bluuebunny/crossref_metadata_2025 huggingface dataset:
Initial extracellular forms of Plasmodium falciparum: their ultrastructure and their definition with monoclonal antibodies.
Authors: Zung J M , Trager W , Gubert E
Published: May 2006

Merozoites of the erythrocytic stage of the human malaria parasite Plasmodium falciparum, when placed under appropriate conditions in a culture medium with erythrocyte extract, differentiate into early trophic forms. These forms have much the same ultrastructure as rings of the same age that have developed intracellularly and have then been freed from their host cells by immune lysis. However, these forms differ in two respects: the extracellular forms have only their single plasma membrane, whereas the forms freed from host cells have, in addition, a surrounding parasitophorous vacuole membrane; the forms that develop extracellularly have fewer ribosomes. Five monoclonal antibodies against the ring stage have been prepared and characterized. Their pattern of immunofluorescence localization differs in merozoites as compared with rings, but their pattern is identical in rings developed extracellularly and those developed intracellularly. These results and the observations on fine structure demonstrate biochemical and morphological differentiation in the extracellular forms.
Decreased adherence of interleukin 6-treated breast carcinoma cells can lead to separation from neighbors after mitosis.
Authors: Tamm I , Cardinale I , Murphy J S
Published: May 2006

Interleukin 6 (IL-6) has been shown to inhibit the proliferation, but increase the motility, of wild-type ZR-75-1 human ductal breast carcinoma cells, a line of cells that resemble closely the malignant cells cultured from the ascitic effusion. IL-6-treated cells lose their epithelial character, become stellate or fusiform in shape, and migrate away from neighbors. In the wild-type ZR-75-1 cells, IL-6 causes cell-cell separation in preformed colonies as well as postmitotically. We have now investigated the action of IL-6 in clone B ZR-75-1 cells, which are morphologically distinct from wild-type ZR-75-1 cells. In the more polygonal rather than cuboidal clone B cells, IL-6 did not cause early inhibition of DNA synthesis and it caused little cell-cell separation in preformed colonies. However, IL-6 treatment markedly prolonged the interval between mitosis and readherence of daughter cells to their neighbors and the substratum. Supernatants from IL-6-treated cultures contained detached viable cells in increased numbers. Intermitotic intervals were prolonged in IL-6-treated cultures. IL-6-treated dividing breast carcinoma cells are characterized by an increased probability of separation from neighbors and the substratum.
Allergen- and bacterial antigen-specific T-cell clones established from atopic donors show a different profile of cytokine production.
Authors: Parronchi P , Macchia D , Piccinni M P , Biswas P , Simonelli C , Maggi E , Ricci M , Ansari A A , Romagnani S
Published: May 2006

We have established a large panel of T-cell clones (TCCs) specific for Dermatophagoides pteronyssinus and Lolium perenne group I grass pollen allergens (total, 61) and for tetanus toxoid and protein purified derivative bacterial antigens (total, 38) from the peripheral blood of two atopic individuals and then analyzed their ability to produce interleukin 4 (IL-4), IL-5, and interferon gamma (IFN-gamma). Upon stimulation with phorbol 12-myristate 13-acetate plus anti-CD3 antibody, the great majority of TCCs specific for bacterial components was able to produce both IL-4 and IFN-gamma, whereas most D. pteronyssinus- and L. perenne group I-specific TCCs produced IL-4, but no, or limited, IFN-gamma. Moreover, the mean amounts of IL-4 and IFN-gamma released by allergen-specific TCCs were significantly higher and lower, respectively, than the mean amounts produced by TCCs specific for bacterial components. Under the same experimental conditions, virtually all allergen-specific TCCs, but only one-third of tested TCCs specific for bacterial components, expressed IL-5 RNA and secreted IL-5 in their supernatants. Eighteen TCCs (nine specific for allergens and nine specific for bacterial components) were also assessed for their ability to induce IgE synthesis by autologous B cells in response to stimulation with the specific antigen. Under these experimental conditions, all allergen-specific TCCs, but only one-third of TCCs specific for bacterial components that produced IL-4 but no, or little, IFN-gamma induced the synthesis of detectable amounts of IgE. The demonstration that most allergen-specific helper T cells in atopic individuals are able to produce high amounts of IL-4 (and IL-5), but no IFN-gamma, may explain why allergens induce production of IgE antibodies and increase eosinophils.
Alternatively spliced products of the maize P gene encode proteins with homology to the DNA-binding domain of myb-like transcription factors.
Authors: Grotewold E , Athma P , Peterson T
Published: May 2006

The Zea mays P gene has been postulated to regulate the biosynthetic pathway of a flavonoid-derived pigment in certain floral tissues [Styles, E. D. & Ceska, O. (1977) Can. J. Genet. Cytol. 19, 289-302]. We have characterized two P transcripts that are alternatively spliced at their 3' ends. One message of 1802 nucleotides encodes a 43.7-kDa protein with an N-terminal region showing approximately 40% homology to the DNA-binding domain of several members of the myb family of protooncogene proteins. A second message of 945 nucleotides encodes a 17.3-kDa protein that contains most of the myb-homologous domain but differs from the first protein at the C terminus. The deduced P-encoded proteins show an even higher homology (70%) in the myb-homologous domain to the maize regulatory gene C1. Additionally, the P and C1 genes are structurally similar in the sizes and positions of the first and second exons and first intron. We show that P is required for accumulation in the pericarp of transcripts of two genes (A1 and C2) encoding enzymes for flavonoid biosynthesis--genes also regulated by C1 in the aleurone.
Innervation and neural regulation of the sex pheromone gland in female Heliothis moths.
Authors: Christensen T A , Itagaki H , Teal P E , Jasensky R D , Tumlinson J H , Hildebrand J G
Published: May 2006

Female Heliothis moths normally produce their species-specific male attractant (sex pheromone blend) during scotophase, and this production is stimulated by pheromone biosynthesis activating neuropeptide (PBAN), presumably carried in the hemolymph. Several lines of evidence indicate that the central nervous system plays another critical role in this regulation. Pheromone biosynthesis was induced during photophase by electrical stimulation of the ventral nerve cord or the peripheral nerves projecting from the terminal abdominal ganglion to the pheromone gland in the tip of the abdomen. Electron microscopy further revealed that axonal branches innervate the gland tissue. Nerve branches associated with pheromone gland cells are enwrapped in glia and contain dense-core vesicles, suggesting that the innervation of the gland might be neurosecretory. Finally, the biogenic monoamine octopamine was nearly as effective as purified Heliothis zea PBAN in stimulating pheromone biosynthesis when injected into intact females during mid-photophase. Furthermore, both octopamine and PBAN stimulated significant increases in the pheromone content of the glands in isolated abdomens lacking a ventral nerve cord but only when abdomens were treated at the onset of scotophase. These data suggest that the regulation of sex pheromone production in Heliothis is more complex than previously thought. Activation of the gland appears to be governed by both neural and hormonal mechanisms, and these control mechanisms depend on photoperiodic cues.
Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor.
Authors: Ferré J , Real M D , Van Rie J , Jansens S , Peferoen M
Published: May 2006

The biochemical mechanism for resistance to Bacillus thuringiensis crystal proteins was studied in a field population of diamondback moths (Plutella xylostella) with a reduced susceptibility to the bioinsecticidal spray. The toxicity and binding characteristics of three crystal proteins [CryIA(b), CryIB, and CryIC] were compared between the field population and a laboratory strain. The field population proved resistant (greater than 200-fold compared with the laboratory strain) to CryIA(b), one of the crystal proteins in the insecticidal formulation. Binding studies showed that the two strains differ in a membrane receptor that recognizes CryIA(b). This crystal protein did not bind to the brush-border membrane of the midgut epithelial cells of the field population, either because of strongly reduced binding affinity or because of the complete absence of the receptor molecule. Both strains proved fully susceptible to the CryIB and CryIC crystal proteins, which were not present in the B. thuringiensis formulation used in the field. Characteristics of CryIB and CryIC binding to brush-border membranes of midgut epithelial cells were virtually identical in the laboratory and the field population.
Parallel pathways of cell cycle control during Xenopus egg activation.
Authors: Bement W M , Capco D G
Published: May 2006

Transit from M phase into interphase in many eukaryotic cells is preceded by an increase in intracellular free calcium ([Ca2+]i), which may act via calcium-dependent enzymes to trigger the M-phase/interphase transition. To test the role of the calcium- and phospholipid-dependent enzyme protein kinase C (PKC) in the M-phase/interphase transition, PKC was activated in M-phase-arrested Xenopus eggs by treatment with the phorbol ester phorbol 12-myristate 13-acetate under conditions that prevent a rise in [Ca2+]i and activation of other calcium-dependent enzymes. Under these conditions, several cellular events characteristic of transit into interphase occur: sperm chromatin decondenses, the Golgi and the nuclear envelope reassemble, and endocytosis resumes. These events are also triggered by treatment of eggs with the diacylglycerol 1,2-dioctanoyl-sn-glycerol. Surprisingly, the activity of M-phase-promoting factor (MPF), a universal regulator of M phase, remains high under these conditions. If [Ca2+]i is subsequently raised, MPF activity is rapidly destroyed. Similarly, lysates made from eggs treated with phorbol 12-myristate 13-acetate support sperm chromatin decondensation in vitro and yet retain high MPF activity, measured either as the ability to induce meiotic resumption in oocytes or as histone H1 kinase activity. These effects are not triggered by the 4 alpha-phorbol ester isomer, which does not activate PKC, and are sensitive to the PKC "pseudosubstrate" peptide. The results suggest that two, parallel signals are generated by the rise in [Ca2+]i both of which contribute to cell cycle regulation. One pathway inactivates MPF; the other pathway activates PKC.
Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions.
Authors: Fasano A , Baudry B , Pumplin D W , Wasserman S S , Tall B D , Ketley J M , Kaper J B
Published: May 2006

Attenuated Vibrio cholerae vaccine strains specifically mutated in genes encoding cholera toxin (CT) are still capable of causing mild to moderate diarrhea. Culture supernatants of V. cholerae strains, both CT-positive and CT-negative, were examined in Ussing chambers, and a toxin was found that increases the permeability of the small intestinal mucosa by affecting the structure of the intercellular tight junction, or zonula occludens. The activity of this toxin is reversible, heat-labile, sensitive to protease digestion, and found in culture supernatant fractions containing molecules between 10 and 30 kDa in size. Production of this factor (named ZOT for zonula occludens toxin) correlates with diarrheagenicity of V. cholerae strains in volunteers and may represent another virulence factor of infectious diarrhea that must be eliminated to achieve a safe and effective live oral vaccine against cholera.
The p15 carboxyl-terminal proteolysis product of the human immunodeficiency virus type 1 reverse transcriptase p66 has DNA polymerase activity.
Authors: Hafkemeyer P , Ferrari E , Brecher J , Hübscher U
Published: May 2006

The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity. The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa. The latter has been characterized as possessing RNase H activity [Hansen, J., Schultze, T., Mellert, W. & Moelling, K. (1988) EMBO J. 7, 239-243]. We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein. On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product. The two reverse transcriptases appear to have different behaviors on various template/primer systems tested. Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication.
Involucrin gene of tarsioids and other primates: alternatives in evolution of the segment of repeats.
Authors: Djian P , Green H
Published: May 2006

The involucrin genes of the prosimian primates and of the anthropoid primates possess nonhomologous segments of repeats located at two different sites, P and M, within the coding region. The involucrin gene of the tarsioids alone contains repeats at both sites, for it derived repeats at site P from a common ancestor of tarsioids and prosimians and a repeat at site M from a later common ancestor of tarsioids and anthropoids. After their divergence from the tarsioids, the anthropoids added many more repeats to site M and excised the older segment of repeats from site P; in contrast, the tarsioids stopped adding repeats at site M, retained the earlier segment of repeats at site P, and enlarged it. In the revision of their involucrin genes, the two lineages followed alternative routes. The mechanisms by which the revisions took place have been subject to abrupt onset or termination.
Mutation and selection during the secondary response to 2-phenyloxazolone.
Authors: Rada C , Gupta S K , Gherardi E , Milstein C
Published: May 2006

The most characteristic feature of the mouse antibody response to the hapten 2-phenyloxazolone is the recurrent expression of the light-chain variable region Igk-VO chi 1 gene in its germ-line or mutated configuration. The analysis of somatic mutants of the Igk-VO chi 1 gene reported here indicates that, as found during the primary response, hypermutation is also activated during the secondary response. Somatic mutations in the Igk-VO chi 1 gene increased in sequences obtained at day 14 and day 21 in the primary response and again in the secondary response at days 3, 5, and 7. The ratio of replacement to silent mutations also increased, particularly between days 5 and 7, suggesting that a stage of negative selection operates on new somatic mutants generated in the secondary response. Most Igk-VO chi 1 mutants isolated in the secondary response had the features of selected memory clones (i.e., they carried mutations known to increase binding affinity for the hapten). However, some clones had chain-termination codons, and others had mutations predicting a nonfunctional light chain. At least three and possibly five of these clones also expressed the mutation characteristic of the memory response to 2-phenyloxazolone (His-34----Asn-34/Gln-34). We conclude that after a second antigenic challenge, new somatic variants, including some leading to the loss of antigen binding, are generated by hypermutation of cells derived from the memory pool.
Highly resolving two-dimensional gels for protein sequencing.
Authors: Hanash S M , Strahler J R , Neel J V , Hailat N , Melhem R , Keim D , Zhu X X , Wagner D , Gage D A , Watson J T
Published: May 2006

Two-dimensional (2D) PAGE, using carrier ampholytes for the first-dimension separation, has provided a tool for the simultaneous analysis of cellular proteins. To extend the utility of 2D PAGE to the preparative level, we have investigated the use of immobilized pH gradients (IPG) for the first-dimension separation. The results we have obtained indicate that as much as 1 mg of cellular protein can be loaded onto a single IPG gel without loss of resolution. Mutant polypeptides previously detected in carrier ampholyte-based 2D gels were equally detectable in IPG-based 2D gels. With IPG gels several hundred cellular polypeptides can be isolated, from as few as 10 gels, in sufficient amount for sequencing with current sequencing technology. We therefore conclude that IPG greatly enhances the prospects for the large-scale sequencing of cellular proteins for the development of 2D gel-related protein data bases and for the identification of new polypeptide gene products, with the attendant implications for a genome sequencing effort.
A "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes.
Authors: Tindle R W , Fernando G J , Sterling J C , Frazer I H
Published: May 2006

We have identified a major T-cell epitope, amino acids 48-54 (DRAHYNI, in one-letter code) in the E7 open reading frame protein of human papillomavirus (HPV) type 16. Lymph node cells from mice immunized with synthetic peptides containing DRAHYNI proliferated and produced interleukin when challenged in vitro with peptide or whole HPV-16 E7 fusion protein. The T epitope was recognized in association with all five major histocompatibility complex class II I-A and I-E alleles tested. Synthetic peptides consisting of DRAHYNI linked to major B-cell epitopes on the E7 molecule formed immunogens capable of eliciting strong antibody responses to HPV-16 E7. The T epitope could provide help for the production of antibody to several B epitopes simultaneously, including a B epitope of HPV-18 E7 protein. Mice immunized with a peptide containing DRAHYNI and B epitope and, at a later date, infected with recombinant vaccinia E7 virus, displayed secondary antibody responses to E7. Because E7 has a role in cell transformation and is the most abundant viral protein in HPV-associated neoplastic cervical epithelial cells, the data have implications for vaccine strategies.
Evidence for non-V3-specific neutralizing antibodies that interfere with gp120/CD4 binding in human immunodeficiency virus 1-infected humans.
Authors: Kang C Y , Nara P , Chamat S , Caralli V , Ryskamp T , Haigwood N , Newman R , Köhler H
Published: May 2006

Total anti-gp120 antibodies (total anti-gp120 Abs) were purified from a pool of four human immunodeficiency virus-positive (HIV+) sera by affinity chromatography on a gp120SF2-Sepharose column and exhibited both type- and group-specific neutralizing activities. To dissect the epitope specificity of the group-specific neutralizing antibodies, CD4 attachment site-specific antibodies (CD4-site Abs) were isolated from total anti-gp120 Abs by using a CD4-blocked gp120SF2-Sepharose column. The CD4-site Abs exhibited group-specific neutralizing activities. Another approach to dissecting type- and group-specific neutralizing activities of total anti-gp120 Abs was to separate the third variable region (V3)-specific antibodies (V3-region Abs) from non-V3-region-specific antibodies (non-V3 Abs). The results indicated that V3-region Abs exhibited type-specific neutralizing activities, whereas non-V3 Abs exhibited group-specific neutralizing activities. By comparing the neutralizing activities of V3-region Abs to those of non-V3 Abs, we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. Collectively, this study indicates that group-specific neutralizing anti-gp120 antibodies are specific for the CD4 attachment site.
The small GTP-binding protein rab4 is associated with early endosomes.
Authors: Van Der Sluijs P , Hull M , Zahraoui A , Tavitian A , Goud B , Mellman I
Published: May 2006

Small GTP-binding proteins of the rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. We demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrin-receptor recycling. An antibody to human rab4p was found to detect a doublet of approximately 24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing 125I-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. We also localized rab4p to transferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4 cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular transport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.
Efficient transformation and regeneration of rice small cell groups.
Authors: Lee N , Wang Y , Yang J , Ge K , Huang S , Tan J , Testa D
Published: May 2006

Transgenic plants have been regenerated from small cell groups of rice using a simpler, faster, and more efficient method than used previously (e.g., protoplast transformation and regeneration methods). Small cell groups of 50-100 cells were prepared from established suspension cultures of rice cell lines. Transformation of small cell groups with intact cell walls was carried out with a plasmid harboring the beta-glucuronidase gene and was mediated by polyethylene glycol. Assay of beta-glucuronidase activity indicated that the frequency of transformation was about 7%. beta-Glucuronidase activity was detected in the roots and leaves of plants regenerated from transformed calli. One or two copies of the beta-glucuronidase gene per cell were determined to be integrated into the rice chromosomal DNA isolated from transformed calli. This method of transformation and regeneration is widely applicable to both dicotyledonous and monocotyledonous plants, especially those varieties that are resistant to regeneration from protoplasts.
E1A oncogene induction of cytolytic susceptibility eliminates sarcoma cell tumorigenicity.
Authors: Walker T A , Wilson B A , Lewis A M , Cook J L
Published: May 2006

The manner in which oncogenes influence tumorigenicity beyond their ability to immortalize cells is uncertain. We tested the hypothesis that, in addition to subverting cellular growth controls, oncogenes can actively determine tumor-inducing capacity by affecting neoplastic cell susceptibility to destruction by the host cellular immune response. The adenovirus type 5 E1A oncogene, which induces susceptibility to lysis by natural killer cells and encodes epitopes recognized by cytotoxic T lymphocytes, was transfected into highly tumorigenic sarcoma cells. E1A expression in these sarcoma cells eliminated their tumorigenicity in recipients with natural killer cell activity that was competent to lyse these E1A-positive targets. Thymus-dependent responses were not required for tumor rejection. These results indicate that oncogene-regulated cellular pathways that affect neoplastic cell susceptibility to natural killer cell lytic mechanisms may influence tumor development in the immunocompetent host.
v-abl causes hematopoietic disease distinct from that caused by bcr-abl.
Authors: Scott M L , Van Etten R A , Daley G Q , Baltimore D
Published: May 2006

v-abl, the oncogene transduced by Abelson murine leukemia virus, was first characterized by its ability to transform lymphoid cells. bcr-abl, the oncogene formed by a t(9;22) translocation thought to occur in human hematopoietic stem cells, is detectable in almost all cases of chronic myelogenous leukemia (CML), a malignancy of granulocytic cells. bcr-abl also causes a CML-like syndrome in mice whose bone-marrow cells are infected with a retrovirus transducing the gene. More recent reports have suggested that v-abl can, however, cause a disease similar to CML. We demonstrate here that v-abl, when transduced in a helper virus-containing system, causes disease similar to, but distinct from, the CML-like syndrome induced by bcr-abl. Animals whose bone marrow has been infected by v-abl virus develop modest splenomegaly, marked granulocytosis, and malignant disease of several hematopoietic cell types. Unlike animals with CML-like disease resulting from bcr-abl, the polymorphonuclear leukocytes from animals infected with a v-abl construct do not contain the v-abl provirus at a significant frequency. Histopathologic analysis also shows significant differences between the diseases caused by v-abl and bcr-abl.
Identification of prolactin and growth hormone binding proteins in rabbit milk.
Authors: Postel-Vinay M C , Belair L , Kayser C , Kelly P A , Djiane J
Published: May 2006

Two distinct soluble proteins that specifically bind 125I-labeled human growth hormone (GH) are identified in the supernatant of ultracentrifuged rabbit milk, using HPLC gel filtration. The higher molecular weight proteins is GH specific, whereas the other one is specific for prolactin (PRL). The PRL-binding protein has a very high affinity for the hormone, almost 10 times higher than the affinity of the mammary gland membrane receptor. The PRL-binding protein is immunoprecipitated by a monoclonal antibody against the PRL receptor; another monoclonal antibody, which inhibits the PRL binding to mammary gland membranes, is a poor competitor for the PRL binding to the milk protein. These findings suggest that the milk PRL-binding protein corresponds to the binding domain of the receptor, but also that the conformation of the receptor and of the binding protein might differ. The milk and the plasma GH-binding proteins have a similar binding affinity. In cross-linking experiments using 125I-labeled human GH, the Mr of the GH-binding protein and of the PRL-binding protein were estimated to be 51,000 and 33,000, respectively. The binding proteins identified in the present work are probably responsible for the transport of their specific ligands in the milk. It is also conceivable that they have a role in the effects of GH and PRL in the mammary gland and/or the intestine of the young.
Allografts of tumor nuclear transplantation embryos: differentiation competence.
Authors: Lust J M , Carlson D L , Kowles R , Rollins-Smith L , Williams J W , McKinnell R G
Published: May 2006

The developmental potential of nuclei can be studied by nuclear transplantation. Although amphibian blastula nuclei and other early embryonic nuclei are totipotent, to our knowledge no nucleus from an adult cell has ever been shown to be totipotent by this procedure. Transfer of Lucké renal carcinoma nuclei into enucleated eggs results in prefeeding swimming tadpoles. Inasmuch as these tadpoles die, rescue of this pluripotential tissue was attempted by grafting fragments of triploid tumor nuclear transplant tadpoles to the tails of normal diploid Rana pipiens hosts. Grafts of tumor nuclear transplant tadpole tissue were histologically indistinguishable from grafts of normally fertilized embryos and developed normal-appearing structures such as complete eyes, well-differentiated neural tissues, kidney tubules, and gut epithelium. Moreover, histological differentiation in tumor nuclear transplant grafts was comparable to that observed in 50-day-old normal larvae. Grafting enhanced the survival of tumor nuclear transplant tissue from no more than 14 days as part of the donor tadpole to 40 days at which time the grafts were harvested as healthy tissue. Thus, both differentiation and survival of tumor nuclear transplant tissue were augmented with the grafting procedure. Cytophotometric analysis of ploidy was used to confirm the tumor origin of the donor tissue.
Two genes encoding 1-aminocyclopropane-1-carboxylate synthase in zucchini (Cucurbita pepo) are clustered and similar but differentially regulated.
Authors: Huang P L , Parks J E , Rottmann W H , Theologis A
Published: May 2006

A 17-kilobase (kb) region of the zucchini (Cucurbita pepo) genome has been sequenced and contains two genes, CP-ACC1A and CP-ACC1B, encoding 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14). The genes are transcribed convergently and are separated by a 5.7-kb intergenic region. Their coding regions are interrupted by four introns located in identical positions. While the DNA identity in their coding regions is 97%, their 5' and 3' flanking regions are highly divergent. Transcription of CP-ACCIA is rapidly induced by wounding in fruit and etiolated hypocotyls and by indoleacetic acid (IAA)/benzyladenine/LiCl only in fruit tissue. Conditions that induce CP-ACC1B expression have not been found. Protein synthesis inhibition derepresses the expression of CP-ACC1A and other unidentified ACC synthase genes, suggesting that they may be under negative control. The amino acid sequences deduced from the nucleotide sequences of the genes are 493 and 494 residues long with 95% identity. The most notable feature of the amino acid sequence is the presence of 11 of the 12 invariant amino acid residues involved in the binding of the substrate and pyridoxal-5'-phosphate in various aminotransferases. We conclude that ACC synthase is encoded by a multigene family of which certain members are differentially induced by auxin in a tissue-specific manner. Furthermore, ACC synthase, a pyridoxal-containing enzyme, may have an evolutionary relationship with the superfamily of aminotransferases.
Selection of unrelated donors for bone marrow transplantation is improved by HLA class II genotyping with oligonucleotide hybridization.
Authors: Tiercy J M , Morel C , Freidel A C , Zwahlen F , Gebuhrer L , Bétuel H , Jeannet M , Mach B
Published: May 2006

As the demand for donors for bone marrow transplantation increases, the use of HLA-matched, genetically unrelated donors represents a promising strategy. It is well documented that the clinical outcome of bone marrow transplantation is directly dependent on optimal matching for HLA class I and class II specificities. Molecular studies have revealed the existence of a much larger number of HLA class II alleles than was anticipated, many of which cannot be recognized by routine serological typing. Currently this "hidden" polymorphism represents a major limitation to the generalized use of unrelated donors for bone marrow transplantation. It has recently become possible, however, to identify HLA allelic polymorphism directly at the DNA level by hybridization with sequence-specific oligonucleotide probes ("HLA oligotyping") after amplification of DNA by polymerase chain reaction. In this study, we have investigated whether donor-recipient pairs that are fully matched for HLA by serology are truly HLA-DR, -DQ, and -DP identical and to what extent class II differences influence the primary mixed lymphocyte culture. We show that HLA oligotyping, performed on 50 pairs of HLA class I and II serologically matched individuals, can indeed reveal phenotypically relevant allelic differences at either DRB or DQB loci in 56% of these pairs and can therefore improve HLA class II typing and the choice of bone marrow donors quite significantly. Oligotyping for DRB/DQB/DPB polymorphism also allows prediction of a positive mixed lymphocyte culture, as established in 31 donor/recipient combinations, and even detection of polymorphic differences that were not revealed by this test. This approach is well suited for accurate HLA typing of large pools of bone marrow donors and was successfully applied to select fully matched donors for bone marrow transplantation.
Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor.
Authors: Tiberi M , Jarvie K R , Silvia C , Falardeau P , Gingrich J A , Godinot N , Bertrand L , Yang-Feng T L , Fremeau R T , Caron M G
Published: May 2006

Multiple D1 dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the D1 dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned D1 receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no D1 dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called D1B.
Differential tissue localization of oviduct and erythroid transferrin receptors.
Authors: Fuernkranz H A , Schwob J E , Lucas J J
Published: May 2006

The tissue distributions of the estrogen-inducible hen oviduct transferrin receptor and the chicken embryonic erythrocyte transferrin receptor were studied. Tissue sections were investigated by immunofluorescence microscopy using specific polyclonal antisera against each receptor. The receptor originally identified and characterized in the oviduct strongly stained liver and ovary; localized staining was observed in the brain (especially endothelial cells). Staining of breast muscle and heart tissue occurred only in occasional interstitial cells. Antiserum against oviduct transferrin receptor did not stain erythrocytes, either from embryos or from mature animals. On the other hand, the antiserum directed against the embryonic erythrocyte receptor stained embryonic erythrocytes; it did not stain any other tissues. The fluorescence microscopy observations were confirmed by Western blot analysis. The strong staining of oviduct, liver, and ovary suggests a major role for the oviduct transferrin receptor in oogenesis.
Analyses of T-cell differentiation from hemopoietic stem cells in the G0 phase by an in vitro method.
Authors: Toki J , Kumamoto T , Ogata H , Kawamura M , Fukumoto M , Cherry  , Yamamoto Y , Than S , Inaba M , Himeno Y
Published: May 2006

Using differential radiation sensitivity of components of mouse embryonal thymus, an in vitro method for studying T-cell differentiation from hemopoietic stem cells (HSCs) in the G0 phase was established. Intrathymic T-cell precursors present in embryonal thymus were found to be quite radioresistant (up to 20 Gy), and consequently 25-Gy-irradiated embryonal thymic lobes were used. Thymic lobes (25-Gy irradiated) taken from mouse fetuses (gestation day 15) were placed in Millipore-HA culture plates supported on squares of gelatin foam sponge in 24-well culture plates in which neonatal thymus stromal cells were cultured. HSCs (10(5) cells per well) in the G0 phase were added to these thymic lobes and cocultured at 37 degrees C in a 5% CO2/95% air incubator. Half the culture medium was changed every week. After 3 weeks, a large number of colonies had formed. Immunohistochemical studies and fluorescence-activated cell sorter analyses revealed that the colonizing cells regularly develop and exhibit surface markers characteristic of T cells (Thy-1, IL-2R, L3T4, Lyt-2, etc.). In situ hybridization analyses revealed that mRNA expression for T-cell receptor (TCR) beta chains occurred within colonizing cells. Using a monoclonal antibody (F23.1), expression of TCR beta-chain variable domain (V beta 8) on the surface of these developing T cells was demonstrated. These cells responded to interleukin 2 and/or anti-CD3 monoclonal antibody, indicating functional T cells. This method will be useful in studying T-cell differentiation pathways from pluripotent HSCs and in clarifying the mechanisms involved in negative and positive selection of T cells within the thymus.
Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase.
Authors: Chen J J , Throop M S , Gehrke L , Kuo I , Pal J K , Brodsky M , London I M
Published: May 2006

We have cloned the cDNA of the heme-regulated eIF-2 alpha kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2 alpha kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of approximately 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2 alpha kinase. This observation suggests that GCN2 protein kinase may be an eIF-2 alpha kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.
Separation of transcriptional activation and silencing functions of the RAP1-encoded repressor/activator protein 1: isolation of viable mutants affecting both silencing and telomere length.
Authors: Sussel L , Shore D
Published: May 2006

The repressor/activator protein 1 (RAP1) binds to the upstream activating sites of many genes, the silencer elements flanking the unexpressed mating-type loci HMR and HML, and the poly(C1-3A) sequences at telomeres, suggesting that RAP1 might have three distinct regulatory functions. To determine the in vivo role of RAP1 in repression of the HMR silent locus, we developed a screen to isolate rap1 mutants specifically defective in silencing. Fifteen independent mutants defining four different rap1 alleles were isolated. These alleles are defective to different extents in repression of an HMR locus containing a mutated, but fully functional, silencer. All four alleles are missense mutations in only three codons within a small C-terminal region of the gene. These silencing-defective mutants have no apparent growth defects, indicating that expression of the large number of essential genes that have promoters containing RAP1-binding sites is normal. A transcriptional silencing function of RAP1 can therefore be genetically separated from its presumably essential activation functions. Surprisingly, three of the silencing-defective rap1 alleles have significantly longer telomeres, suggesting that the function of RAP1 in both transcriptional silencing and telomere-length regulation may be related. In addition, we have demonstrated that increased gene dosage of either SIR1 or SIR4, two other factors required for silencing, suppresses the silencing defect of the rap1 mutants. The properties of SIR4 dosage suppression suggest that SIR4 protein may interact directly with RAP1 at silencers.
Identity of rearranged LINE/c-MYC junction sequences specific for the canine transmissible venereal tumor.
Authors: Amariglio E N , Hakim I , Brok-Simoni F , Grossman Z , Katzir N , Harmelin A , Ramot B , Rechavi G
Published: May 2006

The canine transmissible venereal tumor is a naturally occurring neoplastic disease that affects the external genitalia of both sexes and is transmitted during coitus. Cytogenetic and immunologic studies demonstrated that tumors from different parts of the world are very similar, suggesting that they are transferred from one animal to another by the transplantation of viable cells. We found that the c-MYC oncogene was rearranged in this tumor by the insertion of a transposable genetic element sequence (known as LINE, long interspersed element) 5' to the first exon. The amplification of a DNA segment located in the junction of the LINE genome and c-MYC upstream sequences enabled the testing of the similarity of transmissible venereal tumor samples collected independently in different parts of the world. Oligonucleotide primers flanking the LINE/c-MYC junction were used to amplify a 340-base-pair segment and nested primers amplified a 280-base-pair segment. A fifth oligonucleotide used as a probe contained the actual junction sequence. All of the tumors analyzed revealed the existence of the specific bands, which were absent in normal canine DNA samples. The amplified segments obtained from all of the tumors analyzed were identical in size and nucleotide sequence, suggesting transmission of the original rearranged cell itself, as opposed to independent events of LINE insertion in a "hot spot."
cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia.
Authors: Kanno H , Fujii H , Hirono A , Miwa S
Published: May 2006

cDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-specific primer. The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino acids, the same number as that by rat R-type PK. Compared with human L-type PK, R-type PK was 31 amino acids longer at the amino terminus. We also cloned and characterized R-type PK cDNA clones from patients with hereditary hemolytic anemia from a PK deficiency, PK Tokyo. A single nucleotide substitution (ACG to ATG) was found at nucleotide 1151 of the coding sequence of the R-type PK, which caused an amino acid substitution, Thr384----Met. Dot blot hybridization of PCR-amplified genomic DNA from patients and their parents by allele-specific oligonucleotide probes showed that the parents, who were second cousins, were heterozygous. To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coli and characterized the enzyme. The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris.HCl, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK. From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene.
Enhanced malignant transformation induced by expression of a distinct protein domain of ribonucleotide reductase large subunit from herpes simplex virus type 2.
Authors: Ali M A , McWeeney D , Milosavljevic A , Jurka J , Jariwalla R J
Published: May 2006

The 1.3-kilobase (kb) Pst I DNA fragment C (Pst I-C) of herpes simplex virus type 2 (HSV-2) morphological transforming region III (mtrIII; map unit 0.562-0.570) encodes part of the N-terminal half of the large subunit of ribonucleotide reductase (RR1; amino acid residues 71-502) and induces the neoplastic transformation of immortalized cell lines. To assess directly the role of these RR1 protein sequences in cell transformation, the Pst I-C fragment was cloned in an expression vector (p91023) containing an adenovirus-simian virus 40 promoter-enhancer to generate recombinant plasmid p9-C. Expression of a protein domain (approximately 65 kDa) was observed in p9-C-transfected COS-7 and Rat2 cells but not in those transfected with plasmid pHC-14 (Pst I-C in a promoterless vector). In Rat2 cells, p9-C induced highly transformed foci at an elevated frequency compared with that of pHC-14. Introduction of translation termination (TAG) condons within the RR1 coding sequence and within all three reading frames inactivated RR1 protein expression from p9-C and reduced its transforming activity to the level seen with the standard pHC-14 construct. Wild-type p9-C specified a protein kinase capable of autophosphorylation. Computer-assisted analysis further revealed significant similarity between regions of mtrIII-specific RR1 and amino acid patterns conserved within the proinsulin precursor family and DNA transposition proteins. These results identify a distinct domain of the HSV-2 RR1 protein involved in the induction of enhanced malignant transformation. In addition, the data indicate that the mtrIII DNA itself can induce basal-level transformation in the absence of protein expression.
Theory of symmetry classes.
Authors: Metropolis N , Rota G C , Stein J A
Published: May 2006

Although a great deal of work has gone into construction of the irreducible representations of the symmetric group n (and of the general linear group) a simple, intuitive characterization of the symmetry classes is missing. Relying on a systematic distinction between permutations of variables and permutations of places, we provide two such characterizations, showing that elements belonging to any such symmetry class can be described in one of two ways: (i) as the solutions of explicitly given (though not independent) sets of linear equations or (ii) as linear combinations of "simple" elements of a given symmetry class, a simple element being a generalization to an arbitrary symmetry class of the notion of a decomposable skew-symmetric tensor.