bluuebunny/crossref_metadata_2025 huggingface dataset:
Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.
Authors: Su Z D , Arooz M T , Chen H M , Gross C J , Tsong T Y
Published: July 2002

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.
Aberrant platelet-derived growth factor alpha-receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis.
Authors: Mosselman S , Looijenga L H , Gillis A J , van Rooijen M A , Kraft H J , van Zoelen E J , Oosterhuis J W
Published: July 2002

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.
On the belief that arthritis pain is related to the weather.
Authors: Redelmeier D A , Tversky A
Published: July 2002

There is a widespread and strongly held belief that arthritis pain is influenced by the weather; however, scientific studies have found no consistent association. We hypothesize that this belief results, in part at least, from people's tendency to perceive patterns where none exist. We studied patients (n = 18) for more than I year and found no statistically significant associations between their arthritis pain and the weather conditions implicated by each individual. We also found that college students (n = 97) tend to perceive correlations between uncorrelated random sequences. This departure of people's intuitive notion of association from the statistical concept of association, we suggest, contributes to the belief that arthritis pain is influenced by the weather.
A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation.
Authors: Ikeda M A , Jakoi L , Nevins J R
Published: July 2002

Examination of the interactions involving transcription factor E2F activity during cell growth and terminal differentiation suggests distinct roles for Rb family members in the regulation of E2F accumulation. The major species of E2F in quiescent cells is a complex containing the E2F4 product in association with the Rb-related p130 protein. As cells enter the cell cycle, this complex disappears, and there is a concomitant accumulation of free E2F activity of which E2F4 is a major component. E2F4 then associates with the Rb-related p107 protein as cells enter S phase. Rb can be found in interactions with each E2F species, including E2F4, during G1, but there appears to be a limited amount of Rb with respect to E2F, likely due to the maintenance of most Rb protein in an inactive state by phosphorylation. A contrasting circumstance can be found during the induction of HL60 cell differentiation. As these cells exit the cell cycle, active Rb protein appears to exceed E2F, as there is a marked accumulation of E2F-Rb interactions, involving all E2F species, including E2F4, which is paralleled by the conversion of Rb from a hyperphosphorylated state to a hypophosphorylated state. These results suggest that the specific ability of Rb protein to interact with each E2F species, dependent on concentration of active Rb relative to accumulation of E2F, may be critical in cell-growth decisions.
Homing events in the gyrA gene of some mycobacteria.
Authors: Fsihi H , Vincent V , Cole S T
Published: July 2002

The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.
A mammalian mitochondrial drug receptor functions as a bacterial “oxygen” sensor
Authors: Yeliseev Alexei A. , Krueger Karl E. , Kaplan Samuel
Published: July 2002

The rat mitochondrial outer membrane-localized benzodiazepine receptor (MBR) was expressed in wild-type and TspO
−
(tryptophan-rich sensory protein) strains of the facultative photoheterotroph,
Rhodobacter sphaeroides
2.4.1, and was shown to retain its structure within the bacterial outer membrane as assayed by its binding properties with a variety of MBR ligands. Functionally, it was able to substitute for TspO by negatively regulating the expression of photosynthesis genes in response to oxygen. This effect was reversed pharmacologically with the MBR ligand PK11195. These results suggest a close evolutionary and functional relationship between the bacterial TspO and the MBR. This relationship provides further support for the origin of the mammalian mitochondrion from a “photosynthetic” precursor. Finally, these findings provide novel insights into the physiological role that has been obscure for the MBR
in situ
.
Abnormal dendritic spines in fragile X knockout mice: Maturation and pruning deficits
Authors: Comery Thomas A. , Harris Jennifer B. , Willems Patrick J. , Oostra Ben A. , Irwin Scott A. , Weiler Ivan Jeanne , Greenough William T.
Published: July 2002

Fragile X syndrome arises from blocked expression of the fragile X mental retardation protein (FMRP). Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. Here we describe dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (
Fmr1
) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice were longer than those in wild-type mice and were often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite was greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning.
Novel form of crosstalk between G protein and tyrosine kinase pathways
Authors: Diversé-Pierluissi María , Remmers Ann E. , Neubig Richard R. , Dunlap Kathleen
Published: July 2002

Neuronal Ca
2+
channels are inhibited by a variety of transmitter receptors coupled to G
o
-type GTP-binding proteins. G
o
has been postulated to work via a direct interaction between an activated G protein subunit and the Ca
2+
channel complex. Here we show that the inhibition of sensory neuron N-type Ca
2+
channels produced by γ-aminobutyric acid involves a novel, rapidly activating tyrosine kinase signaling pathway that is mediated by Gα
o
and a src-like kinase. In contrast to other recently described G protein-coupled tyrosine kinase pathways, the Gα
o
-mediated modulation requires neither protein kinase C nor intracellular Ca
2+
. The results suggest that this pathway mediates rapid receptor-G protein signaling in the nervous system and support the existence of a previously unrecognized form of crosstalk between G protein and tyrosine kinase pathways.
Femtosecond time resolution in x-ray diffraction experiments
Authors: Neutze Richard , Hajdu Janos
Published: July 2002

This paper presents the theoretical background for a synthesis of femtosecond spectroscopy and x-ray diffraction. When a diffraction quality crystal with 0.1–0.3 mm overall dimensions is photoactivated by a femtosecond laser pulse (physical length = 0.3 μm), the evolution of molecules at separated points in the crystal will not be simultaneous because a finite time is required for the laser pulse to propagate through the body of the crystal. Utilizing this lack of global crystal synchronization, topographic x-ray diffraction may enable femtosecond temporal resolution to be achieved from reflection profiles in the diffraction pattern with x-ray exposures of picosecond or longer duration. Such x-ray pulses are currently available, and could be used to study femtosecond reaction dynamics at atomic resolution on crystals of both small- and macromolecules. A general treatment of excitation and diffraction geometries in relation to spatial and temporal resolution is presented.
Proliferating cell nuclear antigen promotes DNA synthesis past template lesions by mammalian DNA polymerase δ
Authors: Mozzherin Dmitry Ju. , Shibutani Shinya , Tan Cheng-Keat , Downey Kathleen M. , Fisher Paul A.
Published: July 2002

Consistent with previous observations, proliferating cell nuclear antigen (PCNA) promotes DNA synthesis by calf thymus DNA polymerase δ (pol δ) past several chemically defined template lesions including model abasic sites, 8-oxo-deoxyguanosine (dG) and aminofluorene-dG (but not acetylaminofluorene-dG). This synthesis is potentially mutagenic. The model abasic site was studied most extensively. When all deoxyribonucleoside triphosphates and a template bearing a model abasic site were present, DNA synthesis by pol δ beyond this site was stimulated 53-fold by addition of homologous PCNA. On an unmodified template (lacking any lesions), PCNA stimulated pol δ by 1.3-fold. Product analysis demonstrated that as expected from the “A-rule,” fully and near-fully extended primers incorporated predominantly dAMP opposite the template lesion. Moreover, corollary primer extension studies demonstrated that in the presence (but not the absence) of PCNA, pol δ preferentially elongated primers containing dAMP opposite the model abasic template site. p21, a specific inhibitor of PCNA-dependent DNA replication, inhibits PCNA-stimulated synthesis past model abasic template sites. We propose that DNA synthesis past template lesions by pol δ promoted by PCNA results from the fundamental mechanism by which PCNA stimulates pol δ, i.e., stabilization of the pol δ⋅template-primer complex.
Visual stimuli induce waves of electrical activity in turtle cortex
Authors: Prechtl J. C. , Cohen L. B. , Pesaran B. , Mitra P. P. , Kleinfeld D.
Published: July 2002

The computations involved in the processing of a visual scene invariably involve the interactions among neurons throughout all of visual cortex. One hypothesis is that the timing of neuronal activity, as well as the amplitude of activity, provides a means to encode features of objects. The experimental data from studies on cat [Gray, C. M., Konig, P., Engel, A. K. & Singer, W. (1989)
Nature (London)
338, 334–337] support a view in which only synchronous (no phase lags) activity carries information about the visual scene. In contrast, theoretical studies suggest, on the one hand, the utility of multiple phases within a population of neurons as a means to encode independent visual features and, on the other hand, the likely existence of timing differences solely on the basis of network dynamics. Here we use widefield imaging in conjunction with voltage-sensitive dyes to record electrical activity from the virtually intact, unanesthetized turtle brain. Our data consist of single-trial measurements. We analyze our data in the frequency domain to isolate coherent events that lie in different frequency bands. Low frequency oscillations (<5 Hz) are seen in both ongoing activity and activity induced by visual stimuli. These oscillations propagate parallel to the afferent input. Higher frequency activity, with spectral peaks near 10 and 20 Hz, is seen solely in response to stimulation. This activity consists of plane waves and spiral-like waves, as well as more complex patterns. The plane waves have an average phase gradient of ≈π/2 radians/mm and propagate orthogonally to the low frequency waves. Our results show that large-scale differences in neuronal timing are present and persistent during visual processing.
Disruption of hippocampal development
in vivo
by CR-50 mAb against Reelin
Authors: Nakajima Kazunori , Mikoshiba Katsuhiko , Miyata Takaki , Kudo Chikako , Ogawa Masaharu
Published: July 2002

We previously generated a monoclonal alloantibody, CR-50, by immunizing
reeler
mutant mice with homogenates of normal embryonic brains. This antibody recently was shown to recognize a Reelin protein, which is coded by the recently identified candidate gene for the
reeler
mutation. However, it is still unclear whether Reelin, especially the CR-50 epitope region, is indeed responsible for the
reeler
phenotype
in vivo
. Here we show that Reelin is localized on Cajal–Retzius neurons in the hippocampus and that intraventricular injection of CR-50 at the embryonic stage disrupts the organized development of the hippocampus
in vivo
, converting it to a
reeler
pattern. Labeling experiments with 5-bromodeoxyuridine demonstrated that the labeled cells in the stratum pyramidale of the CR-50-treated mice were distributed in a pattern similar to that of
reeler
. Thus, Cajal–Retzius neurons play a crucial function in hippocampus development, and the CR-50 epitope on Reelin plays a central role in this function.
Quantitative trait loci controlling halothane sensitivity in
Caenorhabditis elegans
Authors: van Swinderen Bruno , Shook David R. , Ebert Robert H. , Cherkasova Vera A. , Johnson Thomas E. , Reis Robert J. Shmookler , Crowder C. Michael
Published: July 2002

Genetic analysis is an essential tool for defining the molecular mechanisms whereby volatile anesthetics (VA) disrupt nervous system function. However, the degree of natural variation of the genetic determinants of VA sensitivity has not been determined nor have mutagenesis approaches been very successful at isolating significantly resistant mutant strains. Thus, a quantitative genetic approach was taken toward these goals. Recombinant-inbred strains derived from two evolutionarily distinct lineages of the nematode
Caenorhabditis elegans
were tested for sensitivity to clinically relevant concentrations (0.3–0.5 mM) of the VA halothane. The halothane sensitivities of coordinated movement and male mating behavior were highly variant among the recombinant-inbred strains with a range of EC
50
values of 13- and 4-fold, respectively. Both traits were highly heritable (
H
2
= 0.82, 0.87, respectively). Several strains were found to be significantly resistant to halothane when compared with the wild-type strain N2. A major locus or loci mapping to the middle of chromosome V accounted for more than 40% of the phenotypic variance for both traits. Five weaker loci, four of which interact, explained most of the remaining variance. None of the halothane-sensitivity quantitative trait loci significantly affected behavior in the absence of halothane or halothane’s potency for
C. elegans
immobilization, which requires 5-fold higher drug concentrations. Thus, the quantitative trait loci are unlikely to result from differences in halothane-independent (native) behavior or differences in halothane metabolism or permeability. Rather, these loci may code for targets and/or downstream effectors of halothane in the
C. elegans
nervous system or for modifiers of such gene products.
CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates
Authors: Kirk Allan D. , Harlan David M. , Armstrong Nicholas N. , Davis Thomas A. , Dong Yinchen , Gray Gary S. , Hong Xuening , Thomas David , Fechner John H. , Knechtle Stuart J.
Published: July 2002

Selective inhibition of T cell costimulation using the B7-specific fusion protein CTLA4-Ig has been shown to induce long-term allograft survival in rodents. Antibodies preventing the interaction between CD40 and its T cell-based ligand CD154 (CD40L) have been shown in rodents to act synergistically with CTLA4-Ig. It has thus been hypothesized that these agents might be capable of inducing long-term acceptance of allografted tissues in primates. To test this hypothesis in a relevant preclinical model, CTLA4-Ig and the CD40L-specific monoclonal antibody 5C8 were tested in rhesus monkeys. Both agents effectively inhibited rhesus mixed lymphocyte reactions, but the combination was 100 times more effective than either drug alone. Renal allografts were transplanted into nephectomized rhesus monkeys shown to be disparate at major histocompatibility complex class I and class II loci. Control animals rejected in 5–8 days. Brief induction doses of CTLA4-Ig or 5C8 alone significantly prolonged rejection-free survival (20–98 days). Two of four animals treated with both agents experienced extended (>150 days) rejection-free allograft survival. Two animals treated with 5C8 alone and one animal treated with both 5C8 and CTLA4-Ig experienced late, biopsy-proven rejection, but a repeat course of their induction regimen successfully restored normal graft function. Neither drug affected peripheral T cell or B cell counts. There were no clinically evident side effects or rejections during treatment. We conclude that CTLA4-Ig and 5C8 can both prevent and reverse acute allograft rejection, significantly prolonging the survival of major histocompatibility complex-mismatched renal allografts in primates without the need for chronic immunosuppression.
Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: Roles for DNA and protein determinants
Authors: Wang Jonathan T. , Syed Adeela , Gralla Jay D.
Published: July 2002

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement
in vitro
. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus −12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP β–γ hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.
Impaired long-term potentiation induction in dentate gyrus of calretinin-deficient mice
Authors: Schurmans Stéphane , Schiffmann Serge N. , Gurden Hirac , Lemaire Martine , Lipp Hans-Peter , Schwam Valérie , Pochet Roland , Imperato Assunta , Böhme Georg Andrees , Parmentier Marc
Published: July 2002

Calretinin (Cr) is a Ca
2+
binding protein present in various populations of neurons distributed in the central and peripheral nervous systems. We have generated Cr-deficient (Cr
−/−
) mice by gene targeting and have investigated the associated phenotype. Cr
−/−
mice were viable, and a large number of morphological, biochemical, and behavioral parameters were found unaffected. In the normal mouse hippocampus, Cr is expressed in a widely distributed subset of GABAergic interneurons and in hilar mossy cells of the dentate gyrus. Because both types of cells are part of local pathways innervating dentate granule cells and/or pyramidal neurons, we have explored in Cr
−/−
mice the synaptic transmission between the perforant pathway and granule cells and at the Schaffer commissural input to CA1 pyramidal neurons. Cr
−/−
mice showed no alteration in basal synaptic transmission, but long-term potentiation (LTP) was impaired in the dentate gyrus. Normal LTP could be restored in the presence of the GABA
A
receptor antagonist bicuculline, suggesting that in Cr
−/−
dentate gyrus an excess of γ-aminobutyric acid (GABA) release interferes with LTP induction. Synaptic transmission and LTP were normal in CA1 area, which contains only few Cr-positive GABAergic interneurons. Cr
−/−
mice performed normally in spatial memory task. These results suggest that expression of Cr contributes to the control of synaptic plasticity in mouse dentate gyrus by indirectly regulating the activity of GABAergic interneurons, and that Cr
−/−
mice represent a useful tool to understand the role of dentate LTP in learning and memory.
A single
in vivo
administration of bombesin antagonist RC-3095 reduces the levels and mRNA expression of epidermal growth factor receptors in MXT mouse mammary cancers
Authors: Szepeshazi Karoly , Schally Andrew V. , Halmos Gabor , Lamharzi Najib , Groot Kate , Horvath Judit E.
Published: July 2002

Epidermal growth factor (EGF) and its receptors (EGFR) play important roles in tumorigenesis. In various experimental cancers, treatment with antagonists of bombesin/gastrin-releasing peptide (BN/GRP) produces a reduction in EGFRs, concomitant to inhibition of tumor growth. To investigate the mechanisms involved, we monitored concentrations of BN/GRP antagonist RC-3095 in serum of mice, rats, and hamsters given a single subcutaneous or intravenous injection of this analog. In parallel studies, we measured levels and mRNA expression of EGFRs in estrogen-dependent and independent MXT mouse mammary cancers, following a single subcutaneous administration of RC-3095 to tumor-bearing mice. Peak values of RC-3095 in serum were detected 2 min after intravenous or 15 min after subcutaneous injection. The levels of RC-3095 declined rapidly and became undetectable after 3–5 hr. In the estrogen-dependent MXT tumors, the concentration of EGF receptors was reduced by about 60% 6 hr following injection and returned to original level after 24 hr. Levels of mRNA for EGFR fell parallel with the receptor number and were nearly normal after 24 hr. In the hormone-independent MXT cancers, the number of EGFRs decreased progressively, becoming undetectable 6 hr after injection of RC-3095, and returned to normal values at 24 hr, but EGFR mRNA levels remained lower for 48 hr. Thus, in spite of rapid elimination from serum, BN/GRP antagonist RC-3095 can induce a prolonged decrease in levels and mRNA expression of EGFRs. These findings may explain how single daily injections of BN/GRP antagonists can maintain tumor growth inhibition.
A yeast sterol auxotroph (
erg
25) is rescued by addition of azole antifungals and reduced levels of heme
Authors: Gachotte D. , Pierson C. A. , Lees N. D. , Barbuch R. , Koegel C. , Bard M.
Published: July 2002

Genetic disruption of the
Saccharomyces cerevisiae
C-4 sterol methyl oxidase
ERG25
gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is
erg11
, which is blocked in 14α-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either
slu1
or
slu2
(suppressor of lanosterol utilization). These mutations are leaky versions of
HEM2
and
HEM4
, respectively; addition of exogenous hemin reverses the suppressing effects of
slu1
and
slu2
. Suppression of
erg25
by
erg11 slu1
(or
erg11 slu2
) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (
ERG6
) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the
ERG11
gene product, the 14α-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs
erg25 slu1
or
erg25 slu2
with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.
D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain
Authors: Huang Lily Jun-shen , Durick Kyle , Weiner Joshua A. , Chun Jerold , Taylor Susan S.
Published: July 2002

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333–372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of R
I
α and R
II
α. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Gα protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.
Glyceraldehyde-3-phosphate dehydrogenase: Nuclear translocation participates in neuronal and nonneuronal cell death
Authors: Sawa Akira , Khan Adil A. , Hester Lynda D. , Snyder Solomon H.
Published: July 2002

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily reflects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism.
Prevention of development of autoimmune disease in BXSB mice by mixed bone marrow transplantation
Authors: Wang Bing-Yan , El-Badri Cherry, Nagwa S. , Good Robert A.
Published: July 2002

Transplantations of fully allogeneic, autoimmune-resistant T-cell-depleted marrow (TCDM) plus syngeneic, autoimmune-prone TCDM into lethally irradiated BXSB mice were carried out to investigate the ability of the mixed bone marrow transplantation (BMT) to prevent development of autoimmune disease and, at the same time, to reconstitute fully the immunity functions of heavily irradiated BXSB recipients. Male BXSB mice were engrafted with mixed TCDM from both allogeneic, autoimmune-resistant BALB/c mice and syngeneic, autoimmune-prone BXSB mice. BMT with mixed TCDM from both resistant and susceptible strains of mice (mixed BMT) prolonged the median life span and inhibited development of glomerulonephritis in BXSB mice. BMT with mixed TCDM also prevented the formation of anti-DNA antibodies that is typically observed in male mice of this strain. Moreover, mixed BMT reconstituted primary antibody production in BXSB recipients, so that no annoying immunodeficiencies that are regularly observed in fully allogeneic chimeras were present in the recipient of the mixed TCDM. These findings indicate that transplanting allogeneic, autoimmune-resistant TCDM plus syngeneic, autoimmune-prone TCDM into lethally irradiated BXSB mice prevents development of autoimmune disease in this strain of mice. In addition, this dual BMT reconstitutes the immunity functions and avoids the immunodeficiencies that occur regularly in fully allogeneic chimeras after total-body irradiation.
Restoration of β-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer
Authors: Akhter Shahab A. , Skaer Christine A. , Kypson Alan P. , McDonald Patricia H. , Peppel Karsten C. , Glower Donald D. , Lefkowitz Robert J. , Koch Walter J.
Published: July 2002

Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β-adrenergic signaling defects including down-regulation of myocardial β-adrenergic receptors (β-ARs), functional β-AR uncoupling, and an up-regulation of the β-AR kinase (βARK1). Adenoviral-mediated gene transfer of the human β
2
-AR or an inhibitor of βARK1 to these failing myocytes led to the restoration of β-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected
in vitro
using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of βARK1 activity in the heart.
Coordinate regulation of lipogenic gene expression by androgens: Evidence for a cascade mechanism involving sterol regulatory element binding proteins
Authors: Swinnen Johannes V. , Ulrix William , Heyns Walter , Verhoeven Guido
Published: July 2002

To gain more insight into the molecular mechanisms by which androgens stimulate lipogenesis and induce a marked accumulation of neutral lipids in the human prostate cancer cell line LNCaP, we studied their impact on the expression of lipogenic enzymes. Northern blot analysis of the steady-state mRNA levels of seven different lipogenic enzymes revealed that androgens coordinately stimulate the expression of enzymes belonging to the two major lipogenic pathways: fatty acid synthesis and cholesterol synthesis. In view of the important role of the recently characterized sterol regulatory element binding proteins (SREBPs) in the coordinate induction of lipogenic genes, we examined whether the observed effects of androgens on lipogenic gene expression are mediated by these transcription factors. Our findings indicate that androgens stimulate the expression of SREBP transcripts and precursor proteins and enhance the nuclear content of the mature active form of the transcription factor. Moreover, by using the fatty acid synthase gene as an experimental paradigm we demonstrate that the presence of an SREBP-binding site is essential for its regulation by androgens. These data support the hypothesis that SREBPs are involved in the coordinate regulation of lipogenic gene expression by androgens and provide evidence for the existence of a cascade mechanism of androgen-regulated gene expression.
Btk dosage determines sensitivity to B cell antigen receptor cross-linking
Authors: Satterthwaite Anne B. , Cheroutre Hilde , Khan Wasif N. , Sideras Pascalis , Witte Owen N.
Published: July 2002

Mutations in Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Btk is a critical component of signaling pathways regulating B cell development and function. We used a genetic approach to determine whether Btk is also limiting for these processes. One allele of a murine Btk transgene expressed a dosage of Btk (25% of endogenous levels in splenic B cells) sufficient to restore normal numbers of phenotypically mature conventional B cells in xid mice. 2,4,6-trinitrophenyl–Ficoll response, anti-IgM-induced proliferation, B1 cell development, and serum IgM and IgG3levels remained significantly impaired in these animals. B cells from Btk −/− transgenic mice also responded poorly to anti-IgM, indicating that the xid mutation does not create a dominant negative form of Btk. Response to 2,4,6-trinitrophenyl–Ficoll and B cell receptor cross-linking were increased 3- to 4-fold in xid mice homozygous for the transgene. These results demonstrate that Btk is a limiting component of B cell antigen receptor signaling pathways and suggest that B cell development and response to antigen may require different levels of Btk activity.
Expression cloning of cDNA encoding a human β-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis
Authors: Sasaki Katsutoshi , Kurata-Miura Kazumi , Ujita Minoru , Angata Kiyohiko , Nakagawa Satoshi , Sekine Susumu , Nishi Tatsunari , Fukuda Minoru
Published: July 2002

The structure and biosynthesis of poly-N-acetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Lex. Herein we describe the isolation and functional expression of a cDNA encoding β-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-N-acetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by fluorescence-activated cell sorting because introduced plasmids containing Epstein–Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAcβ1 → 3Galβ1 → 4GlcNAc → R structure and poly-N-acetyllactosamine extension.
Characterization of the permeability barrier of human skin
in vivo
Authors: Pirot Fabrice , Kalia Yogeshvar N. , Stinchcomb Audra L. , Keating Garrett , Bunge Annette , Guy Richard H.
Published: July 2002

Attenuated-total-reflectance Fourier-transform-infrared
spectroscopy has been used to rapidly and noninvasively quantify
in vivo
the uptake of a chemical into the outermost, and
least permeable, layer of human skin (the stratum corneum). The
objective of the experiment was to develop a general model to predict
the rate and extent of chemical absorption for diverse exposure
scenarios from simple, and safe, short-duration studies. Measurement of
the concentration profile of the chemical in the stratum corneum, and
analysis of the data using the unsteady-state diffusion equation,
enabled estimation of the permeability coefficient and calculation of
the time required to achieve maximal transdermal flux. Validation of
the spectroscopic technique employed was established, and quantitation
of chemical uptake into the stratum corneum was confirmed independently
using trace amounts of radiolabeled chemical in conjunction with liquid
scintillation counting and accelerator mass spectrometry. The results
presented have pharmacological and toxicological implications, as the
technology lends itself both to the prediction of transdermal drug
delivery, and the feasibility of this route of administration, and to
the assessment of risk after dermal contact with toxic chemicals.
Cloning of cDNA and estrogen-induced hepatic gene expression for choriogenin H, a precursor protein of the fish egg envelope (chorion)
Authors: Murata Kenji , Sugiyama Hitoshi , Yasumasu Shigeki , Iuchi Ichiro , Yasumasu Ikuo , Yamagami Kenjiro
Published: July 2002

A cDNA for choriogenin H (Chg H; formerly high-molecular weight spawning female-specific substances, or H-SF), a precursor protein of the inner layer subunits of egg envelope (chorion) of the teleost fish,
Oryzias latipes
, was cloned and analyzed. The clone consisted of 1913 bp and contained an open reading frame encoding a signal peptide of 22 aa and Chg H protein of 569 aa. The Chg protein possessed three potential N-glycosylation sites and Pro-X-Y repeat sequences in the first two-fifths of the N terminus. There were amino acid sequence similarities between Chg H and a gene product expressed in the liver of female winter flounder during vitellogenesis. Moreover, the amino acid sequence of Chg H is similar to that of ZP2 rather than ZP3 of zona pellucida of some mammals. Northern blot analysis indicated that gene expression for Chg H occurred only in the livers of spawning female fish and 17β-estradiol-treated male fish, but not in the ovary of the spawning female fish. Gene expression for Chg H and Chg L (formerly low-molecular weight spawning female-specific substance, or L-SF) was induced and increased in parallel in the male fish liver after 17β-estradiol treatment.
Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long-term gene expression
Authors: Ilan Yaron , Droguett Gustavo , Chowdhury Namita Roy , Li Yongan , Sengupta Krishna , Thummala Narsing R. , Davidson Anne , Chowdhury Jayanta Roy , Horwitz Marshall S.
Published: July 2002

Recombinant adenoviruses (Ads) are highly efficient at transferring foreign genes to the liverin vivo; however, the duration of gene expression is limited by the host antiviral immune response, which prevents expression upon readministration of the virus. To test whether overexpression of the immunomodulatory products of the early Ad genome region 3 (E3) could prevent the antiviral immune response and prolong expression of foreign genes delivered by Ad vectors, we injected a recombinant Ad (Ad-E3-hBUGT), containing both E3 and the human bilirubin-uridine-diphosphoglucuronate-glucuronosyltransferase (BUGT) genes, into BUGT-deficient hyperbilirubinemic Gunn rats. Control Gunn rats received Ad-hBUGT, which expresses human BUGT alone. An initial injection of either virus resulted in hepatic expression of human BUGT as evidenced by excretion of bilirubin glucuronides in bile and a reduction of mean serum bilirubin levels from 7.0 mg/dl to 1.9–2.7 mg/dl within 7 days. In Ad-E3-hBUGT-injected rats, serum bilirubin levels increased to 4.5 mg/dl by 84 days after infection, but a second administration of the virus on that day resulted in a hypobilirubinemic response similar to that seen with the first injection. In contrast, rats receiving Ad-hBUGT had serum bilirubin levels of 7 mg/dl on day 84 after infection, but showed no reduction of serum bilirubin by reinjection of the virus on that day. In the rats injected with Ad-E3-hBUGT, but not in the ones injected with Ad-hBUGT, there was a marked inhibition of the antiviral antibody and Ad-specific cytotoxic T lymphocyte responses. This is the first demonstration that insertion of E3 genes in recombinant Ads facilitates readministration of a functional vector for long-term correction of an inherited metabolic disorder.
recF
and
recR
are required for the resumption of replication at DNA replication forks in
Escherichia coli
Authors: Courcelle Justin , Carswell-Crumpton Catherine , Hanawalt Philip C.
Published: July 2002

Escherichia coli
containing a mutation in
recF
are hypersensitive to UV. However, they exhibit normal levels of conjugational or transductional recombination unless the major pathway (
recBC
) is defective. This implies that the UV sensitivity of
recF
mutants is not due to a defect in recombination such as occurs during conjugation or transduction. Here, we show that when replication is disrupted, at least two genes in the
recF
pathway,
recF
and
recR
, are required for the resumption of replication at DNA replication forks, and that in their absence, localized degradation occurs at the replication forks. Our observations support a model in which
recF
and
recR
are required to reassemble a replication holoenzyme at the site of a DNA replication fork. These results, when taken together with previous literature, suggest that the UV hypersensitivity of
recF
cells is due to an inability to resume replication at disrupted replication forks rather than to a defect in recombination. Current biochemical and genetic data on the conditions under which
recF
-mediated recombination occurs suggest that the recombinational intermediate also may mimic the structure of a disrupted replication fork.
Unique features in the structure of the complex between HIV-1 reverse transcriptase and the bis(heteroaryl)piperazine (BHAP) U-90152 explain resistance mutations for this nonnucleoside inhibitor
Authors: Esnouf Robert M. , Ren Jingshan , Hopkins Andrew L. , Ross Carl K. , Jones E. Yvonne , Stammers David K. , Stuart David I.
Published: July 2002

The viral reverse transcriptase (RT) provides an attractive target in the search for anti-HIV therapies. The nonnucleoside inhibitors (NNIs) are a diverse set of compounds (usually HIV-1 specific) that function by distorting the polymerase active site upon binding in a nearby pocket. Despite being potent and of generally low toxicity, their clinical use has been limited by rapid selection for resistant viral populations. The 2.65-Å resolution structure of the complex between HIV-1 RT and the bis(heteroaryl)piperazine (BHAP) NNI, 1-(5-methanesulfonamido-1
H
-indol-2-yl-carbonyl)-4-[3-(1-methyl-ethylamino)pyridinyl] piperazine (U-90152), reveals the inhibitor conformation and bound water molecules. The bulky U-90152 molecule occupies the same pocket as other NNIs, but the complex is stabilized quite differently, in particular by hydrogen bonding to the main chain of Lys-103 and extensive hydrophobic contacts with Pro-236. These interactions rationalize observed resistance mutations, notably Pro-236-Leu, which occurs characteristically for BHAPs. When bound, part of U-90152 protrudes into the solvent creating a channel between Pro-236 and the polypeptide segments 225–226 and 105–106, giving the first clear evidence of the entry mode for NNIs. The structure allows prediction of binding modes for related inhibitors [(altrylamino)piperidine–BHAPs] and suggests changes to U-90152, such as the addition of a 6 amino group to the pyridine ring, which may make binding more resilient to mutations in the RT. The observation of novel hydrogen bonding to the protein main chain may provide lessons for the improvement of quite different inhibitors.