bluuebunny/crossref_metadata_2025 huggingface dataset:
Adoptive transfer of immunity to Theileria parva inthe CD8+ fraction of responding efferent lymph.
Authors: McKeever D J , Taracha E L , Innes E L , MacHugh N D , Awino E , Goddeeris B M , Morrison W I
Published: May 2006

Evidence that class I major histocompatibility
complex-restricted cytotoxic T lymphocytes (CTL) are involved in immunity to
malaria has highlighted the potential importance of these cells in protection
against intracellular parasites. Parasite-specific CTL are a prominent feature
of the immune response of cattle to Theileria parva, a related apicomplexan
parasite. The relationship between the appearance of these cells in the blood of
immune cattle under challenge and the clearance of infection suggests that they
are involved in the control of infection, but direct evidence is lacking that
CTL can mediate protection. We have made a quantitative kinetic study of CTL
responses in lymph originating from infected lymph nodes in a number of immune
cattle under challenge with T. parva. Direct killing activity and the frequency
of CTL precursors (CTLp) within responding cell populations were evaluated. A
substantial increase in the proportion of CD8+ CTL was observed between days 8
and 11 after challenge. Frequencies of CTLp as high as 1:32 were observed and
activity was essentially confined to the large blasting cell fraction. The
analogous response in peripheral blood was of lower magnitude and delayed by 1-2
days. The high frequency of CTLp in efferent lymph permitted the adoptive
transfer of this activity between immune and naive monozygotic twin calves. In
separate experiments, naive calves lethally infected with T. parva were
protected by inoculation of up to 10(10) responding CD8+ T cells derived from
their immune twins. Elimination of CD8+ T cells within the inoculum abrogated
this effect. These findings provide direct evidence that CD8+ T cells can
control T. parva infections in immune cattle.
Relocation of the plastid rbcL gene to the nucleusyields functional ribulose-1,5-bisphosphate carboxylase in tobaccochloroplasts.
Authors: Kanevski I , Maliga P
Published: May 2006

The conserved plastid localization of rbcL
suggests that biosynthesis of the large subunit of ribulose-1,5-bisphosphate
carboxylase [Rubisco; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC
4.1.1.39] in chloroplasts is required to obtain functional enzyme. To examine
the validity of this hypothesis, we relocated the plastid rbcL gene to the
nucleus. First, we deleted the rbcL gene from the tobacco plastid genome by
targeted insertion of a selectable aadA gene encoding spectinomycin resistance.
The rbcL coding region was then inserted into an expression cassette and
introduced into the nuclear genome of these plants by Agrobacterium-mediated
transformation. We report that the nuclear rbcL functionally complements the
defective plastids when the Rubisco large subunit is targeted to chloroplasts by
a transit peptide. Therefore, the evolutionary process that relocates functional
plastid genes to the nucleus has not yet occurred in the case of the rbcL gene.
Targeted deletion of plastid genes, combined with their allotopic expression,
will provide opportunities for studying the function of plastid enzyme
complexes.
Multiple isoforms of CD46 (membrane cofactor protein) serve as receptors for measles virus.
Authors: Manchester M , Liszewski M K , Atkinson J P , Oldstone M B
Published: May 2006

Measles virus (MV) causes a productive infection in humans and certain simian hosts. Rodent cells such as Chinese hamster ovary (CHO) and murine cell lines normally resist MV infection. Human CD46, or membrane cofactor protein, a complement regulatory protein, recently has been reported as the cellular receptor for MV. Multiple isoforms of the CD46 protein exist; four of these isoforms are commonly expressed on human cells. Expression of each of the four isoforms in CHO cells followed by exposure to MV led to the appearance of viral proteins within the cells and on the cell surface as detected by immunofluorescence. Syncytium formation also was observed in the cultures. CHO cells expressing any of the four isoforms and exposed to MV formed infectious centers when plated on Vero cell monolayers, indicating that the cells can transmit virus to uninfected cells. The murine cell line MC57 expressing the BC1 isoform of CD46 also stained positively for MV antigens and was positive in the infectious center assay after exposure to MV. Treatment of CD46-expressing cells with antibody to human CD46 inhibited MV binding in a dose-dependent manner. These observations indicate that any of the four primary isoforms of CD46 are able to serve as a receptor for MV.
PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.
Authors: Barnes W M
Published: May 2006

A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.
Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3.
Authors: Wang X W , Forrester K , Yeh H , Feitelson M A , Gu J R , Harris C C
Published: May 2006

Chronic active hepatitis caused by infection with hepatitis B virus, a DNA virus, is a major risk factor for human hepatocellular carcinoma. Since the oncogenicity of several DNA viruses is dependent on the interaction of their viral oncoproteins with cellular tumor-suppressor gene products, we investigated the interaction between hepatitis B virus X protein (HBX) and human wild-type p53 protein. HBX complexes with the wild-type p53 protein and inhibits its sequence-specific DNA binding in vitro. HBX expression also inhibits p53-mediated transcriptional activation in vivo and the in vitro association of p53 and ERCC3, a general transcription factor involved in nucleotide excision repair. Therefore, HBX may affect a wide range of p53 functions and contribute to the molecular pathogenesis of human hepatocellular carcinoma.
Cloning and expression of a distinctive class of self-incompatibility (S) gene from Papaver rhoeas L.
Authors: Foote H C , Ride J P , Franklin-Tong V E , Walker E A , Lawrence M J , Franklin F C
Published: May 2006

We present the identification, cloning, and characterization of a self-incompatibility (S) gene from Papaver rhoeas that has no significant homology to any previously reported gene sequences, including S genes from other species. This result suggests that a different self-incompatibility mechanism may be operating in this species and has important implications for the evolutionary relationships between the S genes. The S1 cDNA was cloned by using an oligonucleotide based upon N-terminal amino acid sequence data from stigmatic proteins that show complete linkage with the S1 gene. The single-copy gene has been expressed in Escherichia coli to test biological activity. Although the recombinant S1 protein (S1e) is not processed in the same way as the protein produced in the plant, it exhibits, in vitro, the specific pollen inhibitory activity expected of an S gene product; pollen carrying the S1 allele is inhibited, whereas pollen not carrying S1 is not inhibited. These results provide definitive demonstration that the product of a cloned S gene has S-specific pollen inhibitory activity.
Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle.
Authors: Marty I , Robert M , Villaz M , De Jongh K , Lai Y , Catterall W A , Ronjat M
Published: May 2006

Membrane vesicles enriched in both ryanodine receptor and dihydropyridine receptor were obtained from rabbit skeletal muscle and solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Analysis of the sedimentation behavior of the solubilized proteins showed the existence of a population of alpha 1 subunits of the dihydropyridine receptor which cosedimented with the ryanodine receptor. Solubilized proteins were immunoprecipitated with antibodies directed against either the ryanodine receptor or the alpha 1, alpha 2, or beta subunits of the dihydropyridine receptor. Immunoprecipitated proteins were identified by Western blot analysis and by specific labeling with [3H]ryanodine or [3H]PN200-110. Immunoprecipitation of the solubilized proteins with antibodies directed against the dihydropyridine receptor led to the coimmunoprecipitation of the ryanodine receptor. Conversely, immunoprecipitation with antibodies directed against the ryanodine receptor led to an immune complex containing both receptors, but these antibodies were unable to precipitate purified dihydropyridine receptor. These results demonstrate that ryanodine receptor and dihydropyridine receptor are present in the triad membrane preparation in a complex which may play an important role in excitation-contraction coupling.
Hospital-acquired infections: diseases with increasingly limited therapies.
Authors: Swartz M N
Published: May 2006

About 5% of patients admitted to acute-care hospitals acquire nosocomial infections. A variety of factors contribute: increasing age of patients; availability, for treatment of formerly untreatable diseases, of extensive surgical and intensive medical therapies; and frequent use of antimicrobial drugs capable of selecting a resistant microbial flora. Nosocomial infections due to resistant organisms have been a problem ever since infections due to penicillinase-producing Staphylococcus aureus were noted within a few years of the introduction of penicillin. By the 1960s aerobic Gram-negative bacilli had assumed increasing importance as nosocomial pathogens, and many strains were resistant to available antimicrobials. During the 1980s the principal organisms causing nosocomial bloodstream infections were coagulase-negative staphylococci, aerobic Gram-negative bacilli, S. aureus, Candida spp., and Enterococcus spp. Coagulase-negative staphylococci and S. aureus are often methicillin-resistant, requiring parenteral use of vancomycin. Prevalence of vancomycin resistance among enterococcal isolates from patients in intensive care units has increased, likely due to increased use of this drug. Plasmid-mediated gentamicin resistance in up to 50% of enterococcal isolates, along with enhanced penicillin resistance in some strains, leaves few therapeutic options. The emergence of Enterobacteriaceae with chromosomal or plasmid-encoded extended spectrum beta-lactamases presents a world-wide problem of resistance to third generation cephalosporins. Control of these infections rests on (i) monitoring infections with such resistant organisms in an ongoing fashion, (ii) prompt institution of barrier precautions when infected or colonized patients are identified, and (iii) appropriate use of antimicrobials through implementation of antibiotic control programs.
Mutation of His-105 in the beta 1 subunit yields a nitric oxide-insensitive form of soluble guanylyl cyclase.
Authors: Wedel B , Humbert P , Harteneck C , Foerster J , Malkewitz J , Böhme E , Schultz G , Koesling D
Published: May 2006

Soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.2] is a hemoprotein that exists as a heterodimer; the heme moiety has been proposed to bind nitric oxide, resulting in a dramatic activation of the enzyme. Mutation of six conserved His residues reduced but did not abolish nitric oxide stimulation whereas a change of His-105 to Phe in the beta 1 subunit yielded a heterodimer that retained basal cyclase activity but failed to respond to nitric oxide. Heme was not detected as a component of the mutant heterodimer and protophorphyrin IX failed to stimulate enzyme activity. The activity of the His mutant was almost identical to that of the wild-type enzyme in the presence of KCN, suggesting that disruption of heme binding is the principal effect of the mutation. Thus, the mutation provides a means to inhibit the nitric oxide-sensitive guanylyl cyclase signaling pathway.
Transport of newly synthesized glucosylceramide to the plasma membrane by a non-Golgi pathway.
Authors: Warnock D E , Lutz M S , Blackburn W A , Young W W , Baenziger J U
Published: May 2006

High-gradient magnetic affinity chromatography (HIMAC) has been used to obtain highly enriched plasma membranes, free of intracellular membrane contaminants, from cultured Chinese hamster ovary (CHO) cells in yields of > or = 80%. Using this procedure we have characterized the transport of glucosylceramide (GlcCer) and the ganglioside GM3 to the plasma membrane. Newly synthesized GlcCer reaches the plasma membrane in 7.2 min, whereas GM3 requires 21.5 min to reach the plasma membrane. Brefeldin A prevents transport of newly synthesized GM3 and sphingomyelin to the plasma membrane but has no effect on the transport of GlcCer. Similarly, incubation of CHO cells at 15 degrees C blocks transport of GM3 and sphingomyelin to the plasma membrane but has no effect on GlcCer movement. We propose that carrier-mediated transport accounts for a major fraction of the plasma membrane GlcCer. Pulse-chase studies with either [3H]glucose or [3H]palmitate indicate that newly synthesized GlcCer which has reached the plasma membrane is not utilized for the synthesis of GM3 but is instead rapidly either degraded or converted into an as yet unidentified product. Our results indicate that in addition to serving as a precursor for higher glycosylation in the Golgi, a major fraction of newly synthesized GlcCer is rapidly transported to the plasma membrane by a non-Golgi pathway and then rapidly turned over.
Functional dependence of Ca(2+)-activated K+ current on L- and N-type Ca2+ channels: differences between chicken sympathetic and parasympathetic neurons suggest different regulatory mechanisms.
Authors: Wisgirda M E , Dryer S E
Published: May 2006

The influx of Ca2+ ions controls many important processes in excitable cells, including the regulation of the gating of Ca(2+)-activated K+ channels (the current IK[Ca]). Various IK[Ca] channels contribute to the regulation of the action-potential waveform, the repetitive discharge of spikes, and the secretion of neurotransmitters. It is thought that large-conductance IK[Ca] channels must be closely colocalized with Ca2+ channels (ICa) to be gated by Ca2+ influx. We now report that IK[Ca] channels can be preferentially colocalized with pharmacologically distinct subtypes of voltage-activated Ca2+ channel and that this occurs differently in embryonic chicken sympathetic and parasympathetic neurons. The effects of various dihydropyridines and omega-conotoxin on voltage-activated Ca2+ currents (ICa) and Ca(2+)-activated K+ currents (IK[Ca]) were examined by using perforated-patch whole-cell recordings from embryonic chicken ciliary and sympathetic ganglion neurons. Application of nifedipine or omega-conotoxin each caused a 40-60% reduction in ICa, whereas application of S-(-)-BAY K 8644 potentiated ICa in ciliary ganglion neurons. But application of omega-conotoxin had little or no effect on IK[Ca], whereas nifedipine and S-(-)-BAY K 8644 inhibited and potentiated IK[Ca], respectively. These results indicate that IK[Ca] channels are preferentially coupled to L-type, but not to N-type, Ca2+ channels on chicken ciliary ganglion neurons. Chicken sympathetic neurons also express dihydropyridine-sensitive and omega-conotoxin-sensitive components of ICa. However, in those cells, application of omega-conotoxin caused a 40-60% reduction in IK[Ca], whereas nifedipine reduced IK[Ca] but only in a subpopulation of cells. Therefore, IK[Ca] in sympathetic neurons is either coupled to N-type Ca2+ channels or is not selectively coupled to a single Ca(2+)-channel subtype. The preferential coupling of IK[Ca] channels with distinct ICa subtypes may be part of a mechanism to allow for selective modulation of neurotransmitter release. Preferential coupling may also be important for the differentiation and development of vertebrate neurons.
Telomerase activity in human ovarian carcinoma.
Authors: Counter C M , Hirte H W , Bacchetti S , Harley C B
Published: May 2006

Telomeres fulfill the dual function of protecting eukaryotic chromosomes from illegitimate recombination and degradation and may aid in chromosome attachment to the nuclear membrane. We have previously shown that telomerase, the enzyme which synthesizes telomeric DNA, is not detected in normal somatic cells and that telomeres shorten with replicative age. In cells immortalized in vitro, activation of telomerase apparently stabilizes telomere length, preventing a critical destabilization of chromosomes, and cell proliferation continues even when telomeres are short. In vivo, telomeres of most tumors are shorter than telomeres of control tissues, suggesting an analogous role for the enzyme. To assess the relevance of telomerase and telomere stability in the development and progression of tumors, we have measured enzyme activity and telomere length in metastatic cells of epithelial ovarian carcinoma. We report that extremely short telomeres are maintained in these cells and that tumor cells, but not isogenic nonmalignant cells, express telomerase. Our findings suggest that progression of malignancy is ultimately dependent upon activation of telomerase and that telomerase inhibitors may be effective antitumor drugs.
Role of glutamine-117 in the ribonucleolytic activity of human angiogenin.
Authors: Russo N , Shapiro R , Acharya K R , Riordan J F , Vallee B L
Published: May 2006

The crystal structure of human angiogenin (reported in the preceding paper in this issue) reveals that the site that corresponds to the pyrimidine binding site of RNase A is obstructed by Gln-117. Mutation of this residue to Ala and Gly is here found to increase activity 11- to 18-fold and 21- to 30-fold, respectively, toward dinucleotide, polynucleotide, and cyclic nucleotide substrates, but without changing specificity. The enhanced activity of Q117G toward CpA is due to a 5-fold decrease in Km and a 6-fold increase in kcat. Its Ki value for 2'-CMP is 5-fold lower than that of native angiogenin, whereas its Ki value for 5'-AMP is unchanged. It has been reported previously that mutating Asp-116 to Ala increases activity 15-fold. The double mutant D116A/Q117A is shown to be only slightly more active than each individual mutant. The present results demonstrate that Gln-117 impedes the ribonucleolytic activity of angiogenin, as predicted by x-ray crystallography. Moreover, they suggest that prior to or during catalysis angiogenin must undergo a conformational change to reorient the C-terminal segment that contains this residue, and that a similar reorganization is required for the mutants as well. This view is supported by molecular modeling of an angiogenin-uridine vanadate complex. These in vitro findings have implications for the angiogenic activity of angiogenin in vivo.
Mice expressing both B7-1 and viral glycoprotein on pancreatic beta cells along with glycoprotein-specific transgenic T cells develop diabetes due to a breakdown of T-lymphocyte unresponsiveness.
Authors: Harlan D M , Hengartner H , Huang M L , Kang Y H , Abe R , Moreadith R W , Pircher H , Gray G S , Ohashi P S , Freeman G J
Published: May 2006

T lymphocytes have been implicated in the onset of many autoimmune diseases; however, the mechanisms underlying T-cell activation toward self antigens are poorly understood. To study whether T-lymphocyte costimulation can overcome the immunologic unresponsiveness observed in an in vivo model, we have created transgenic mice expressing the costimulatory mouse molecule B7-1, a ligand for the CD28 receptor, on pancreatic beta cells. We now report that triple-transgenic mice expressing both B7-1 and a viral glycoprotein on their beta cells, along with T cells expressing the viral-glycoprotein-specific transgenic T-cell receptor, all develop insulitis (lymphocytic infiltration of the pancreatic islets) and diabetes. In striking contrast, the T cells in double-transgenic mice expressing the same viral glycoprotein (but no B7) on their pancreatic beta cells and the transgenic T-cell receptor on their T cells, reported earlier, remain indifferent to the glycoprotein-expressing beta cells. In fact, all three transgenes are required to initiate immune-mediated destruction of the beta cells. Mice expressing any of the transgenes alone, or any two in combination, maintain normal islet architecture and never spontaneously develop insulitis or diabetes. Our results show that aberrant B7 expression on peripheral tissues may play an important role in the activation of self-reactive T cells and further suggest that abnormal expression of costimulatory receptors may be involved in various T-cell-mediated autoimmune diseases.
The insect tracheal system: a conduit for the systemic spread of Autographa californica M nuclear polyhedrosis virus.
Authors: Engelhard E K , Kam-Morgan L N , Washburn J O , Volkman L E
Published: May 2006

Baculoviruses establish systemic infections within susceptible insect hosts, even though host tissues are surrounded by basal laminae, extracellular matrices that exclude particles smaller than these viruses. Using a recombinant Autographa californica M nuclear polyhedrosis virus containing a lacZ reporter gene under the control of a constitutive promoter, we followed the progression of infection in Trichoplusia ni larvae. We discovered that infection of the larval insect tracheal system (and not hemocytes, as thought previously) provides the major conduit for this virus to pass through basal laminae and to spread throughout the host. Tracheal epidermal cells, the only known cellular components of the tracheal system, share a common lymph system. Locally these cells contact one another by interdigitating cytoplasmic extensions called epidermal feet. These two features of the tracheal system are likely to facilitate the rapid systemic spread of the virus. The findings reported here have major implications for the fields of insect pathology and biological control and usher in an important consideration regarding host-range factors.
Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody.
Authors: Conley A J , Kessler J A , Boots L J , Tung J S , Arnold B A , Keller P M , Shaw A R , Emini E A
Published: May 2006

The antiviral characteristics of monoclonal antibody IAM-41-2F5 (2F5) were determined in cell culture. The antibody had been previously shown to bind a specific sequence, ELDKWA, within the external domain of the gp41 envelope glycoprotein human immunodeficiency virus type 1 (HIV-1). Selection by 2F5 of recombinant phage from an epitope library confirmed the identification of the antibody's binding determinant. The antibody was found to be capable of neutralizing a broad range of lymphoid cell culture-adapted HIV-1 variants as well as HIV-1 primary isolates. Sequence analysis of the latter showed that neutralization was related to the presence of the antibody binding site. From kinetic measurements using an epitope-containing peptide or gp41, the half-time of dissociation for 2F5 was determined to be 122 min for the peptide and 156 min for gp41. The region of gp41 expressing this sequence exhibits greater conservation among HIV-1 isolates than do the variable domains of gp120.
Clonal variants of hybridoma cells that switch isotype at a high frequency.
Authors: Spira G , Gregor P , Aguila H L , Scharff M D
Published: May 2006

As B cells differentiate under the influence of antigen and T cells, they frequently switch from the expression of IgM antibody to the expression of other isotypes. This is accomplished by rearranging the expressed variable region gene to downstream constant region genes and deleting the intervening sequences. Some B-cell lines that represent early stages in development switch constitutively in culture at frequencies that approach those of lipopolysaccharide- or lymphokine-stimulated normal B cells. Hybridoma cells represent a later stage of development and rarely switch in culture. In contrast to early B-cell lines, hybridomas produce large amounts of immunoglobulin, and single cells can be assayed easily for the expression of new isotypes. We have used the ELISA spot assay and fluctuation analysis to determine the rate of switching of two hybridoma cell lines. By identifying subclones that switched more frequently, we have progressively enriched for cells that switch spontaneously at higher rates. These cells, like normal cells, switch by rearrangement and deletion, and the frequency of switched cells in some of the clones is comparable to that which has been observed in less differentiated B-cell lines and in normal B cells.
p53 and E2F-1 cooperate to mediate apoptosis.
Authors: Wu X , Levine A J
Published: May 2006

The tumor-suppressor protein p53 appears to function at the G1 phase of the cell cycle as a checkpoint in response to DNA damage. Mutations in the p53 gene lead to an increased rate of genomic instability and tumorigenesis. The E2F-1 transcription factor is a protein partner of the retinoblastoma-susceptibility gene product, RB. E2F-1 appears to function as a positive regulator or signal for entry into S phase. To explore possible interactions of p53 and E2F-1 in the cell cycle, a human E2F-1 expression plasmid was introduced into a murine cell line containing a temperature-sensitive p53 allele which produces a p53 protein in the wild-type conformation at 32 degrees C and the mutant form at 37.5 degrees C. Coexpression of the wild-type p53 protein and E2F-1 in these cells resulted in a rapid loss of cell viability through a process of apoptosis. Thus, the cell cycle utilizes an interacting or communicative pathway between RB-E2F-1 and p53.
Evidence that an amoeba acquired a chloroplast by retaining part of an engulfed eukaryotic alga.
Authors: McFadden G I , Gilson P R , Hofmann C J , Adcock G J , Maier U G
Published: May 2006

Chlorarachniophytes are amoeboid algae with unusual chloroplasts. Instead of the usual two membranes that surround the chloroplasts of plants, green algae, and red algae, the chloroplasts of chlorarachniophytes have four bounding membranes. The extra membranes may reflect an unusual origin of chlorarachniophyte chloroplasts. Rather than inheriting the organelle directly from their ancestors, chlorarachniophytes may have adopted the chloroplast of an algal cell ingested as prey. Parts of the algal cell are postulated to remain within the amoeba as a reduced eukaryotic endosymbiont [Hibberd, D. J. & Norris, R. E. (1984) J. Phycol. 20, 310-330]. A small nucleus-like structure, proposed to be a vestige of the endosymbiont's nucleus, is located in a space between the second and third chloroplast membranes. We cloned and sequenced nuclear-type rRNA genes from chlorarachniophytes and found two highly divergent genes. In situ hybridization shows that one gene is expressed by the amoebal (host) nucleus and the other is expressed by the putative endosymbiont nucleus, suggesting that the latter is indeed a foreign genome. Transcripts from the endosymbiont gene accumulate in the small cytoplasmic compartment between the second and third chloroplast membranes, which we believe to be the remnant cytoplasm of the endosymbiont. Using the endosymbiont gene as a probe, we identified three small chromosomes belonging to the endosymbiont nucleus. This knowledge should allow a detailed molecular analysis of the role of the endosymbiont's genome and cytoplasm in the partnership.
Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia.
Authors: Nakayama K , Nakayama K , Negishi I , Kuida K , Sawa H , Loh D Y
Published: May 2006

Mice carrying ablated coding regions of the bcl-2 alpha and bcl-2 beta transcripts have been made. bcl-2-/- mutants are smaller but viable, although about half of them die by 6 weeks of age. As shown earlier with somatic bcl-2 gene-targeted mice, the number of lymphocytes markedly decreased within few weeks after birth while other hematopoietic lineages remained unaffected. Among lymphocytes, CD8+ T cells disappeared most quickly followed by CD4+ T cells, whereas B cells were least affected. bcl-2-/- lymphocytes, however, could respond normally to various stimuli including anti-CD3, Con A, phorbol 12-myristate 13-acetate plus ionomycin, interleukin 2, lipopolysaccharide, and anti-IgM antibody. Abnormalities among nonlymphoid organs include smaller auricles, hair color turning gray at 4-5 weeks of age, and polycystic kidney disease-like change of renal tubules. These results suggest that Bcl-2 may be involved during morphogenesis where inductive interactions between epithelium and mesenchyme are important such as in the kidneys, hair follicles, and perichondrium of auricles. Surprisingly, the nervous system, intestines, and skin appear normal despite the fact that these organs show high levels of endogenous Bcl-2 expression in normal mice.
The nature of the autoimmune antibody repertoire in human immunodeficiency virus type 1 infection.
Authors: Ditzel H J , Barbas S M , Barbas C F , Burton D R
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) seropositive donors typically have high serum antibody titers to a range of autoantigens, and the corresponding autoantibodies have been suggested to be of importance in the pathogenesis of HIV-1 infection. We have prepared 38 IgG human monoclonal autoantibodies from asymptomatic HIV-1 seropositive donors with elevated serum titers to autoantigens by construction of Fab combinatorial libraries on the surface of phage and affinity selection using a range of autoantigens, including double-stranded DNA, major histocompatibility complex class II, CD14, epidermal growth factor receptor, and ganglioside GD2. The autoantibodies are shown to be of moderate affinity and exhibit marked cross-reactivity with a range of antigens. This contrasts with the specific high-affinity antibodies selected (i) against infectious agents using the same libraries and (ii) against one of the autoantigens using a library from a donor with established autoimmune disease. The results lend no support to the presence of specific autoantibodies in HIV-1 infection and instead suggest attention should be focused on the pathological significance of high serum levels of antibodies capable of interacting with multiple molecular species.
Allelotyping of butadiene-induced lung and mammary adenocarcinomas of B6C3F1 mice: frequent losses of heterozygosity in regions homologous to human tumor-suppressor genes.
Authors: Wiseman R W , Cochran C , Dietrich W , Lander E S , Söderkvist P
Published: May 2006

To identify the potential involvement of tumor-suppressor gene inactivation during neoplastic development in B6C3F1 mice, genetic losses were determined from allelotypes of butadiene-induced lung and mammary adenocarcinomas. By using length polymorphisms in restriction fragments and simple sequence repeats, or "microsatellites," markers on each autosome were analyzed for allele losses in tumor DNAs. Losses of heterozygosity on chromosome 11 were observed at several loci surrounding the p53 tumor-suppressor gene (Trp53) in 12 of 17 mammary tumors and 2 of 8 lung tumors. Although most of these alterations appeared to result from nondisjunction, at least two examples of somatic recombination or deletion were also observed. Southern analysis revealed a homozygous deletion of the remaining Trp53 allele of one of these mammary tumors. Losses of heterozygosity were also detected at the Rb-1 tumor-suppressor gene in 7 of 17 mammary tumors and 1 lung tumor. Finally, frequent allele losses were observed on chromosome 4 in lung tumors. Analysis of nine chromosome 4 loci defined an interstitial deletion containing the Ifa gene cluster in one of the lung tumors. A tumor-suppressor gene was previously mapped to this region of chromosome 4 in studies with somatic cell hybrids. In addition, homozygous deletions have been reported in a homologous region of human chromosome 9p for acute lymphocytic leukemias, glioblastomas, melanomas, and lung carcinomas. These findings suggest that the inactivation of tumor-suppressor genes including Trp53, Rb-1, and an unidentified gene on chromosome 4 plays a significant role during carcinogenesis in mice.
Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice.
Authors: Guidotti L G , Ando K , Hobbs M V , Ishikawa T , Runkel L , Schreiber R D , Chisari F V
Published: May 2006

During hepatitis B virus (HBV) infection, distinct host-virus interactions may establish the patterns of viral clearance and persistence and the extent of virus-associated pathology. It is generally thought that HBV-specific class I-restricted cytotoxic T lymphocytes (CTLs) play a critical role in this process by destroying infected hepatocytes. This cytopathic mechanism, however, could be lethal if most of the hepatocytes are infected. In the current study, we demonstrate that class I-restricted HBV-specific CTLs profoundly suppress hepatocellular HBV gene expression in HBV transgenic mice by a noncytolytic process, the strength of which greatly exceeds the cytopathic effect of the CTLs in magnitude and duration. We also show that the regulatory effect of the CTLs is initially mediated by interferon gamma and tumor necrosis factor alpha, is delayed in onset, and becomes independent of these cytokines shortly after it begins. The data indicate that the anti-viral CTL response activates a complex regulatory cascade that inhibits hepatocellular HBV gene expression without killing the cell. The extent to which this mechanism contributes to viral clearance or viral persistence during HBV infection remains to be determined.
Diffusivity of myoglobin in intact skeletal muscle cells.
Authors: Jürgens K D , Peters T , Gros G
Published: May 2006

We report a method that allows us to determine the diffusion coefficient of native myoglobin in intact and mechanically unaffected red muscle fibers. The method is based on an optical recording of intracellular diffusion of metmyoglobin, which is produced inside the cells by photooxidation of oxymyoglobin with a UV light pulse. We find a myoglobin diffusivity of 1.2 x 10(-7) cm2/s (22 degrees C), which is only 1/10th of the value measured in very dilute myoglobin solutions and 1/5th of the value obtained from measurements in solutions of myoglobin at 18 g/dl. The latter value often has been used in model calculations of oxygen transport to tissue incorporating myoglobin-facilitated oxygen diffusion. Recalculating facilitated diffusion with the value obtained by us implies that its contribution to total intracellular oxygen transport is of minor importance. Furthermore, it shows that sterical hindrance to myoglobin diffusion is dominated by the muscle-cell architecture rather than by the overall protein concentration of the muscle fiber.
PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro.
Authors: Graña X , De Luca A , Sang N , Fu Y , Claudio P P , Rosenblatt J , Morgan D O , Giordano A
Published: May 2006

Members of the cell division cycle 2 (CDC2) family of kinases play a pivotal role in the regulation of the eukaryotic cell cycle. In this communication, we report the isolation of a cDNA that encodes a CDC2-related human protein kinase temporarily designated PITALRE for the characteristic Pro-Ile-Thr-Ala-Leu-Arg-Glu motif. Its deduced amino acid sequence is 47% identical to that of the human cholinesterase-related cell division controller (CHED) kinase, which is required during hematopoiesis, and 42% identical to the Saccharomyces cerevisiae SGV1 gene product, a putative kinase involved in the response to pheromone via its guanine nucleotide-binding protein alpha subunit. PITALRE expression is ubiquitous, but its expression levels are different in various human tissues. PITALRE is an approximately 43-kDa protein that associates with three cellular polypeptides of 80, 95, and 155 kDa. PITALRE is localized primarily to the nucleus. In addition, we have identified a retinoblastoma protein kinase activity associated with PITALRE immunocomplexes that cannot phosphorylate histone H1, suggesting that the target phosphorylation site of PITALRE differs from that of CDC2 kinase. Interestingly, the retinoblastoma kinase activity associated with PITALRE does not oscillate during the cell cycle.
Timely immunization subverts the development of peripheral nonresponsiveness and suppresses tumor development in simian virus 40 tumor antigen-transgenic mice.
Authors: Ye X , McCarrick J , Jewett L , Knowles B B
Published: May 2006

Tolerance to tumor cell-expressed molecules and selection of cells that evade immune surveillance during tumor progression create effective barriers to immunotherapy. We investigated the cytotoxic T-lymphocyte response to simian virus 40 (SV40) tumor (T/t) antigen in two lineages of transgenic mice bearing the same rat insulin promoter-SV40 T/t antigen (RIP Tag) hybrid gene. RIP1-Tag2 mice, which express Tag as embryos, are tolerant to Tag, whereas RIP1-Tag4 mice, which express the transgene in pancreatic islet beta cells several weeks after birth and develop insulinomas, can be immunized to generate active Tag-specific cytotoxic T lymphocytes as determined by in vitro assays. Indeed, RIP1-Tag4 mice immunized with Tag by SV40 infection prior to the time of endogenous transgene expression also mount an effective in vivo cellular immune response to the Tag-expressing pancreatic beta cells, and Tag-induced tumor growth is significantly delayed (up to 1 year). However, after the transgene is expressed, RIP1-Tag4 mice are unable to mount a tumor-inhibiting response upon immunization, although Tag-specific cytotoxic T cells can still be demonstrated in vitro. Our data suggest that Tag-specific T cells are rendered unresponsive in vivo in RIP1-Tag4 mice and that the establishment of this unresponsiveness to Tag can be prevented by SV40 immunization only before the onset of the transgene expression. In the older, successfully immunized mouse, decreased immune surveillance and selection of cells with down-regulation of major histocompatibility complex class I expression most likely set the stage for insulinoma development.
Yeast RAD3 protein binds directly to both SSL2 and SSL1 proteins: implications for the structure and function of transcription/repair factor b.
Authors: Bardwell L , Bardwell A J , Feaver W J , Svejstrup J Q , Kornberg R D , Friedberg E C
Published: May 2006

The RAD3 and SSL2 gene products are essential proteins that are also required for the nucleotide excision repair pathway. We have recently demonstrated that the RAD3 gene product along with the SSL1 and TFB1 gene products are subunits of RNA polymerase II basal transcription factor b. Additionally, the SSL2 gene product physically interacts with purified factor b. Here we combine an in vitro immunoprecipitation assay and an in vivo genetic assay to demonstrate a series of pairwise protein-protein interactions involving these components. RAD3 protein binds directly to both SSL2 protein and SSL1 protein in vitro. SSL1 protein interacts with itself and with RAD3 and TFB1 proteins in living yeast cells. An N-terminal, possibly noncatalytic, domain of SSL2 protein is sufficient for the factor b-SSL2 interaction, and a product of a DNA repair-defective allele of SSL2 is not defective in binding to factor b. We present genetic evidence suggesting that the DNA-repair function of SSL2 protein is not dependent on its essential function.
Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting.
Authors: Rom E , Kahana C
Published: May 2006

We provide here an example of a mammalian cellular gene expressed by frame-shifting. Conventional reading of the sequence of ornithine decarboxylase-antizyme mRNA (a protein that modulates the rate of ornithine decarboxylase degradation) results in premature termination at an in-frame termination codon (stop-1), located shortly after the initiation codon. By translating, in vitro in reticulocyte lysate, antizyme mRNA with a full coding capacity and various mutants derived from it, we demonstrate that antizyme expression requires that ribosomes shift from the first open reading frame (termed ORF0) to a second +1 open reading frame (ORF1). Our studies show that this frame-shifting, which occurs at maximal efficiency of approximately 20%, is stimulated by polyamines and requires the functional integrity of the stop codon (stop-1) of ORF0. By introducing in-frame deletions, we have shown that an 87-nt segment surrounding stop-1 enhances frame-shifting efficiency, whereas the 6 nt located just upstream to stop-1 are absolutely essential for this process. Because this segment does not contain sequences that were previously characterized as shifty segments, our results suggest that another mechanism of frame-shifting is involved in mediating antizyme expression.
Lyn tyrosine kinase signals cell cycle arrest but not apoptosis in B-lineage lymphoma cells.
Authors: Scheuermann R H , Racila E , Tucker T , Yefenof E , Street N E , Vitetta E S , Picker L J , Uhr J W
Published: May 2006

Signal transduction initiated by binding of antibodies to cell surface molecules can have an important impact on the growth of tumor cells. The malignant behavior of the murine lymphoma BCL1 can be suppressed and the neoplastic cells can be induced to enter a dormant state by in vivo ligation of membrane immunoglobulin. Anti-CD19 antibodies can prolong the survival of SCID mice challenged with the human Burkitt lymphoma cell line, Daudi. Here, we show that cross-linking of membrane immunoglobulin on both murine BCL1 and human Daudi cells initiates a cascade of signals leading to the induction of both apoptosis and cell cycle arrest in vitro. Using antisense oligonucleotides, we demonstrate that the immunoglobulin-associated Lyn tyrosine kinase is required for anti-immunoglobulin-mediated cell cycle arrest but is not required for the signal leading to apoptosis. These results define a branch point in the cytosolic signaling pathways mediating cell cycle arrest and apoptosis. In Daudi cells, Lyn is also critical for cell cycle arrest induced by anti-CD19 signaling. Thus, the Lyn tyrosine kinase may be an important mediator of cell cycle arrest in neoplastic B lymphocytes and, perhaps, other cell types.
Apoptosis mediates and thymidine prevents erythroblast destruction in folate deficiency anemia.
Authors: Koury M J , Horne D W
Published: May 2006

Deficiency of the vitamin folic acid causes pancytopenia by decreasing the production of new blood cells. Although impaired DNA synthesis and destruction of hematopoietic cells have been implicated, the mechanism by which folate deficiency decreases blood cell production is uncertain. An in vitro model of folate-deficient erythropoiesis was developed by using proerythroblasts isolated from folate-deficient mice that were infected with Friend leukemia virus. Proerythroblasts from folate-deficient mice had one-tenth the total folate as did proerythroblasts from control mice. The folate-deficient proerythroblasts underwent apoptosis, a form of programmed cell death, after 20-32 h in culture in folate-deficient medium. At the time of apoptosis the cells had differentiated into the later erythroblast stages and some had begun hemoglobin synthesis. Addition of either folic acid or thymidine, but not deoxycytidine or inosine, to the folate-deficient medium prevented the apoptosis and permitted proliferation and differentiation of the proerythroblasts into reticulocytes. The prevention of apoptosis by thymidine indicates (i) that decreased thymidylate synthesis plays a role in erythroblast apoptosis and the anemia of folate deficiency and (ii) that DNA cleavage is likely to be a primary event in the apoptosis of folate-deficient erythroblasts. Apoptosis of erythroblasts in the late stages of differentiation leads to decreased erythrocyte production and to anemia. The increased erythropoietin produced in response to the anemia increases the number of erythroid progenitor cells in the differentiation stages preceding those in which the cells undergo apoptosis. This population shift to earlier stage erythroblasts and proerythroblasts is characteristic of bone marrows of individuals with folate deficiency anemia.
Urea unfolding of peptide helices as a model for interpreting protein unfolding.
Authors: Scholtz J M , Barrick D , York E J , Stewart J M , Baldwin R L
Published: May 2006

To provide a model system for understanding how the unfolding of protein alpha-helices by urea contributes to protein denaturation, urea unfolding was measured for a homologous series of helical peptides with the repeating sequence Ala-Glu-Ala-Ala-Lys-Ala and chain lengths varying from 14 to 50 residues. The dependence of the helix propagation parameter of the Zimm-Bragg model for helix-coil transition theory (s) on urea molarity ([urea]) was determined at 0 degree C with data for the entire set of peptides, and a linear dependence of In s on [urea] was found. The results were fitted by the binding-site model and by the solvent-exchange model for the interaction of urea with the peptides. Each of these thermodynamic models is able to describe the data quite well and we are not able to discern any difference between the ability of each model to fit the data. Thus a linear relation, ln s = ln s0 - (m/RT).[urea], fits the data for alpha-helix unfolding, just as others have found for protein unfolding. When the m value determined here for alpha-helix unfolding is multiplied by the number of helical residues in partly helical protein molecules, the resulting values agree within a factor of 2 with observed m values for these proteins. This result indicates that the interaction between urea and peptide groups accounts for a major part of the denaturing action of urea on proteins, as predicted earlier by some model studies with small molecules.
Brain-derived neurotrophic factor promotes the survival of neurons arising from the adult rat forebrain subependymal zone.
Authors: Kirschenbaum B , Goldman S A
Published: May 2006

Neuronal precursor cells persist in the adult forebrain ependymal/subependymal zone (SZ) and have been found to produce neurons in cultures derived from birds, rodents, and humans. We postulated that the survival of neurons generated from these cells might be constrained in adulthood by the local absence of trophic support. To test this hypothesis, we established explant cultures of adult rat forebrain SZ and assessed the effect of defined neurotrophins on the survival of new neurons arising from these explants. We found that microtubule-associated protein 2+ neurons arose from explants derived from a wide area of the SZ, spanning the rostral 6 mm of the ventricular system. In cultures exposed to brain-derived neurotrophic factor (BDNF), > 35% of new neurons survived at 22 days in vitro (DIV), and > 25% survived at 42 DIV, concurrent with the virtually complete loss of neurons in unsupplemented controls. The surviving cells expressed trkB, the high-affinity receptor for BDNF. In contrast, neither nerve growth factor nor neurotrophic factor 3 enhanced neuronal survival. Thus, BDNF supports the survival of neurons produced by the adult rat forebrain and may act as a permissive factor for neuronal recruitment in adulthood.