bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermoprotection of a functional epithelium: heat stress effects on transepithelial transport by flounder renal tubule in primary monolayer culture.
Authors: Brown M A , Upender R P , Hightower L E , Renfro J L
Published: May 2006

Primary monolayer cultures of winter flounder renal proximal-tubule cells were used to determine whether transepithelial transport could be protected from the damaging effects of extreme temperature by previous mild heat shock. Renal tubule epithelial cells were enzymatically dispersed and reorganized as confluent monolayer sheets on native rat tail collagen. Transepithelial electrical properties (potential difference, resistance, short-circuit current, and Na(+)-dependent glucose current) and unidirectional [35S]sulfate fluxes were measured in Ussing chambers at 22 degrees C. Examination of transepithelial electrical properties following acute 1-hr elevation of temperature over a range of 22-37 degrees C provided the basis for the "mild" versus "severe" thermal stress protocols. Severe elevation from 22 degrees C to 32 degrees C for 1.5 hr followed by 1.5 hr at 22 degrees C significantly decreased glucose current (7 +/- 0.7 to 3 +/- 0.8 microA/cm2) as well as net sulfate secretion [131 +/- 11 to 33 +/- 11 nmol/(cm2.hr)]. Mild heat shock of 27 degrees C for 6 hr prior to this severe heat shock completely protected both glucose transport (6 +/- 0.7 microA/cm2) and sulfate flux (149 +/- 13 nmol/(cm2.hr)]. Scanning electron microscopy showed that the number of microvilli on the apical (luminal) surface of the epithelium was decreased after a 32 degrees C heat shock. Monolayers exposed to 27 degrees C for 6 hr prior to incubation at 32 degrees C showed no loss of microvilli. SDS/PAGE analysis of protein patterns from the cultures showed that three classes of heat shock proteins were maximally induced at 27 degrees C. Inhibition of protein synthesis by cycloheximide prevented the thermoprotective effect of mild heat shock. This suggests that certain renal transport functions can be protected from sublethal but debilitating thermal stress by prior mild heat shock and that heat shock proteins may play a role in this protection.
Altering the antigenicity of proteins.
Authors: Alexander H , Alexander S , Getzoff E D , Tainer J A , Geysen H M , Lerner R A
Published: May 2006

To better understand the binding interaction between antigen and antibody we need to distinguish protein residues critical to the binding energy and mechanism from residues merely localized in the interface. By analyzing the binding of monoclonal antibodies to recombinant wild-type and mutant myohemerythrin (MHr) proteins, we were able to test the role of individual critical residues at the highly antigenic site MHr-(79-84), within the context of the folded protein. The results directly show the existence of antigenically critical residues, whose mutations significantly reduce antibody binding to the folded protein, thus verifying peptide-based assignments of these critical residues and demonstrating the ability of buried side chains to influence antigenicity. Taken together, these results (i) distinguish the antigenic surface from the solvent-exposed protein surface before binding, (ii) support a two-stage interaction mechanism allowing inducible changes in protein antigens by antibody binding, and (iii) show that protein antigenicity can be significantly reduced by alteration of single critical residues without destroying biological activity.
Mendelian inheritance of familial prostate cancer.
Authors: Carter B S , Beaty T H , Steinberg G D , Childs B , Walsh P C
Published: May 2006

Previous studies have demonstrated familial clustering of prostate cancer. To define the nature of this familial aggregation and to assess whether Mendelian inheritance can explain prostate cancer clustering, proportional hazards and segregation analyses were performed on 691 families ascertained through a single prostate cancer proband. The proportional hazards analyses revealed that two factors, early age at onset of disease in the proband and multiple affected family members, were important determinants of risk of prostate cancer in these families. Furthermore, segregation analyses revealed that this clustering can be best explained by autosomal dominant inheritance of a rare (q = 0.0030) high-risk allele leading to an early onset of prostate cancer. The estimated cumulative risk of prostate cancer for carriers revealed that the allele was highly penetrant: by age 85, 88% of carriers compared to only 5% of noncarriers are projected to be affected with prostate cancer. The best fitting autosomal dominant model further suggested that this inherited form of prostate cancer accounts for a significant proportion of early onset disease but overall is responsible for a small proportion of prostate cancer occurrence (9% by age 85). These data provide evidence that prostate cancer is inherited in Mendelian fashion in a subset of families and provide a foundation for gene mapping studies of heritable prostate cancer. Characterization of genes involved in inherited prostate cancer could provide important insight into the development of this disease in general.
Brain transplants of cells expressing the carboxyl-terminal fragment of the Alzheimer amyloid protein precursor cause specific neuropathology in vivo.
Authors: Neve R L , Kammesheidt A , Hohmann C F
Published: May 2006

PC12 cells transfected with retroviral recombinants expressing the carboxyl-terminal 104 amino acids of the Alzheimer amyloid protein precursor (beta APP-C104) or PC12 cells transfected with the retroviral vector (DO) alone were transplanted into the brains of newborn mice. At 20 days after grafting, transplants could be detected in all of the mouse brains examined. At 4 months after transplantation, experimental animals exhibited significant cortical atrophy. Some also revealed immunoreactivity with Alz-50, an antibody that detects an Alzheimer disease-related protein, in the somatodendritic domain of neurons in the cortex surrounding the transplants. In addition, disorganization of the neuropil in the CA2/3 region of the hippocampus ipsilateral to the transplant was revealed by staining with an antibody to the carboxyl-terminal end of the amyloid protein precursor. A decrease in cell body immunoreactivity for this portion of the amyloid protein precursor was also detected with this antibody. Together, these results suggest that the carboxyl-terminal fragment of beta APP may cause specific neuropathology and neurodegeneration in vivo.
Two types of chloroplast gene promoters in Chlamydomonas reinhardtii.
Authors: Klein U , De Camp J D , Bogorad L
Published: May 2006

Structures of the promoters of Chlamydomonas reinhardtii plastid atpB and 16S rRNA-encoding genes were analyzed in vivo. Chimeric constructs, containing the Chlamydomonas chloroplast atpB or 16S rRNA-encoding gene promoter coupled to the Escherichia coli uidA (beta-glucuronidase, GUS) reporter gene and bordered by C. reinhardtii chloroplast sequences, were stably introduced into the chloroplast of Chlamydomonas by microprojectile bombardment. Activity of the promoters in the chloroplast of GUS gene-positive transformants was assayed by measuring the abundance of GUS transcripts and determining the relative rates of GUS transcription in vivo. Deletion analyses of the 16S rRNA gene and atpB promoter fragments showed that the two promoters differ structurally. The 16S rRNA gene promoter resembles the bacterial sigma 70 type with typical -10 and -35 elements. The atpB promoter, on the other hand, lacks a conserved motif in the -35 region but contains, in the -10 region, a characteristic octameric palindrome (TATAATAT) that is conserved in the promoter sequences of some other C. reinhardtii chloroplast genes. For maximum activity, the atpB promoter requires sequences of approximately 22 base pairs upstream and approximately 60 base pairs downstream of the transcription start site.
Altered binding of regulatory factors to HLA class I enhancer sequence in human tumor cell lines lacking class I antigen expression.
Authors: Blanchet O , Bourge J F , Zinszner H , Israel A , Kourilsky P , Dausset J , Degos L , Paul P
Published: May 2006

Class I antigens encoded in the major histocompatibility complex (MHC) (HLA in man, H-2 in the mouse) play a key role in the recognition of target cells by cytolytic T lymphocytes. Tumor cells frequently do not express class I MHC molecules, which strongly suggests that down-regulation of the latter facilitates escape of tumor cells from immune surveillance. The expression of class I MHC genes is tightly regulated. An enhancer element, conserved in the promoters of mouse and human MHC genes, has been shown to be important for mouse class I MHC gene expression. At least two related regulatory factors (KBF1 and NF-kappa B) bind to this regulatory element. We have analyzed the binding of these factors in cellular extracts of 23 human tumor cell lines displaying various levels of class I mRNA and surface expression. In this panel, combined deficiency of KBF1- and NF-kappa B-like DNA-binding activities was frequent among the class I-negative cell lines and correlated with the absence of class I mRNA. A few cell lines that lack KBF1 binding activity still display NF-kappa B-like activity and express normal levels of MHC class I mRNA. These results suggest (i) that, in the absence of KBF1, NF-kappa B or a related factor promotes MHC class I gene transcription; and (ii) that a combined defect in KBF1/NF-kappa B DNA-binding activity can cause a pleiotropic defect in class I gene expression, which may facilitate tumor progression.
Are the stray 60-Hz electromagnetic fields associated with the distribution and use of electric power a significant cause of cancer?
Authors: Jackson J D
Published: May 2006

The putative causal relation between ambient low-frequency (50 or 60 Hz) electromagnetic fields (necessarily present in living and working environments because of our ever increasing use of electrical devices) and cancer, especially leukemia, can be tested on the large scale by examining historical data on the growth of the generation and consumption of electric power since 1900 and corresponding data on cancer death and incidence rates. The United States per capita generation and residential consumption of electric power have grown roughly exponentially since 1900; total per capita generation has increased by a factor of 10 since 1940, and per capita residential consumption has increased by a factor of 20 in the same period. The ubiquitous stray fields from power distribution lines and internal and external wiring in buildings have grown in the same proportions. In contrast to the explosive increase in the generation and use of electricity, the age-adjusted cancer death rate for the population as a whole shows only a slight rise since 1900. When respiratory cancers (largely caused by tobacco use) are subtracted, the remaining death rate has actually fallen since 1940. That the death rate may have fallen because of better diagnosis and treatment, despite a rising incidence rate, is not substantiated, especially for leukemia, including childhood leukemia, where the incidence rate has been constant or declining slightly for the past 25 yr. The absence of any appreciable change in the national cancer incidence rates during a period in which residential use of electric power has increased dramatically shows that the associated stray 50- or 60-Hz electromagnetic fields pose no significant hazard to the average individual.
Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.
Authors: Jayasena S D , Johnston B H
Published: May 2006

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo.
Role of a vitronectin-like molecule in embryo adhesion of the brown alga Fucus.
Authors: Wagner V T , Brian L , Quatrano R S
Published: May 2006

The rhizoid cell of the two-celled embryo of the brown alga Fucus is structurally and functionally differentiated from the thallus cell. The rhizoid cell is highly polar and transports directionally components of the cell wall to its elongating tip, which attaches the developing embryo to the substratum. Polyclonal antibodies to human vitronectin (Vn) recognize a vitronectin-like glycoprotein (Vn-F) in extracts of zygotes and two-celled embryos of Fucus, with a molecular mass (approximately 62 kDa) similar to that of human Vn. The specificity of the immunological cross-reactivity of Vn-F to rabbit polyclonal antibodies made to human Vn is demonstrated by competition experiments using pure human Vn and monospecific antibodies generated toward Vn-F. Vn-F possesses affinities for glass and heparin that are identical to those of human Vn. Immunolocalization and subcellular fractionation results demonstrate that Vn-F is localized first in the cytoplasm of the zygote, which is followed by the polar transport of Vn-F to its exclusive localization in the cell wall of the elongating rhizoid tip. Vn does not localize to the rhizoid tip under culture conditions that prevent two-celled embryos from attaching. Furthermore, an adhesion assay demonstrates that two-celled Fucus embryos do not adhere to the substratum in the presence of the Vn antibody, suggesting that the Vn-F in this brown alga not only possesses structural similarity to mammalian Vn but may also have a similar functional role in adhesion. The presence of Vn-F in brown algae suggests a high degree of evolutionary conservation of its structural and functional characteristics.
Transient expression from cab-m1 and rbcS-m3 promoter sequences is different in mesophyll and bundle sheath cells in maize leaves.
Authors: Bansal K C , Viret J F , Haley J , Khan B M , Schantz R , Bogorad L
Published: May 2006

Cell-specific and light-regulated expression of the beta-glucuronidase (GUS) reporter gene from maize cab-m1 and rbcS-m3 promoter sequences was studied in maize leaf segments by using an in situ transient expression microprojectile bombardment assay. The cab-m1 gene is known to be strongly photoregulated and to be expressed almost exclusively in mesophyll cells (MC) but not in bundle sheath cells (BSC). Expression of GUS from a 1026-base-pair 5' promoter fragment of cab-m1 is very low in dark-grown leaves; GUS expression is increased about 10-fold upon illumination of dark-grown leaves. In illuminated leaves, the ratio of GUS expression in MC vs. BSC is about 10:1. The cab-m1 region between 868 and 1026 base pairs 5' to the translation start confers strong MC-preferred expression on the remainder of the chimeric gene in illuminated leaves, but a region between -39 and -359 from the translation start is required for photoregulated expression. Transcripts of rbcS-m3 are found in BSC but not in MC and are about double in BSC of greening dark-grown seedlings. In contrast to the behavior of the cab-m1-GUS construct, GUS expression driven by 2.1 kilobase pairs of the rbcS-m3 5' region was about twice as high in MC as in BSC of unilluminated dark-grown maize leaves. The number of BSC, but not MC, expressing GUS nearly doubled upon greening of bombarded etiolated leaves. These data suggest that the 5' region of rbcS-m3 used here could be responsible for most of the light-dependent increase in rbcS-m3 transcripts observed in BSC of greening leaves and that transcriptional or posttranscriptional mechanisms are responsible for the lack of rbcS-m3 transcripts in MC.
What drives the translocation of proteins?
Authors: Simon S M , Peskin C S , Oster G F
Published: May 2006

We propose that protein translocation across membranes is driven by biased random thermal motion. This "Brownian ratchet" mechanism depends on chemical asymmetries between the cis and trans sides of the membrane. Several mechanisms could contribute to rectifying the thermal motion of the protein, such as binding and dissociation of chaperonins to the translocating chain, chain coiling induced by pH and/or ionic gradients, glycosylation, and disulfide bond formation. This helps explain the robustness and promiscuity of these transport systems.
Coordinated antiinflammatory effects of interleukin 4: interleukin 4 suppresses interleukin 1 production but up-regulates gene expression and synthesis of interleukin 1 receptor antagonist.
Authors: Vannier E , Miller L C , Dinarello C A
Published: May 2006

Interleukin 1 receptor antagonist (IL-1ra), a naturally occurring polypeptide with amino acid sequence homology to interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta), prevents Escherichia coli-induced shock and death. Both IL-1 and IL-1ra are produced by monocytes stimulated with lipopolysaccharide (LPS). Because interleukin 4 (IL-4) suppresses IL-1 production, we investigated whether IL-4 modulated IL-1ra synthesis in LPS-stimulated human peripheral blood mononuclear cells. IL-1 beta and IL-1ra were measured by specific RIAs. IL-4 alone (0.01-100 ng/ml) did not stimulate IL-1 beta synthesis but rather induced IL-1ra (4.82 +/- 0.94 ng/ml). LPS induced synthesis of both IL-1 beta (6.67 +/- 1.06 ng/ml) and IL-1ra (10.77 +/- 2.79 ng/ml). IL-4 suppressed LPS-induced IL-1 beta mRNA accumulation and synthesis. However, IL-4 acted synergistically with LPS in inducing IL-1ra. IL-4 enhanced LPS-induced IL-1ra mRNA accumulation 4-fold and IL-1ra protein synthesis nearly 2-fold. Moreover, IL-1ra mRNA levels were maximal after 6 hr of exposure to LPS but peaked within the first 3 hr in the presence of IL-4. IL-4 added as late as 12 hr after LPS stimulation still enhanced IL-1ra synthesis. In human peripheral blood mononuclear cells stimulated with IL-1 alpha, IL-4 markedly suppressed IL-1 beta production but enhanced IL-1ra synthesis greater than 2-fold. Because IL-4 favors synthesis of the natural antagonist IL-1ra over synthesis of the agonist IL-1, IL-4 may exert potent antiinflammatory effects on host responses to Gram-negative infections.
Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution.
Authors: Embretson J , Zupancic M , Beneke J , Till M , Wolinsky S , Ribas J L , Burke A , Haase A T
Published: May 2006

Latent and productive viral infections are at the extremes of the spectrum of virus-cell interactions that are thought to play a major role in the ability of such important human pathogens as human immunodeficiency virus (HIV) to elude host defenses and cause disease. The recent development of PCR-based methods to amplify target sequences in individual cells in routinely fixed tissues affords opportunities to directly examine the subtle and covert virus-cell relationships at the latent end of the spectrum that are inaccessible to analysis by conventional in situ hybridization techniques. We have now used PCR in situ with in situ hybridization to document latent and permissive HIV infection in routinely fixed and paraffin-embedded tissue. In one of the first specimens we examined, a tumor biopsy from an HIV-infected individual, we found many of the lymphocytes and lymphocytes infiltrating the tumor had HIV DNA that was detectable only by PCR in situ. The fraction of positive cells varied regionally, but there were foci where most of the cells contained HIV DNA. Most of these lymphocytes and macrophages are latently infected, as we could detect HIV RNA in fewer than one in a thousand of these cells. We also detected HIV RNA, surprisingly, in 6% of the tumor cells, where the number of copies of viral RNA per cell was equivalent to productively infected cell lines. The alternative states of HIV-gene expression and high local concentration of latently infected lymphocytes and monocytes revealed by these studies conceptually supports models of lentiviral pathogenesis that attribute persistence to the reservoir of latently infected cells and disease to the consequences of viral-gene expression in this population. The magnitude of infection of lymphocytes documented in this report is also consistent with the emerging view that HIV infection per se could contribute substantially to depletion of immune cells in AIDS.
Hypervariable-control-region sequences reveal global population structuring in a long-distance migrant shorebird, the Dunlin (Calidris alpina).
Authors: Wenink P W , Baker A J , Tilanus M G
Published: May 2006

Hypervariable segments of the control region of mtDNA as well as part of the cytochrome b gene of Dunlins were amplified with PCR and sequenced directly. The 910 base pairs (bp) obtained for each of 73 individuals complete another of the few sequencing studies that examine the global range of a vertebrate species. A total of 35 types of mtDNA were detected, 33 of which were defined by the hypervariable-control-region segments. Thirty of the latter were specific to populations of different geographic origin in the circumpolar breeding range of the species. The remaining three types indicate dispersal between populations in southern Norway and Siberia, but female-mediated flow of mtDNA apparently is too low to overcome the effects of high mutation rates of the control-region sequences, as well as population subdivision associated with historical range disjunctions. A genealogical tree relating the types grouped them into five populations: Alaska, West Coast of North America, Gulf of Mexico, western Europe, and the Taymyr Peninsula. The Dunlin is thus highly structured geographically, with measures of mutational divergence approaching 1.0 for fixation of alternative types in different populations. High diversity of types within populations as well as moderate long-term effective population sizes argue against severe population bottlenecks in promoting this differentiation. Instead, population fragmentation in Pleistocene refuges is the most plausible mechanism of mtDNA differentiation but at a much earlier time scale than suggested previously with morphometric data.
Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control.
Authors: Amsterdam A , Pitzer F , Baumeister W
Published: May 2006

We describe the isolation and characterization of proteasomes from recently established immortalized ovarian granulosa cell lines and their intracellular distribution during mitosis and during cAMP-induced differentiation, as revealed by immunofluorescence microscopy. In interphase, proteasomes were localized in small clusters throughout the cytoplasm and the nuclear matrix. In prophase, a substantial increase in proteasomal staining was observed in the perichromosomal area. A dramatic increase occurred in metaphase and in early anaphase; the chromosomes remained unstained. In late anaphase, intensive staining remained associated mainly with the spindle fibers. In telophase and early interphase of the daughter cells, intensive staining of proteasomes persisted in the nuclei. In contrast, in cells stimulated to differentiate by forskolin, which substantially elevates intracellular cAMP in these cell lines, only a weak staining of proteasomes was revealed in both the nucleus and the cytoplasm. Double staining of nondividing cells with antibodies to proteasomes and to tubulin did not show colocalization of proteasomes and microtubules. In contrast, dividing cells show a preferential concentration of proteasomes around spindle microtubules during metaphase and anaphase. The observed spatial and temporal distribution pattern of proteasomes during mitosis is highly reminiscent of the behavior of cyclins [Pines, J. & Hunter, T. (1991) J. Cell Biol. 115, 1-17]. Since proteasome accumulation appears to coincide with disappearance of cyclins A and B1 from the spindle apparatus, it is suggested that proteasomes may play a role in termination of mitosis by degrading the cyclins, which act as regulatory elements.
Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site.
Authors: Philippe C , Eyermann F , Bénard L , Portier C , Ehresmann B , Ehresmann C
Published: May 2006

From genetic and biochemical evidence, we previously proposed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops. Here, we use "toeprint" experiments with Moloney murine leukemia virus reverse transcriptase to analyze the effect of S15 on the formation of the ternary mRNA-30S-tRNA(fMet) complex. We show that the binding of the 30S subunit on the mRNA stops reverse transcriptase near position +10, corresponding to the 3' terminus of the pseudoknot, most likely by stabilizing the pseudoknot conformation. Furthermore, S15 is found to stabilize the binary 30S-mRNA complex. When the ternary 30S-mRNA-tRNA(fMet) complex is formed, a toeprint is observed at position +17. This toeprint progressively disappears when the ternary complex is formed in the presence of increasing concentrations of S15, while a shift from position +17 to position +10 is observed. Beside, RNase T1 footprinting experiments reveal the simultaneous binding of S15 and 30S subunit on the mRNA. Otherwise, we show by filter binding assays that initiator tRNA remains bound to the 30S subunit even in the presence of S15. Our results indicate that S15 prevents the formation of a functional ternary 30S-mRNA-tRNA(fMet) complex, the ribosome being trapped in a preternary 30S-mRNA-tRNA(fMet) complex.
In vivo evidence against the existence of antiprogestins disrupting receptor binding to DNA.
Authors: Delabre K , Guiochon-Mantel A , Milgrom E
Published: May 2006

The binding of a steroid hormone to its receptor elicits a sequence of events: activation of the receptor (probably through dissociation from a complex of heat shock proteins), dimerization, binding to hormone responsive elements, and finally modulation of gene transcription. RU 486, the first antiprogestin studied, has been shown to act at the last step of this sequence: provoking an inefficient binding of the receptor to hormone responsive elements. Recently, based on in vitro studies, it has been proposed that ZK 98299 was the prototype of a second class of antiprogestins that were supposed to act through disruption of the binding to DNA. We have devised methods allowing us to study the various steps of agonist or antagonist action in vivo. We show here that RU 486 and ZK 98299 have the same effects on receptor activation, dimerization, and binding to hormone responsive elements; differences in their action are explained by the 10-fold difference in their affinity for the receptor (ZK 98299 having the lower affinity).
Systemic immunological effects of cytokine genes injected into skeletal muscle.
Authors: Raz E , Watanabe A , Baird S M , Eisenberg R A , Parr T B , Lotz M , Kipps T J , Carson D A
Published: May 2006

Somatic gene therapy is an interesting approach for the delivery of cytokines for prolonged periods. The present experiments show that direct injections into mouse skeletal muscle of cDNA expression vectors encoding interleukin 2 (IL-2), IL-4, or type beta 1 transforming growth factor (TGF-beta 1) induce biological effects characteristic of these cytokines in vivo. Mice injected intramuscularly with a vector encoding IL-2 had enhanced humoral and cellular immune responses to an exogenous antigen, transferrin, that was delivered at a separate site. These IL-2 effects were abolished by coadministration of a vector directing synthesis of TGF-beta 1. The TGF-beta 1 vector by itself depressed the anti-transferrin antibody response and caused an 8-fold increase in plasma TGF-beta 1 activity. The TGF-beta 1 plasmid injection did not cause muscle infiltration with monocytes or neutrophils and there was no evidence for fibrotic changes. Muscle injection with a cDNA encoding IL-4 selectively increased IgG1 levels but did not alter the cellular immune response to transferrin. In lupus-prone mice (MRL/lpr/lpr), injection with IL-2 expression vectors increased and TGF-beta 1 vectors decreased auto-antibodies to chromatin. These results demonstrate that intramuscular injection of cytokine genes, in the absence of infectious viral vectors, can regulate humoral and cellular immune responses in vivo.
bcl-2 inhibits death of central neural cells induced by multiple agents.
Authors: Zhong L T , Sarafian T , Kane D J , Charles A C , Mah S P , Edwards R H , Bredesen D E
Published: May 2006

The protooncogene bcl-2, which has been implicated in B-cell lymphoma development, inhibits apoptosis due to growth factor withdrawal in some, but not all, hematopoietic cells. Recently we found that bcl-2 also inhibits apoptosis in PC12 pheochromocytoma cells. We now report that bcl-2 inhibits the death of a central neural cell line due to serum and growth factor withdrawal, the calcium ionophore A23187, glucose withdrawal, membrane peroxidation, and, in some cases, free radical-induced damage. This broad range of protective effects of BCL-2 protein suggests that BCL-2 may interact with a central step in neural cell death. Measurements of intracellular free calcium suggest that BCL-2 alters the transduction of neural death signals at a point distal to the rise in intracellular free calcium.
Gating currents of the cloned delayed-rectifier K+ channel DRK1.
Authors: Taglialatela M , Stefani E
Published: May 2006

Gating currents of the cloned delayed-rectifier K+ channel DRK1 expressed in Xenopus oocytes were measured with the open-oocyte Vaseline-gap voltage-clamp technique. DRK1 gating charge had the following salient properties: (i) gating-charge amplitude correlated positively with size of the expressed ionic K+ currents; (ii) the time integral of ON and OFF gating currents was similar, indicating charge conservation and lack of charge immobilization; (iii) the gating-charge activation curve was shallower and had a half-activation potential 15 mV more negative than the activation curve for K+ conductance; (iv) effective valence for the gating current was about two electronic charges per gating subunit; (v) for large depolarizations (to > 0 mV) prominent rising phases were observed during the ON and OFF gating charge, which appeared as shoulders in unsubtracted traces; (vi) for small depolarizing pulses (to < 0 mV) ionic-current activation and deactivation had time constants similar to ON and OFF gating-current decay, respectively; (vii) negative prepulses made more prominent the ON rising phase and delayed ionic and gating currents. The results are consistent with a model for K+ channel activation that has an early slow and/or weakly voltage-dependent transition between early closed states followed by more voltage-dependent transitions between later closed states and a final voltage-independent closed-open transition.
Galaxy dynamics and the mass density of the universe.
Authors: Rubin V C
Published: May 2006

Dynamical evidence accumulated over the past 20 years has convinced astronomers that luminous matter in a spiral galaxy constitutes no more than 10% of the mass of a galaxy. An additional 90% is inferred by its gravitational effect on luminous material. Here I review recent observations concerning the distribution of luminous and nonluminous matter in the Milky Way, in galaxies, and in galaxy clusters. Observations of neutral hydrogen disks, some extending in radius several times the optical disk, confirm that a massive dark halo is a major component of virtually every spiral. A recent surprise has been the discovery that stellar and gas motions in ellipticals are enormously complex. To date, only for a few spheroidal galaxies do the velocities extend far enough to probe the outer mass distribution. But the diverse kinematics of inner cores, peripheral to deducing the overall mass distribution, offer additional evidence that ellipticals have acquired gas-rich systems after initial formation. Dynamical results are consistent with a low-density universe, in which the required dark matter could be baryonic. On smallest scales of galaxies [10 kiloparsec (kpc); Ho = 50 km.sec-1.megaparsec-1] the luminous matter constitutes only 1% of the closure density. On scales greater than binary galaxies (i.e., >/=100 kpc) all systems indicate a density approximately 10% of the closure density, a density consistent with the low baryon density in the universe. If large-scale motions in the universe require a higher mass density, these motions would constitute the first dynamical evidence for nonbaryonic matter in a universe of higher density.
The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis.
Authors: Appel L F , Prout M , Abu-Shumays R , Hammonds A , Garbe J C , Fristrom D , Fristrom J
Published: May 2006

The Stubble-stubbloid (Sb-sbd) gene is required for hormone-dependent epithelial morphogenesis of imaginal discs of Drosophila, including the formation of bristles, legs, and wings. The gene has been cloned by using Sb-sbd-associated DNA lesions in a 20-kilobase (kb) region of a 263-kb genomic walk. The region specifies an approximately 3.8-kb transcript that is induced by the steroid hormone 20-hydroxyecdysone in imaginal discs cultured in vitro. The conceptually translated protein is an apparent 786-residue type II transmembrane protein (N terminus in, C terminus out), including an intracellular N-terminal domain of at least 35 residues and an extracellular C-terminal trypsin-like serine protease domain of 244 residues. Sequence analyses indicate that the Sb-sbd-encoded protease could activate itself by proteolytic cleavage. Consistent with the cell-autonomous nature of the Sb-sbd bristle phenotype, a disulfide bond between cysteine residues in the noncatalytic N-terminal fragment and the C-terminal catalytic fragment could tether the protease to the membrane after activation. Both dominant Sb and recessive sbd mutations affect the organization of microfilament bundles during bristle morphogenesis. We propose that the Sb-sbd product has a dual function. (i) It acts through its proteolytic extracellular domain to detach imaginal disc cells from extracellular matrices, and (ii) it transmits an outside-to-inside signal to its intracellular domain to modify the cytoskeleton and facilitate cell shape changes underlying morphogenesis.
Intracellular Ca2+ pool content is linked to control of cell growth.
Authors: Short A D , Bian J , Ghosh T K , Waldron R T , Rybak S L , Gill D L
Published: May 2006

A close correlation was observed between intracellular Ca2+ pool depletion and refilling and the onset of DNA synthesis and proliferation of DDT1MF-2 smooth muscle cells. The intracellular Ca2+ pump inhibitors 2,5-di-tert-butyl-hydroquinone (DBHQ) and thapsigargin (TG) specifically emptied identical inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools and both arrested cell growth at concentrations corresponding to Ca2+ pump blockade. However, an important distinction was observed between the two inhibitors with respect to their reversibility of action. Upon removal of DBHQ from DBHQ-arrested cells, Ca2+ pools immediately refilled, and 14 hr later cells entered S phase followed by normal cell proliferation; the time for entry into S phase was identical to that for cells released from confluence arrest. Although TG irreversibly blocked Ca2+ pumping and emptied Ca2+ pools, high serum treatment of TG-arrested cells induced recovery of functional Ca2+ pools in 6 hr (via probable synthesis of new pump); thereafter cells proceeded to S phase and normal cell proliferation within the same time period (14 hr) as that following release of DBHQ-arrested cells. The precise relationship between Ca2+ pump blockade and growth arrest indicates that Ca2+ pool emptying maintains cells in a G0-like quiescent state; upon refilling of pools, normal progression into the cell cycle is resumed. It is possible that a specific cell cycle event necessary for G0 to G1 transition depends upon signals generated from the InsP3-sensitive Ca2+ pool.
The interaction of bride of sevenless with sevenless is conserved between Drosophila virilis and Drosophila melanogaster.
Authors: Hart A C , Harrison S D , Van Vactor D L , Rubin G M , Zipursky S L
Published: May 2006

An inductive interaction between the sevenless (sev) transmembrane tyrosine kinase receptor and the bride of sevenless (boss) transmembrane ligand is required for the development of the R7 photoreceptor neuron in the compound eye of Drosophila melanogaster. The boss protein is proposed to contain a large N-terminal extracellular domain, seven transmembrane segments, and a C-terminal cytoplasmic tail. The boss protein from Drosophila virilis (bossvir) retains strong amino acid identity with loss from D. melanogaster (bossmel): 73% identity in the N-terminal extracellular domain and 91% identity in the seven-transmembrane domain, including the cytoplasmic tail. By using P-element-mediated DNA transformation, the bossmel and bossvir genes were shown to rescue the D. melanogaster boss1 mutation. The expression of bossvir protein in D. melanogaster is indistinguishable from that of bossmel protein. Noncoding sequences which may regulate boss expression were identified based on their conservation during evolution. The predicted sev protein from D. virilis (sevvir) was previously shown to be 63% identical to sev from D. melanogaster (sevmel). A chimeric gene, (sevvir/mel), encoding the extracellular domain of sevvir and the cytoplasmic domain of sevmel rescues the D. melanogaster sevd2 mutation through interaction with either bossvir or bossmel.
Molecular evolution of the Adh1 locus in the genus Zea.
Authors: Gaut B S , Clegg M T
Published: May 2006

We sampled DNA sequences from the locus encoding alcohol dehydrogenase 1 (alcohol:NAD+ oxidoreductase, EC 1.1.1.1). Our sample represents Adh1 alleles from a wide geographic distribution of Zea mays (maize) and two species of teosinte (Zea luxurians and Zea diploperennis). Using these and previously published sequences, we analyze the molecular evolution of Adh1 in the genus Zea. We perform tests to characterize recombination and identify the putative parents of the recombinant Adh1-Cm allele. We also perform tests for selection but are unable to detect either a selective sweep or strong balancing selection at the Adh1 locus. We estimate that divergence times between teosinte and some maize alleles are approximately 1 million years, whereas divergence times between distantly related maize alleles are approximately 2 million years. We conclude that the common ancestor to the genus Zea was polymorphic at the Adh1 locus. On the basis of previous estimates of nucleotide diversity at other maize loci, it appears that the common ancestor to the genus Zea was polymorphic at many loci.
Blockade of tolerance to morphine but not to kappa opioids by a nitric oxide synthase inhibitor.
Authors: Kolesnikov Y A , Pick C G , Ciszewska G , Pasternak G W
Published: May 2006

The nitric oxide synthase inhibitor NG-nitro-L-arginine (NO2Arg) blocks morphine tolerance in mice. After implantation of morphine pellets the analgesic response decreases from 100% on the first day to 0% on the third. Coadministration of NO2Arg along with the pellets markedly retards the development of tolerance; 60% of mice are analgesic after 3 days, and 50% of mice are analgesic after 5 days. In a daily injection paradigm the analgesic response to morphine is reduced from 60% to 0% by 5 days. Concomitant administration of morphine along with NO2Arg at doses of 2 mg/kg per day prevents tolerance for 4 weeks. A single NO2Arg dose retards morphine tolerance for several days, and dosing every 4 days is almost as effective as daily NO2Arg. NO2Arg slowly reverses preexisting tolerance over 5 days despite the continued administration of morphine along with NO2Arg. NO2Arg also reduces dependence and reverses previously established dependence. NO2Arg does not prevent tolerance to analgesia mediated by the kappa 1 agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolindinyl)cyclohexyl]- benzene-acetamide (U50,488H) or the kappa 3 agent naloxone benzoylhydrazone, indicating a selective action of NO in the mechanisms of mu tolerance and dependence.
Priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces CD8+ T-cell-mediated protective immunity against malaria.
Authors: Li S , Rodrigues M , Rodriguez D , Rodriguez J R , Esteban M , Palese P , Nussenzweig R S , Zavala F
Published: May 2006

Live vectors expressing foreign antigens have been used to induce immunity against several pathogens. However, for the virulent rodent malaria parasite Plasmodium yoelii, the use of recombinant vaccinia virus, pseudorabies virus, or Salmonella, expressing the circumsporozoite protein of this parasite, failed to induce protection. We generated a recombinant influenza virus expressing an epitope from the circumsporozoite protein of P. yoelii known to be recognized by CD8+ T cells and demonstrated that this vector induced class I major histocompatibility complex-restricted cytotoxic T cells against this foreign epitope. Immunization of mice with this recombinant influenza virus, followed by a recombinant vaccinia virus expressing the entire circumsporozoite protein, induced protective immunity against sporozoite-induced malaria. The sequence of immunization appears to be crucial, since a primer injection with recombinant vaccinia virus, followed by a booster injection with recombinant influenza virus, failed to induce protection. The protection induced by immunization with these recombinant viruses is mostly mediated by CD8+ T cells, as treatment of mice with anti-CD8 monoclonal antibody abolishes the anti-malarial immunity. The use of different live vectors for primer and booster injections has a synergistic effect on the immune response and might represent an effective general strategy for eliciting protective immune responses to key antigens of microbial pathogens.
Electron self-exchange in azurin: calculation of the superexchange electron tunneling rate.
Authors: Mikkelsen K V , Skov L K , Nar H , Farver O
Published: May 2006

Electronic coupling between the copper atoms in an azurin dimer has been calculated in this conformationally well-defined system by using many-electronic wave functions. When one of the two water molecules forming intermolecular hydrogen bonds between the copper-ligating His-117 of the two azurins is removed, the calculated coupling element is reduced from 2.5 x 10(-6) to 1.1 x 10(-7) eV (1 eV = 1.602 x 10(-19) J). Also, the effects of the relative orientations of the two water molecules have been analyzed. The results show that water molecules may play an important role as switches for biological electron transfer. The rate of electron self-exchange between two azurins has been calculated, and the result is in very good agreement with the rate found experimentally.
On the directional specificity of ribosome frameshifting at a "hungry" codon.
Authors: Lindsley D , Gallant J
Published: May 2006

Limitation for aminoacyl-tRNA promotes ribosome frameshifting at certain sites. We have previously demonstrated ribosome frameshifting to the right (3') at an AAG site in one context, and to the left (5') at an AAG site in a different context. Here, we demonstrate that the "rightwing" context is largely specific for frameshifting to the right, and the "leftwing" context is largely specific for frameshifting to the left. Analysis of these context rules, and the conversion of a sequence that promotes leftward frameshifting to one that promotes rightward frameshifting, demonstrated here, permits us to define a minimal heptanucleotide sequence sufficient for shiftiness in each direction at an AAG codon whose lysyl-tRNA is in short supply.
Cloning and expression of GRK5: a member of the G protein-coupled receptor kinase family.
Authors: Kunapuli P , Benovic J L
Published: May 2006

Guanine nucleotide binding protein (G-protein)-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G-protein-coupled receptors such as the beta 2-adrenergic receptor and rhodopsin. The best characterized members of this family include the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase. To identify additional members of the GRK family, the polymerase chain reaction was used to amplify human heart cDNA using degenerate oligonucleotide primers from highly conserved regions unique to the GRK family. Here we report the isolation of a cDNA that encodes a 590-amino acid protein kinase, termed GRK5, which has 34.8% and 47.2% amino acid identities with beta ARK and rhodopsin kinase, respectively. Interestingly, GRK5 has an even higher homology with Drosophila GPRK-2 (71.0% identity) and the recently identified human IT11 (69.1% identity). Northern blot analysis of GRK5 with selected human tissues reveals a message of approximately 3 kilobases with highest levels in heart, placenta, lung > skeletal muscle > brain, liver, pancreas > kidney. GRK5, overexpressed in Sf9 insect cells using the baculovirus system, was able to phosphorylate rhodopsin in a light-dependent manner. In addition, GRK5 neither contains a consensus sequence for isoprenylation like rhodopsin kinase nor is activated by G-protein beta gamma subunits like beta ARK1. Thus, GRK5 represents a member of the GRK family that likely has a unique physiological role.
Peroxidase-dependent metal-independent oxidation of low density lipoprotein in vitro: a model for in vivo oxidation?
Authors: Wieland E , Parthasarathy S , Steinberg D
Published: May 2006

Oxidative modification of low density lipoprotein is believed to be an important pathway by which the lipoprotein becomes atherogenic. The in vitro systems for oxidative modification of low density lipoprotein thus far described all appear to depend upon the presence in the medium of free transition metal ions (copper or iron). In vivo, on the other hand, these metals are present almost exclusively in tightly complexed forms that do not catalyze oxidative modification. The present studies describe oxidation of low density lipoprotein in a simple system that does not depend upon the presence of added free metal ions. It requires the presence of horseradish peroxidase and either hydrogen peroxide or lipid hydroperoxides.