamped R15 neurons. Both electrical discharge of the bag cells, which releases endogenous ELH, and application of synthetic ELH on cell R15 result in an increase in two discrete ionic currents. One of these currents activates with hyperpolarization, reverses near the K+ equilibrium potential, is sensitive to the external K+ concentration, and is blocked by addition of 5 mM Rb+ or 1 mM Ba2+ to the bathing medium. This current appears to be identical to the inwardly rectifying K+ current IR. The other current activates with depolarization and is blocked by replacement of external Ca2+ with Co2+ or Mn2+. This current appears to be a voltage-gated Ca2+ current ICa. Both ICa and IR in R15 have previously been shown to be enhanced by the neurotransmitter serotonin, acting via intracellular cyclic AMP. We now report that increasing cyclic AMP in R15, by applying either serotonin or the adenylate cyclase activator forskolin together with a phosphodiesterase inhibitor, mimics and occludes the action of ELH on neuron R15. Furthermore, application of ELH increases the cyclic AMP content of single R15 neurons, as measured by radioimmunoassay. Finally, the effects of ELH are potentiated by a phosphodiesterase inhibitor. These results suggest that ELH augments bursting activity in R15 by causing cyclic AMP-mediated increases in IR and ICa.
Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: precursors and role of the respiratory burst.
Authors: Iyengar R , Stuehr D J , Marletta M A
Published: May 2006

The macrophage cell line RAW 264.7 when activated with Escherichia coli lipopolysaccharide and interferon-gamma synthesized nitrite (NO3-) and nitrate (NO3-). Medium change after the activation showed that L-arginine was the only amino acid essential for this synthesis. D-Arginine would not substitute for L-arginine. Other analogues that could replace L-arginine were L-homoarginine, L-arginine methyl ester, L-arginamide, and the peptide L-arginyl-L-aspartate. L-Argininic acid, L-agmatine, L-ornithine, urea, L-citrulline, and ammonia were among the nonprecursors, while L-canavanine inhibited this L-arginine-derived NO2-/NO3- synthesis. When morpholine was added to the culture medium of the activated RAW 264.7 macrophages, N-nitrosation took place, generating N-nitrosomorpholine. GC/MS experiments using L-[guanido-15N2]arginine established that the NO2-/NO3- and the nitrosyl group of N-nitrosomorpholine were derived exclusively from one or both of the terminal guanido nitrogens of arginine. Chromatographic analysis showed that the other product of the L-arginine synthesis of NO2-/NO3- was L-citrulline. The role of the respiratory burst in NO2-/NO3- synthesis was examined using the macrophage cell lines J774.16 and J774 C3C. Both cell lines synthesized similar amounts of NO2-/NO3-. However, J774 C3C cells do not produce superoxide and hence do not exhibit the respiratory burst. Additional experiments also ruled out the involvement of the respiratory burst in NO2-/NO3- synthesis.
Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors.
Authors: Park M , Dean M , Kaul K , Braun M J , Gonda M A , Vande Woude G
Published: May 2006

We isolated overlapping cDNA clones corresponding to the major MET protooncogene transcript. The cDNA nucleotide sequence contained an open reading frame of 1408 amino acids with features characteristic of the tyrosine kinase family of growth factor receptors. These features include a putative 24-amino acid signal peptide and a candidate, hybrophobic, membrane-spanning segment of 23 amino acids, which defines an extracellular domain of 926 amino acids that could serve as a ligand-binding domain. A putative intracellular domain 435 amino acids long shows high homology with the SRC family of tyrosine kinases and within the kinase domain is most homologous with the human insulin receptor (44%) and v-abl (41%). Despite these similarities, however, we found no apparent sequence homology to other growth factor receptors in the putative ligand-binding domain. We conclude from these results that the MET protooncogene is a cell-surface receptor for an as-yet-unknown ligand.
Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor.
Authors: Cheresh D A
Published: May 2006

Human umbilical vein endothelial cells express a heterodimeric adhesion receptor complex consisting of noncovalently associated alpha and beta subunits that under reducing conditions have molecular masses of 135 kDa and 115 kDa, respectively. This complex can be isolated in pure form from an affinity matrix consisting of an Arg-Gly-Asp-containing heptapeptide and is specifically immunoprecipitated with monoclonal antibodies (mAbs) directed against the vitronectin receptor of human melanoma cells. These data suggest that this complex is one member of a large family of cell adhesion receptors. One of the mAbs, LM609, inhibits the attachment of human endothelial cells to fibrinogen, von Willebrand factor, and vitronectin yet has no effect on the attachment of these cells to fibronectin, collagen, or laminin. In addition, mAb LM609 inhibits attachment of endothelial cells to an immobilized synthetic peptide containing the Arg-Gly-Asp sequence. This adhesion receptor appears structurally similar to the IIb/IIIa glycoprotein complex expressed on platelets yet is antigenically distinct, since mAb LM609 fails to recognize IIb/IIIa glycoproteins. This receptor organizes in clusters on endothelial cells during their attachment to von Willebrand factor, vitronectin, or the Arg-Gly-Asp-containing heptapeptide. The data presented in this report suggest that Arg-Gly-Asp recognition may play a significant role in biological events associated with vascular proliferation.
Evidence for a mosaic representation of the body surface in area 3b of the somatic cortex of cat.
Authors: Favorov O V , Diamond M E , Whitsel B L
Published: May 2006

A discontinuous representation of the forelimb body surface in area 3b is proposed. Two different methods were used: single-neuron receptive-field (RF) mapping in unanesthetized cats (maximal RF) and multiunit RF mapping in deeply anesthetized cats (minimal RF). Ten or more maximal RFs were sampled in each of 14 near-radial microelectrode penetrations. In 6 penetrations, the maximal RFs of all sampled neurons (despite prominent variations in RF size and shape) shared in common a small skin area--termed the "RF center." Each of the remaining penetrations had to be divided into at least two segments (6 penetrations) or three segments (2 penetrations), for all maximal RFs mapped in a segment to include a common skin site. In six penetrations, after maximal RFs were mapped, deep general anesthesia was induced and minimal RFs were mapped in the same penetration at cortical sites separated by 150 microns. Minimal RFs closely matched the RF centers defined by maximal RFs in the same penetration. In penetrations that mapped two or three RF centers, a rapid transition in minimal RF position was detected at the same cortical site where the shift in RF center was detected. Closely spaced penetrations revealed discrete cortical columns, having the size and shape of 350- to 400-microns-diameter irregular hexagons, such that the identical minimal RF was mapped at any site within a column. The forelimb body surface in cat 3b thus appears to be represented by a mosaic of discrete columns--an organization similar to the whisker representation in rodent primary somatosensory cortex.
Spectroscopic analysis of the cytochrome c oxidase-cytochrome c complex: circular dichroism and magnetic circular dichroism measurements reveal change of cytochrome c heme geometry imposed by complex formation.
Authors: Weber C , Michel B , Bosshard H R
Published: May 2006

Binding of cytochrome c to cytochrome c oxidase induces a conformational change in both proteins as well as a change of the electronic structure of the heme of cytochrome c, indicating an altered heme c-protein interaction. This follows from the observation that the induced circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the oxidase-cytochrome c complex in the Soret region differ from the summed spectra of oxidase plus cytochrome c. Spectral changes occur in the complex composed of either the two ferric or the two ferrous hemoproteins. The difference CD and MCD signals saturate at a ratio of 1 heme c per heme aa3. The difference spectra are specific to the cognate complex. The results are interpreted to reflect a direct relationship between the recognition/binding step and the electron-transfer reaction. The conformational rearrangement induced in cytochrome c by cytochrome c oxidase consists of a structural rearrangement of the heme environment and possibly a change of the geometry of the heme iron-methionine-80 sulfur axial bond. This rearrangement may decrease the reorganizational free energy of electron transfer by adjusting the heme c geometry to a state between that of ferri- and ferrocytochrome c.
Carboxyl-terminal sequences influence the import of mitochondrial protein precursors in vivo.
Authors: Ness S A , Weiss R L
Published: May 2006

The large subunit of carbamoyl phosphate synthase A [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] from Neurospora crassa is encoded by a nuclear gene but is localized in the mitochondrial matrix. We have utilized N. crassa strains that produce both normal and carboxyl-terminal-truncated forms of carbamoyl phosphate synthase A to ask whether the carboxyl terminus affects import of the carbamoyl phosphate synthase A precursor. We found that carboxyl-terminal-truncated precursors were directed to mitochondria but that they were imported less efficiently than full-length proteins that were synthesized in the same cytoplasm. Our results suggest that effective import of proteins into mitochondria requires appropriate combinations of targeting sequences and three-dimensional structure.
The heavy chain of Acanthamoeba myosin IB is a fusion of myosin-like and non-myosin-like sequences.
Authors: Jung G , Korn E D , Hammer J A
Published: May 2006

Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. We now present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which we deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approximately 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approximately 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an alpha-helical, coiled-coil structure. We conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, we find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. We discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure.
Biological macromolecules as gels: functional similarities.
Authors: Douzou P
Published: May 2006

Functional biological macromolecules arising from folding, cross-connection, and solvation of long-chain biopolymers forming three-dimensional networks can be compared to gels. Both involve identical internal competitive forces that are selectively influenced by external conditions and conspire to adjust conformations and modulate functions. In spite of important differences in size, chemical composition, polymer density, and configuration, biological macromolecules indeed manifest some of the essential physical-chemical properties of gels. This result represents presumptive evidence for common underlying mechanisms in functional molecules and gels. Thus, the present and highly perfectible model explains why and how, depending on initial conditions, a system may respond differently to an external parameter and similarly to different parameters. Moreover, the fact that any localized change in one of the competitive forces gives rise to a pressure in the system as a whole provides one explanation for the mechanism of the transmission of information.
Isolation and characterization of a cDNA clone encoding wheat germ agglutinin
Authors: Raikhel Natasha V. , Wilkins Thea A.
Published: May 2006

Two sets of synthetic oligonucleotides coding for amino acids in the amino- and carboxyl-terminal portions of wheat germ agglutinin were synthesized and used as hybridization probes to screen cDNA libraries derived from developing embryos of tetraploid wheat. The nucleotide sequence for a cDNA clone recovered from the cDNA library was determined by dideoxynucleotide chain-termination sequencing in vector M13. The amino acid sequence deduced from the DNA sequence indicated that this cDNA clone (pNVR1) encodes isolectin 3 of wheat germ agglutinin. Comparison of the deduced amino acid sequence of clone pNVR1 with published sequences indicates isolectin 3 differs from isolectins 1 and 2 by 10 and 8 amino acid changes, respectively. In addition, the protein encoded by pNVR1 extends 15 amino acids beyond the carboxyl terminus of the published amino acid sequence for isolectins 1 and 2 and includes a potential site for N-linked glycosylation. Utilizing the insert of pNVR1 as a hybridization probe, we have demonstrated that the expression of genes for wheat germ agglutinin is modulated by exogenous abscisic acid. Striking homology is observed between wheat germ agglutinin and chitinase, both of which are proteins that bind chitin.
T-cell-receptor beta- and I-A beta-chain genes of normal SWR mice are linked with the development of lupus nephritis in NZB x SWR crosses.
Authors: Ghatak S , Sainis K , Owen F L , Datta S K
Published: May 2006

The incidence of nephritis in autoimmune New Zealand Black (NZB) mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed the association of the I-A beta-chain gene of Ia-encoding region, the T-cell-receptor beta (TcR beta)-chain gene, and immunoglobulin heavy-chain allotype (IgH) with the development of lupus nephritis in 165 NZB X SWR crosses. We found that genes linked to the TcR and Ir gene loci of the normal SWR mice interacted with NZB-derived genes, leading to the development of accelerated and severe nephritis in the NZB X SWR crosses.
A pancreas specificity results from the combination of polyomavirus and Moloney murine leukemia virus enhancer.
Authors: Rochford R , Campbell B A , Villarreal L P
Published: May 2006

An infectious recombinant polyomavirus was constructed in which a regulatory region of its genome, the B enhancer region (nucleotides 5128-5265) has been replaced with the 72- or 73-base-pair repeat enhancer from the Moloney murine leukemia virus genome. We show that this recombinant polyomavirus displays a strong tissue specificity for the pancreas of mice. This organ was not permissive for either the parental polyomavirus, which is predominantly kidney and salivary gland specific, or the Moloney murine leukemia virus, which is lymphotropic. This result indicated that tissue specificity can be achieved by a combination of apparently modular elements. Some of the implications of a modular mechanism of tissue specificity are considered.
Genes for the catabolism and synthesis of an opine-like compound in
Rhizobium meliloti
are closely linked and on the Sym plasmid
Authors: Murphy Peter J. , Heycke Nina , Banfalvi Zsofia , Tate Max E. , de Bruijn Frans , Kondorosi Adam , Tempé Jacques , Schell Jeff
Published: May 2006

In alfalfa nodules induced by
Rhizobium meliloti
strain L5-30 the compound L-3-
O
-methyl-
scyllo
-inosamine (3-
O
-M
S
I) is synthesized. This compound is also catabolized specifically by this strain. Its biological properties are therefore similar to the
Agrobacterium
opines. To answer the question whether opine-like compounds (“Rhizopines”) play a role in a plant symbiotic interaction, we isolated the genes for the catabolism of 3-
O
-M
S
I (
moc
genes) and for the induction of its synthesis in the nodule [
mos
gene(s)].
moc
and
mos
genes were shown to be closely linked and located on the Sym plasmid of L5-30, suggesting that they have co-evolved and may be important in symbiosis. These genes have been cloned into a broad host-range vector that can be mobilized into other
R. meliloti
strains where they are expressed. The location of the
mos
genes in the bacteria extends the opine concept, initially developed for a plant pathological interaction, to a symbiotic one.
Recognition of solubilized apoproteins from delipidated, oxidized low density lipoprotein (LDL) by the acetyl-LDL receptor.
Authors: Parthasarathy S , Fong L G , Otero D , Steinberg D
Published: May 2006

Macrophages express a specific receptor that recognizes acetylated low density lipoprotein (LDL) and certain other chemically modified forms of LDL but not native LDL. LDL oxidatively modified either by incubation with endothelial cells in Ham's F-10 medium or by incubation with 5 microM copper(II) ion in the absence of cells is recognized by this same receptor. This oxidative modification, whether cell-induced or copper-catalyzed, is accompanied by many changes in the physical and chemical properties of LDL, including an increase in density, conversion of phosphatidylcholine to lysophosphatidylcholine, generation of lipid peroxides, and degradation of apolipoprotein B-100. Which changes are essential for eliciting the recognition by the receptor is not known. In the present paper it is shown that fragments of the degraded apolipoprotein from delipidated, oxidized LDL can be almost quantitatively resolubilized using n-octyl beta-D-glucopyranoside. These 125I-labeled, solubilized apoproteins were degraded rapidly by mouse peritoneal macrophages, and that degradation was competitively inhibited by unlabeled acetyl-LDL and endothelial cell-modified LDL but not by native LDL. These results show that the acetyl-LDL receptor recognizes an epitope on the apoprotein moiety, either newly generated or exposed as a result of oxidative modification, rather than some oxidized lipid moiety. Further, the results suggest that the lipids of oxidatively modified LDL do not play an obligatory role in determining the conformation of that epitope.
Intestinal brush border membranes contain regulatory subunits of adenylyl cyclase.
Authors: Domínguez P , Velasco G , Barros F , Lazo P S
Published: May 2006

Cholera toxin alters intestinal function by stimulation of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing) or adenylate cyclase, EC 4.6.1.1]. The mechanism of this activation is unknown and particularly puzzling because adenylyl cyclase is confined to the basal lateral membrane of enterocytes, whereas it is the brush border membrane that binds the toxin and contains proteins that undergo cholera toxin-catalyzed ADP ribosylation. It is shown that cholate extracts from cholera toxin-treated brush border membranes can efficiently reconstitute adenylyl cyclase activity in the guanine nucleotide-binding regulatory component (Gs)-deficient cyc- variant of the S49 mouse lymphoma cell line (cyc- cells lack the alpha subunit of Gs needed to activate the catalytic subunit of adenylyl cyclase). Moreover, NaF (in the presence of Al3+) and guanyl-5'-yl imidodiphosphate mediate strong activation of cyc- adenylyl cyclase provided the cholate extracts of brush border membranes are also present. Therefore, it appears that brush border membranes contain high levels of regulatory subunits of adenylyl cyclase in the absence of catalytic subunits. This represents a previously unrecognized feature of this transduction system that presumably plays an important role in the derangement of intestinal cell function by cholera toxin.
Molecular cloning and characterization of the insecticidal crystal protein gene of
Bacillus thuringiensis
var.
tenebrionis
Authors: Sekar Vaithilingam , Thompson David V. , Maroney Michael J. , Bookland Roger G. , Adang Michael J.
Published: May 2006

The insecticidal crystal protein gene of the coleopteran-toxic
Bacillus thuringiensis
var.
tenebrionis
has been isolated, and the nucleotide sequence has been determined. A total DNA library from var.
tenebrionis
was made in the plasmid vector pUC12. By using a synthetic 27-base oligonucleotide corresponding to a stretch of nine N-terminal amino acids of a tryptic fragment of purified crystal protein of var.
tenebrionis
as a probe, recombinant colonies were screened by
in situ
hybridization for the presence of the crystal protein gene. Positive clones obtained from this screening were further tested for toxicity. One recombinant, NSBP544 (which contained a 5.9-kilobase
Bam
HI insert), was toxic to larvae of Colorado potato beetle. Immunoblot analysis revealed that this clone produces two crystal-specific antigens of 65 and 73 kDa as do sporulating var.
tenebrionis
cells. However, purified crystal inclusions from var.
tenebrionis
contain a primary peptide component of 65 kDa. A 1932-base-pair open reading frame with a coding capacity of 73,119 Da has been identified by nucleotide sequencing analysis of the cloned crystal protein. In addition, mung bean nuclease mapping indicates that transcription of the crystal protein of var.
tenebrionis
initiates 130 base pairs upstream from the translational start site. Southern blot analysis using an internal 0.7-kilobase
Eco
RI fragment of pNSBP544 as a probe revealed that the crystal protein gene is located on a 90-MDa plasmid.
Multiple tissue-specific sites of transcriptional initiation of the mouse anion antiport gene in erythroid and renal cells.
Authors: Kopito R R , Andersson M A , Lodish H F
Published: May 2006

Primer extension and nuclease protection analysis were used to map the 5' end of mRNA transcripts of the single gene encoding the mouse erythroid anion-exchange protein, band 3. RNA from induced Friend murine erythroleukemia cells contains transcripts whose 5' ends are heterogeneous but that map to five clustered sites between 146 and 189 nucleotides upstream of the initiator AUG codon. The steady-state level of band 3 mRNA increases markedly following dimethyl sulfoxide-induced differentiation, but the pattern of transcription initiation does not vary. mRNA from anemic and normal mouse spleen exhibits the same pattern, with the addition of another transcript whose 5' end maps to position -260. In contrast, mRNA from mouse kidney has a single predominant transcript, mapping to -260. These data establish that the band 3 gene is expressed in kidney as well as in erythroid cells and suggest the presence of tissue-specific alternate promoter elements within the first exon of the band 3 gene. The nucleotide sequence of 1.7 kilobases of genomic DNA 5' to the first intron of the single-copy mouse band 3 gene, although moderately (G + C)-rich, has no "TATA" or "CAAT" boxes or other homologies with globin or other eukaryotic polymerase II promoter regions.
Somatostatin analogs as adjuncts to agonists of luteinizing hormone-releasing hormone in the treatment of experimental prostate cancer.
Authors: Schally A V , Redding T W
Published: May 2006

The combination of a long-acting delivery system for the agonist [D-Trp6]luteinizing hormone-releasing hormone ([D-Trp6]LH-RH) with modern somatostatin analogs was studied in the Dunning R-3327H rat prostate cancer model. Microcapsules of [D-Trp6]LH-RH releasing 25 micrograms/day were injected once a month. In the first experiment the adjunct was the somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (RC-121), administered at a dose of 2.5 micrograms twice a day, and the therapy was continued for 70 days. Tumor volume was significantly decreased by [D-Trp6]LH-RH microcapsules or RC-121 given alone. The combination of microcapsules and analog RC-121 caused a greater inhibition of tumor growth than the single agents. Similar effects were seen when the percent increase in the tumor volume was examined. The inhibition of tumor growth caused by the [D-Trp6]LH-RH microcapsules was greater than that caused by RC-121. The combination of the two agents was again the most effective, resulting in the smallest increase in tumor volume. Tumor weights were much lower in the groups treated with microcapsules or RC-121 alone than in controls. The lowest tumor weights were obtained in the group that received the combination of [D-Trp6]LH-RH microcapsules and RC-121. Similar results were obtained in the second experiment, in which the animals were treated for a period of 83 days with microcapsules containing the somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) that released 5 micrograms/day and were injected twice a month alone or in combination with microcapsules of [D-Trp6]LH-RH. Microcapsules of analog RC-160 given alone significantly decreased tumor growth as measured by the final tumor volume, the percentage change from the initial tumor volume, and the reduction in tumor weight. The inhibition of tumor growth induced by [D-Trp6]LH-RH microcapsules was greater than that caused by RC-160. The most striking decrease in tumor weight and volume was obtained in animals treated with microcapsules of [D-Trp6]LH-RH combined with the delayed delivery system for RC-160. The overall response to the combination therapy could reflect the inhibition by somatostatin analogs of the proliferation of prostate cancer cells through a decrease in growth hormone and prolactin release and interference with endogenous growth factors, in addition to the main effect, which is the suppression by [D-Trp6]LH-RH of the growth of androgen-dependent tumor cells. Our results indicate that somatostatin analogs enhance the inhibitory effects of [D-Trp6]LH-RH on the growth of prostate tumors. The administration of somatostatin analogs in combination with microcapsules of [D-Trp6]LH-RH might improve clinical response in patients with advanced prostate carcinoma.
Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.
Authors: Felgner P L , Gadek T R , Holm M , Roman R , Chan H W , Wenz M , Northrop J P , Ringold G M , Danielsen M
Published: May 2006

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.
Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression.
Authors: Murray M T , White K , Munro H N
Published: May 2006

Ferritin stores iron within a protein shell consisting of 24 subunits of two types, heavy (H) and light (L). According to Southern blotting, the rat genome contains four copies homologous to the H-subunit cDNA (H cDNA). To determine whether only one of these is expressed, H cDNAs isolated from rat liver and heart mRNAs were compared and found to share identical nucleotide sequences. Next, genomic clones for three of the four rat H-subunit loci were isolated. Two were classical processed pseudogenes, whereas the third contained an expressed gene. RNase intron mapping of this expressed gene generated the same exon protection pattern when total RNA from rat liver or heart was used, indicating that this gene accounts for most or all of the H-subunit mRNAs (H mRNAs) in these tissues. Comparison of the expressed rat H-subunit gene (H gene) structure with published sequences for other species displays considerable conservation. The coding sequence of the rat H gene predicts 95% similarity to the human amino acid sequence, thus being more highly conserved than the L-subunit sequence of these species. Near the cap region of the 5' untranslated region, the rat H mRNA displays a 28-nucleotide sequence that is almost totally conserved in the corresponding region of the human, bullfrog, and chicken H mRNA and is also faithfully represented in the rat and human L-subunit mRNAs (L mRNAs), thus making this sequence a prime candidate for involvement in the known translational regulation of both subunits by iron. In the 5' flanking region, partially conserved sequences common to H gene and L-subunit gene (L gene) of the rat may be involved in transcriptional regulation by iron, whereas those conserved only in the H gene of man and the rat imply that other factors may independently control H-subunit regulation.
Mouse and frog violate the paradigm of species-specific transcription of ribosomal RNA genes.
Authors: Culotta V C , Wilkinson J K , Sollner-Webb B
Published: May 2006

Transcription of ribosomal RNA genes by RNA polymerase I is generally accepted as being highly species specific, a conclusion based on numerous reports that rRNA genes of one species are not transcribed by factors of even closely related species. It thus was striking to find that cloned rDNA from the frog Xenopus laevis is specifically transcribed in extracts prepared from mouse cells. The data in this paper demonstrate that this heterologous transcription is due to a normal initiation process and not to a fortuitous event. Transcription of Xenopus rDNA in the mouse cell extract is directed by the same large promoter (residue-141 to +6) that is utilized to promote the synthesis of frog rRNA in homologous Xenopus systems. Moreover, the same factors of the mouse cell extract that transcribe the homologous mouse rDNA also catalyze transcription from the X. laevis rDNA promoter. We conclude that polymerase I transcriptional machinery does not evolve as rapidly as prior studies would suggest.
Model for intermediate steps in monocytic differentiation: c-myc, c-fms, and ferritin as markers.
Authors: Cayre Y , Raynal M C , Darzynkiewicz Z , Dorner M H
Published: May 2006

A mature hematopoietic cell represents the end product of a stepwise differentiation process. As a model system for studying differentiation, the human promyelocytic leukemia cell line HL-60 undergoes terminal monocytic/macrophagic differentiation following exposure to either phorbol 12-myristate 13-acetate or 1,25-dihydroxyvitamin D3. We have derived and analyzed a variant HL-60 cell line, 1F10, that permits the study of several intermediate steps in the myeloid differentiation process. These intermediate steps are documented by cell cycle data and phenotype analysis as well as markers such as c-myc, c-fms, and both subunits of ferritin.
Myristoylation and the post-translational acquisition of hydrophobicity by the membrane immunoglobulin heavy-chain polypeptide in B lymphocytes.
Authors: Pillai S , Baltimore D
Published: May 2006

Membrane immunoglobulin heavy chain in pre-B and in B cells is initially synthesized as a relatively hydrophilic protein that is nonetheless stably anchored in the endoplasmic reticulum membrane. In B cells, but not in pre-B cells, the membrane immunoglobulin heavy chain is post-translationally converted to a relatively hydrophobic form that partitions into the oil phase when solubilized with the phase-separating detergent Triton X-114. Covalent myristoylation of the membrane and secretory forms of immunoglobulin heavy chains as well as of light chains was observed in B cells. Myristoylation of the membrane immunoglobulin heavy chain correlates with its transport to the cell surface and its post-translational conversion to a relatively hydrophobic form. This post-translational modification is hydroxylamine resistant and may be responsible for the assembly and transport of membrane immunoglobulin to the cell surface in B cells.
Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus.
Authors: Matsukura M , Shinozuka K , Zon G , Mitsuya H , Reitz M , Cohen J S , Broder S
Published: May 2006

Nuclease-resistant phosphorothioate analogs of certain oligodeoxynucleotides have been tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Phosphorothioate analogs complementary to HIV sequences, as well as noncomplementary analogs including homooligomers, exhibited potent antiviral activity. The antiviral activity was related to the base composition of the analogs, and longer phosphorothioates were more effective than shorter ones. A 28-mer phosphorothioate oligodeoxycytidine (S-dC28) at a concentration of 1 microM exhibited potent antiviral activity and inhibited de novo viral DNA synthesis as shown by Southern blot analysis. However, S-dC28 failed to inhibit gag expression in chronically infected T cells assessed by immunofluorescent assay at concentrations up to 25 microM. An N3-methylthymidine-containing phosphorothioate analog, which does not hybridize efficiently in vitro to complementary normal DNA, showed no antiviral activity. A 14-mer phosphorothioate oligodeoxycytidine (S-dC14) synergistically enhanced the antiviral activity of 2',3'-dideoxyadenosine, an anti-HIV nucleoside. Therefore, phosphorothioate analogs of oligodeoxynucleotides could represent a unique class of experimental therapeutic agents against the acquired immunodeficiency syndrome and related diseases. However, their mechanism of action is likely to be complex.