bluuebunny/crossref_metadata_2025 huggingface dataset:
The endoribonucleolytic N-terminal half of
Escherichia coli
RNase E is evolutionarily conserved in
Synechocystis
sp. and other bacteria but not the C-terminal half, which is sufficient for degradosome assembly
Authors: Kaberdin Vladimir R. , Miczak Andras , Jakobsen Jimmy S. , Lin-Chao Sue , McDowall Kenneth J. , von Gabain Alexander
Published: July 2002

Escherichia coli
RNase E, an essential single-stranded specific endoribonuclease, is required for both ribosomal RNA processing and the rapid degradation of mRNA. The availability of the complete sequences of a number of bacterial genomes prompted us to assess the evolutionarily conservation of bacterial RNase E. We show here that the sequence of the N-terminal endoribonucleolytic domain of RNase E is evolutionarily conserved in
Synechocystis
sp. and other bacteria. Furthermore, we demonstrate that the
Synechocystis
sp. homologue binds RNase E substrates and cleaves them at the same position as the
E. coli
enzyme. Taken together these results suggest that RNase E-mediated mechanisms of RNA decay are not confined to
E. coli
and its close relatives. We also show that the C-terminal half of
E. coli
RNase E is both sufficient and necessary for its physical interaction with the 3′–5′ exoribonuclease polynucleotide phosphorylase, the RhlB helicase, and the glycolytic enzyme enolase, which are components of a “degradosome” complex. Interestingly, however, the sequence of the C-terminal half of
E. coli
RNase E is not highly conserved evolutionarily, suggesting diversity of RNase E interactions with other RNA decay components in different organisms. This notion is supported by our finding that the
Synechocystis
sp. RNase E homologue does not function as a platform for assembly of
E. coli
degradosome components.
Genetic relationship of populations in China
Authors: Chu J. Y. , Huang W. , Kuang S. Q. , Wang J. M. , Xu J. J. , Chu Z. T. , Yang Z. Q. , Lin K. Q. , Li P. , Wu M. , Geng Z. C. , Tan C. C. , Du R. F. , Jin L.
Published: July 2002

Despite the fact that the continuity of morphology of fossil specimens of modern humans found in China has repeatedly challenged the Out-of-Africa hypothesis, Chinese populations are underrepresented in genetic studies. Genetic profiles of 28 populations sampled in China supported the distinction between southern and northern populations, while the latter are biphyletic. Linguistic boundaries are often transgressed across language families studied, reflecting substantial gene flow between populations. Nevertheless, genetic evidence does not support an independent origin of
Homo sapiens
in China. The phylogeny also suggested that it is more likely that ancestors of the populations currently residing in East Asia entered from Southeast Asia.
Apoptosis regulator Bcl-w is essential for spermatogenesis but appears otherwise redundant
Authors: Print Cristin G. , Loveland Kate L. , Gibson Leonie , Meehan Terri , Stylianou Anna , Wreford Nigel , de Kretser David , Metcalf Donald , Köntgen Frank , Adams Jerry M. , Cory Suzanne
Published: July 2002

Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolatedbcl-wgene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, we have inactivated thebcl-wgene in the mouse by homologous recombination. Mice that lack Bcl-w were viable, healthy, and normal in appearance. Most tissues exhibited typical histology, and hematopoiesis was unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function was normal, the males were infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis was largely unaffected. The seminiferous tubules of adult males, however, were disorganized, contained numerous apoptotic cells, and produced no mature sperm. Both Sertoli cells and germ cells of all types were reduced in number, the most mature germ cells being the most severely depleted. Thebcl-w−/−mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.
Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice
Authors: Eilam Raya , Peter Yakov , Elson Ari , Rotman Galit , Shiloh Yosef , Groner Yoram , Segal Menahem
Published: July 2002

Ataxia-telangiectasia (AT) is a human disease caused by mutations in the
ATM
gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction. Surprisingly, mice in which the
Atm
gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease. To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum. A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor
l
-dopa. In addition, these mice were hypersensitive to the dopamine releasing drug
d
-amphetamine. These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.
Direct molecular level measurements of the electrostatic properties of a protein surface
Authors: Sivasankar S. , Subramaniam S. , Leckband D.
Published: July 2002

In this work, we used direct measurements with the surface force apparatus to determine the pH-dependent electrostatic charge density of a single binding face of streptavidin. Mean field calculations have been used with considerable success to model electrostatic potential fields near protein surfaces, but these models and their inherent assumptions have not been tested directly at the molecular level. Using the force apparatus and immobilized, oriented monolayers of streptavidin, we measured a pI of 5–5.5 for the biotin-binding face of the protein. This differs from the pI of 6.3 for the soluble protein and confirms that we probed the local electrostatic features of the macromolecule. With finite difference solutions of the linearized Poisson–Boltzmann equation, we then calculated the pH-dependent charge densities adjacent to the same face of the protein. These calculated values agreed quantitatively with those obtained by direct force measurements. Although our study focuses on the pH-dependence of surface electrostatics, this direct approach to probing the electrostatic features of proteins is applicable to investigations of any perturbations that alter the charge distribution of the surfaces of immobilized molecules.
Ionizing radiation and short wavelength UV activate NF-κB through two distinct mechanisms
Authors: Li Nanxin , Karin Michael
Published: July 2002

We examined the mechanisms by which two different types of photonic radiation, short wavelength UV (UV-C) and γ radiation, activate transcription factor NF-κB. Exposure of mammalian cells to either form of radiation resulted in induction with similar kinetics of NF-κB DNA binding activity, nuclear translocation of its p65(RelA) subunit, and degradation of the major NF-κB inhibitor IκBα. In both cases, induction of NF-κB activity was attenuated by proteasome inhibitors and a mutation in ubiquitin-activating enzyme, suggesting that both UV-C and γ radiation induce degradation of IκBs by means of the ubiquitin/proteasome pathway. However, although the induction of IκBα degradation by γ rays was dependent on its phosphorylation at Ser-32 and Ser-36, UV-C-induced IκBα degradation was not dependent on phosphorylation of these residues. Even the “super repressor” IκBα mutant, which contains alanines at positions 32 and 36, was still susceptible to UV-C-induced degradation. Correspondingly, we found that γ radiation led to activation of IKK, the protein kinase that phosphorylates IκBα at Ser-32 and Ser-36, whereas UV-C radiation did not. Furthermore, expression of a catalytically inactive IKKβ mutant prevented NF-κB activation by γ radiation, but not by UV-C. These results indicate that γ radiation and UV-C activate NF-κB through two distinct mechanisms.
Nitrosative stress: Metabolic pathway involving the flavohemoglobin
Authors: Hausladen Alfred , Gow Andrew J. , Stamler Jonathan S.
Published: July 2002

Nitric oxide (NO) biology has focused on the tightly regulated enzymatic mechanism that transforms
l
-arginine into a family of molecules, which serve both signaling and defense functions. However, very little is known of the pathways that metabolize these molecules or turn off the signals. The paradigm is well exemplified in bacteria where
S
-nitrosothiols (SNO)—compounds identified with antimicrobial activities of NO synthase—elicit responses that mediate bacterial resistance by unknown mechanisms. Here we show that
Escherichia coli
possess both constitutive and inducible elements for SNO metabolism. Constitutive enzyme(s) cleave SNO to NO whereas bacterial hemoglobin, a widely distributed flavohemoglobin of poorly understood function, is central to the inducible response. Remarkably, the protein has evolved a novel heme-detoxification mechanism for NO. Specifically, the heme serves a dioxygenase function that produces mainly nitrate. These studies thus provide new insights into SNO and NO metabolism and identify enzymes with reactions that were thought to occur only by chemical means. Our results also emphasize that the reactions of SNO and NO with hemoglobins are evolutionary conserved, but have been adapted for cell-specific function.
Physical interaction between components of DNA mismatch repair and nucleotide excision repair
Authors: Bertrand Pascale , Tishkoff Daniel X. , Filosi Nicole , Dasgupta Ruchira , Kolodner Richard D.
Published: July 2002

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown.
Saccharomyces cerevisiae
RAD14 was identified in a two-hybrid screen using MSH2 as “bait,” and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in
msh3 msh6
and
mlh1 pms1
double mutants, suggesting a direct interaction with MSH2. Mutations in
MSH2
increased the UV sensitivity of NER-deficient yeast strains, and
msh2
mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.
Reciprocal interactions between β1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology
Authors: Wang Fei , Weaver Valerie M. , Petersen Ole W. , Larabell Carolyn A. , Dedhar Shoukat , Briand Per , Lupu Ruth , Bissell Mina J.
Published: July 2002

Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of β1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4–2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a three-dimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of β1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogen-activated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant down-regulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Cross-modulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and β1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of β1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be “normalized” by manipulating either pathway.
Phenotypic switching in the human pathogenic fungusCryptococcus neoformansis associated with changes in virulence and pulmonary inflammatory response in rodents
Authors: Goldman David L. , Fries Bettina C. , Franzot Sarah P. , Montella Lya , Casadevall Arturo
Published: July 2002

High-frequency reversible changes in colony morphology were observed in three strains ofCryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as 10−3. Mice infected with colony type W died faster than those infected with other colony types. The rat inflammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inflammation with caseous necrosis for infection with type C to minimal inflammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains ofC. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate thatC. neoformansundergoes phenotypic switching and that this process can affect virulence and host inflammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.
Opening-up of liposomal membranes by talin
Authors: Saitoh Akihiko , Takiguchi Kingo , Tanaka Yohko , Hotani Hirokazu
Published: July 2002

Morphological changes of liposomes caused by interactions between liposomal membranes and talin, a cytoskeletal submembranous protein, were studied by direct, real-time observation by using high-intensity dark-field microscopy. Surprisingly, when talin was added to a liposome solution, liposomes opened stable holes and were transformed into cup-shaped liposomes. The holes became larger with increasing talin concentration, and finally the cup-shaped liposomes were transformed into lipid bilayer sheets. These morphological changes were reversed by protein dilution, i.e., the sheets could be transformed back into closed spherical liposomes. We demonstrated that talin was localized mainly along the membrane verges, presumably avoiding exposure of its hydrophobic portion at the edge of the lipid bilayer. This is the first demonstration that a lipid bilayer can stably maintain a free verge in aqueous solution. This finding refutes the established dogma that all lipid bilayer membranes inevitably form closed vesicles and suggests that talin is a useful tool for manipulating liposomes.
Homology and functional similarity of an
hrp
-linked pathogenicity locus,
dspEF
, of
Erwinia amylovora
and the avirulence locus
avrE
of
Pseudomonas syringae
pathovar tomato
Authors: Bogdanove Adam J. , Kim Jihyun F. , Wei Zhongmin , Kolchinsky Peter , Charkowski Amy O. , Conlin Alison K. , Collmer Alan , Beer Steven V.
Published: July 2002

The “disease-specific” (
dsp
) region next to the
hrp
gene cluster of
Erwinia amylovora
is required for pathogenicity but not for elicitation of the hypersensitive reaction. A 6.6-kb apparent operon,
dspEF
, was found responsible for this phenotype. The operon contains genes
dspE
and
dspF
and is positively regulated by
hrpL
. A
blast
search revealed similarity in the
dspE
gene to a partial sequence of the
avrE
locus of
Pseudomonas syringae
pathovar tomato. The entire
avrE
locus was sequenced. Homologs of
dspE
and
dspF
were found in juxtaposed operons and were designated
avrE
and
avrF
. Introduced on a plasmid, the
dspEF
locus rendered
P. syringae
pv. glycinea race 4 avirulent on soybean. An
E. amylovora dspE
mutant, however, elicited a hypersensitive reaction in soybean. The
avrE
locus
in trans
restored pathogenicity to
dspE
strains of
E. amylovora
, although restored strains were low in virulence. DspE and AvrE are large (198 kDa and 195 kDa) and hydrophilic. DspF and AvrF are small (16 kDa and 14 kDa) and acidic with predicted amphipathic α helices in their C termini; they resemble chaperones for virulence factors secreted by type III secretion systems of animal pathogens.
Functional analysis of primary visual cortex (V1) in humans
Authors: Tootell Roger B. H. , Hadjikhani Nouchine K. , Vanduffel Wim , Liu Arthur K. , Mendola Janine D. , Sereno Martin I. , Dale Anders M.
Published: July 2002

Human area V1 offers an excellent opportunity to study, using
functional MRI, a range of properties in a specific cortical visual
area, whose borders are defined objectively and convergently by
retinotopic criteria. The retinotopy in V1 (also known as primary
visual cortex, striate cortex, or Brodmann’s area 17) was defined in
each subject by using both stationary and phase-encoded polar
coordinate stimuli. Data from V1 and neighboring retinotopic areas were
displayed on flattened cortical maps. In additional tests we revealed
the paired cortical representations of the monocular “blind
spot.” We also activated area V1 preferentially (relative to other
extrastriate areas) by presenting radial gratings alternating between
6% and 100% contrast. Finally, we showed evidence for orientation
selectivity in V1 by measuring transient functional MRI increases
produced at the change in response to gratings of differing
orientations. By systematically varying the orientations presented, we
were able to measure the bandwidth of the orientation
“transients” (45°).
Dynamics of surfactant release in alveolar type II cells
Authors: Haller Thomas , Ortmayr Jörg , Friedrich Franz , Völkl Harald , Dietl Paul
Published: July 2002

Pulmonary surfactant, secreted via exocytosis of lamellar bodies (LB) by alveolar type II (AT II) cells, maintains low alveolar surface tension and is therefore essential for normal lung function. Here we describe real-time monitoring of exocytotic activity in these cells by visualizing and quantifying LB fusion with the plasma membrane (PM). Two approaches were used. First, fluorescence of LysoTracker Green DND-26 (LTG) in LB disappeared when the dye was released after exocytosis. Second, phospholipid staining by FM 1–43 resulted in bright fluorescence when this dye entered the LB through the fusion pore. Both processes were restricted to and colocalized with LB and occurred simultaneously. In AT II cells, FM 1–43 offered the unique advantage to independently define the moment and cellular location of single exocytotic events as well as the amount of material released, and to monitor its extracellular fate. Furthermore, both dyes could be used in combination with fura-2. The results indicate considerable diversity in the dynamics of LB exocytosis. In the majority of cells stimulated with ATP and isoproterenol, the first fusion of LB coincided with the rise of [Ca
2+
]
i
, but subsequent response of other LB in the same cell considerably outlasted this signal. In other cells, however, the onset of exocytosis was delayed by several minutes. After LB fusion, release of surfactant from LB into an aqueous solution was slow. In summary, stimulated exocytosis in AT II cells occurs at a much slower rate than in most other secretory cells but is still a more dynamic process than predicted from conventional measurements of surfactant released into cell supernatants.
Synthesis and biological evaluation of cytotoxic analogs of somatostatin containing doxorubicin or its intensely potent derivative, 2-pyrrolinodoxorubicin
Authors: Nagy Attila , Schally Andrew V. , Halmos Gábor , Armatis Patricia , Cai Ren-Zhi , Csernus Valér , Kovács Magdolna , Koppán Miklós , Szepesházi Károly , Kahán Zsuzsanna
Published: July 2002

To create cytotoxic hybrid analogs of somatostatin (SST), octapeptides RC-160 (
d
-Phe-C
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s-Trp-NH
2
) and RC-121 (
d
-Phe-C
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s-Thr-NH
2
) were linked to doxorubicin (DOX) or its superactive derivative, 2-pyrrolino-DOX (AN-201). The conjugation was performed by coupling
N
-9-fluorenylmethoxycarbonyl (
N
-Fmoc)-DOX-14-
O
-hemiglutarate or 2-pyrrolino-DOX-14-
O
-hemiglutarate to the amino terminus of [Lys(Fmoc)
5
]RC-160 yielding AN-163 and AN-258, respectively, after deprotection. The respective cytotoxic conjugates of RC-121 (AN-162 and AN-238) were prepared similarly.
In vitro
tests on human cancer cell lines—MKN-45 gastric cancer, MDA-MB-231 breast cancer, PC-3 prostate cancer, and MIA PaCa-2 pancreatic cancer—demonstrated that the antiproliferative activity of the cytotoxic radicals in these conjugates was virtually retained. In H-345 human small cell lung carcinoma cell line, conjugates of RC-121 preserved the cytotoxic activity of their radicals, but the hybrids with RC-160 showed ≈10 times lower activity. The ability of the carriers and the hybrids to inhibit the binding of
125
I-labeled RC-160 to receptors for SST on rat pituitary membrane preparation was also determined. The cytotoxic conjugates inhibited 50% of the specific binding of the radioligand in the nanomolar concentration range (IC
50
< 80 nM). When SST-like activities of AN-238 and its carrier, RC-121, were compared in the rat pituitary superfusion system, both compounds were found to suppress a stimulated growth hormone release at nanomolar concentrations. Preliminary studies in animal models of breast and prostate cancers showed that AN-238 is less toxic than AN-201 and more potent in inhibiting tumor growth. These highly active cytotoxic analogs of SST have been designed as targeted antitumor agents for the treatment of various cancers expressing receptors for SST octapeptides.
A splicing variant of a death domain protein that is regulated by a mitogen-activated kinase is a substrate for c-Jun N-terminal kinase in the human central nervous system
Authors: Zhang Yan , Zhou Li , Miller Carol A.
Published: July 2002

The mitogen-activated kinase activating death domain protein (MADD) that is differentially expressed in neoplastic vs. normal cells (DENN) was identified as a substrate for c-Jun N-terminal kinase 3, the first demonstration of such an activity for this stress-activated kinase that is predominantly expressed in the brain. A splice isoform was identified that is a variant of MADD. A protein identical to MADD has been reported to be expressed differentially in neoplastic vs. normal cells and is termed “DENN.” We demonstrated differential effects on DENN/MADD in a stressed vs. basal environment. Using
in situ
hybridization, we localized both the substrate and the kinase to large pyramidal neurons in the human hippocampus. It was interesting that, in four of four patients with neuropathologically confirmed acute hypoxic changes, we detected a unique translocation of DENN/MADD to the nucleolus. These changes were apparent only in neurons sensitive to hypoxia. Moreover, in those cells, translocation of the substrate was accompanied by nuclear translocation of JNK3. These findings place DENN/MADD and JNK in important hypoxia insult-induced intracellular signaling pathways. Our conclusions are important for future studies for understanding these stress-activated mechanisms.
Self-consistently optimized energy functions for protein structure prediction by molecular dynamics
Authors: Koretke Kristin K. , Luthey-Schulten Zaida , Wolynes Peter G.
Published: July 2002

The protein energy landscape theory is used to obtain optimal energy functions for protein structure prediction via simulated annealing. The analysis here takes advantage of a more complete statistical characterization of the protein energy landscape and thereby improves on previous approximations. This schema partially takes into account correlations in the energy landscape. It also incorporates the relationships between folding dynamics and characteristic energy scales that control the collapse of the proteins and modulate rigidity of short-range interactions. Simulated annealing for the optimal energy functions, which are associative memory hamiltonians using a database of folding patterns, generally leads to quantitatively correct structures. In some cases the algorithm achieves “creativity,” i.e., structures result that are better than any homolog in the database.
Calbindin D28k blocks the proapoptotic actions of mutant presenilin 1: Reduced oxidative stress and preserved mitochondrial function
Authors: Guo Qing , Christakos Sylvia , Robinson Nic , Mattson Mark P.
Published: July 2002

Mutations in the presenilin 1 (PS-1) gene account for many cases of early-onset autosomal dominant inherited forms of Alzheimer’s disease. Recent findings suggest that PS-1 mutations may sensitize neurons to apoptosis induced by trophic factor withdrawal and exposure to amyloid β-peptide (Aβ). We now report that overexpression of the calcium-binding protein calbindin D28k prevents apoptosis in cultured neural cells expressing mutant PS-1 (L286V and M146V missense mutations). Elevations of the intracellular Ca
2+
concentration and generation of reactive oxygen species induced by Aβ, and potentiated by mutant PS-1, were suppressed in calbindin-overexpressing cells. Impairment of mitochondrial function by Aβ (which preceded apoptosis) was exacerbated by PS-1 mutations and was largely prevented by calbindin. These findings suggest that PS-1 mutations render neurons vulnerable to apoptosis by a mechanism involving destabilization of cellular calcium homeostasis, which leads to oxidative stress and mitochondrial dysfunction.
Sequence-specific polypeptoids: A diverse family of heteropolymers with stable secondary structure
Authors: Kirshenbaum Kent , Barron Annelise E. , Goldsmith Richard A. , Armand Philippe , Bradley Erin K. , Truong Kiet T. V. , Dill Ken A. , Cohen Fred E. , Zuckermann Ronald N.
Published: July 2002

We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide α-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the α-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions.
Cardiac arrest in rodents: Maximal duration compatible with a recovery of neuronal activity
Authors: Charpak S. , Audinat E.
Published: July 2002

We report here that during a permanent cardiac arrest, rodent brain tissue is “physiologically” preserved
in situ
in a particular quiescent state. This state is characterized by the absence of electrical activity and by a critical period of 5–6 hr during which brain tissue can be reactivated upon restoration of a simple energy (glucose/oxygen) supply. In rat brain slices prepared 1–6 hr after cardiac arrest and maintained
in vitro
for several hours, cells with normal morphological features, intrinsic membrane properties, and spontaneous synaptic activity were recorded from various brain regions. In addition to functional membrane channels, these neurons expressed mRNA, as revealed by single-cell reverse transcription–PCR, and could synthesize proteins
de novo
. Slices prepared after longer delays did not recover. In a guinea pig isolated whole-brain preparation that was cannulated and perfused with oxygenated saline 1–2 hr after cardiac arrest, cell activity and functional long-range synaptic connections could be restored although the electroencephalogram remained isoelectric. Perfusion of the isolated brain with the γ-aminobutyric acid A receptor antagonist picrotoxin, however, could induce self-sustained temporal lobe epilepsy. Thus, in rodents, the duration of cardiac arrest compatible with a short-term recovery of neuronal activity is much longer than previously expected. The analysis of the parameters that regulate this duration may bring new insights into the prevention of postischemic damages.
A primitive pathway of porphyrin biosynthesis and enzymology in
Desulfovibrio vulgaris
Authors: Ishida Tetsuo , Yu Ling , Akutsu Hideo , Ozawa Kiyoshi , Kawanishi Shosuke , Seto Akira , Inubushi Toshiro , Sano Seiyo
Published: July 2002

Culture of
Desulfovibrio vulgaris
in a medium supplemented with 5-aminolevulinic acid and
l
-methionine-
methyl
-
d
3
resulted in the formation of porphyrins (sirohydrochlorin, coproporphyrin III, and protoporphyrin IX) in which the methyl groups at the C-2 and C-7 positions were deuterated. A previously unknown hexacarboxylic acid was also isolated, and its structure was determined to be 12,18-didecarboxysirohydrochlorin by mass spectrometry and
1
H NMR. These results indicate a primitive pathway of heme biosynthesis in
D. vulgaris
consisting of the following enzymatic steps: (
i
) methylation of the C-2 and C-7 positions of uroporphyrinogen III to form precorrin-2 (dihydrosirohydrochlorin); (
ii
) decarboxylation of acetate groups at the C-12 and C-18 positions of precorrin-2 to form 12,18-didecarboxyprecorrin-2; (
iii
) elimination of acetate groups of the C-2 and C-7 positions of 12,18-didecarboxyprecorrin-2 to form coproporphyrinogen III; and (
iv
) conversion of coproporphyrinogen III to protoporphyrin IX via protoporphyrinogen IX. We isolated the following three enzymatic activities involved in steps
i–iii
from the soluble fraction of the cells by anion-exchange chromatography:
S
-adenosyl-
l
-methionine:uroporphyrinogen III methyltransferase, precorrin-2 12,18-acetate decarboxylase, and 12,18-didecarboxyprecorrin-2 2,7-decarboxymethylase; all enzymic products were converted into autooxidized methyl esters and analyzed by thin-layer chromatography, UV–visible (UV-VIS) absorption, and mass spectrometry. The enzymatic reactions in
D. vulgaris
shed new light on porphyrin biosynthesis at an early stage in the evolution of prokaryotes.
Tumor necrosis factor α is a determinant of pathogenesis and disease progression in mycobacterial infection in the central nervous system
Authors: Tsenova Liana , Bergtold Amy , Freedman Victoria H. , Young Richard A. , Kaplan Gilla
Published: July 2002

The pathogenesis of tuberculous meningitis, a devastating complication of tuberculosis in man, is poorly understood. We previously reported that rabbits with experimental tuberculous meningitis were protected from death by a combination of antibiotics and thalidomide therapy. Survival was associated with inhibition of tumor necrosis factor α (TNF-α) production by thalidomide. To test whether cerebrospinal fluid (CSF) levels of TNF-α correlated with pathogenesis, the response of rabbits infected in the central nervous system (CNS) with various mycobacterial strains was studied. CNS infection with
Mycobacterium bovis
Ravenel,
M. bovis
bacillus Calmette–Guérin (BCG) Pasteur, and
M. bovis
BCG Montreal were compared.
M. bovis
Ravenel induced the highest levels of TNF-α in the CSF in association with high leukocytosis, protein accumulation, and severe meningeal inflammation. BCG Pasteur had intermediate effects, and BCG Montreal was the least virulent. In addition,
M. bovis
Ravenel numbers were highest in the brain and CSF and the bacilli also disseminated more efficiently to distant organs, compared with BCG Pasteur and BCG Montreal. In subsequent experiments, rabbits were infected with either recombinant
M. bovis
BCG Montreal (vector), or BCG Montreal expressing the murine gene for TNF-α (BCG mTNF-α). BCG Montreal was rendered virulent by the expression of murine TNF-α, as demonstrated by high CSF leukocytosis, high protein accumulation, severe meningeal inflammation, persistent bacillary load, and progressive clinical deterioration. Taken together, these results demonstrate that the level of TNF-α produced during mycobacterial CNS infection determines, at least in part, the extent of pathogenesis.
Effect of juvenile hormone on the central nervous processing of sex pheromone in an insect
Authors: Anton Sylvia , Gadenne Christophe
Published: July 2002

Behavioral sex pheromone responsiveness in the male moth
Agrotis ipsilon
was previously shown to be controlled by juvenile hormone (JH). However, this morphogenetic hormone did not change the sensitivity of antennae to sex pheromones. To analyze the possible involvement of JH in the central integration of the female-produced sex pheromone, we investigated the pheromone response of olfactory antennal lobe (AL) interneurons in male
A. ipsilon
as a function of age and JH status by using intracellular recordings. When the antennae were stimulated with the sex pheromone blend, the sensitivity of olfactory AL neurons increased with age, as does the JH-dependent behavioral and physiological development of
A. ipsilon
males. Furthermore, males surgically deprived of JH showed a significant decrease in the sensitivity of the AL neurons. JH injection in operated or in young males restored or induced, respectively, a high sensitivity of the AL neurons. JH seems likely to be involved in the plasticity of the adult insect brain by modulating the central nervous processing of olfactory information, thus allowing mate recognition and reproduction at the optimal time.
Trading force for speed: Why superfast crossbridge kinetics leads to superlow forces
Authors: Rome Lawrence C. , Cook Chris , Syme Douglas A. , Connaughton Martin A. , Ashley-Ross Miriam , Klimov Andrei , Tikunov Boris , Goldman Yale E.
Published: July 2002

Superfast muscles power high-frequency motions such as sound production and visual tracking. As a class, these muscles also generate low forces. Using the toadfish swimbladder muscle, the fastest known vertebrate muscle, we examined the crossbridge kinetic rates responsible for high contraction rates and how these might affect force generation. Swimbladder fibers have evolved a 10-fold faster crossbridge detachment rate than fast-twitch locomotory fibers, but surprisingly the crossbridge attachment rate has remained unchanged. These kinetics result in very few crossbridges being attached during contraction of superfast fibers (only ≈1/6 of that in locomotory fibers) and thus low force. This imbalance between attachment and detachment rates is likely to be a general mechanism that imposes a tradeoff of force for speed in all superfast fibers.
Genetic engineering of the unsaturation of fatty acids in membrane lipids alters the tolerance ofSynechocystisto salt stress
Authors: Allakhverdiev Suleyman I. , Nishiyama Yoshitaka , Suzuki Iwane , Tasaka Yasushi , Murata Norio
Published: July 2002

The role of unsaturated fatty acids in membrane lipids in the tolerance of the photosynthetic machinery to salt stress was studied by comparing thedesA−/desD−mutant ofSynechocystissp. PCC 6803, which contained monounsaturated fatty acids, with the wild-type strain, which contained a full complement of polyunsaturated fatty acids. In darkness, the loss of oxygen-evolving photosystem II activity in the presence of 0.5 M NaCl or 0.5 M LiCl was much more rapid indesA−/desD−cells than in wild-type cells. Oxygen-evolving activity that had been lost during incubation with 0.5 M NaCl in darkness returned when cells were transferred to conditions that allowed photosynthesis or respiration. Recovery was much greater in wild-type than indesA−/desD−cells, and it was prevented by lincomycin. Thus, the unsaturation of fatty acids is important in the tolerance of the photosynthetic machinery to salt stress. It appears also that the activity and synthesis of the Na+/H+antiporter system might be suppressed under high-salt conditions and that this effect can be reversed, in part, by the unsaturation of fatty acids in membrane lipids.
On the structure of endogenous ouabain
Authors: Kawamura Akira , Guo Jinsong , Itagaki Yasuhiro , Bell Charles , Wang Yi , Haupert Garner T. , Magil Sheila , Gallagher Rex T. , Berova Nina , Nakanishi Koji
Published: July 2002

The ouabain-like sodium pump inhibitor in mammals (so-called “endogenous ouabain”) has been considered a subtle structural isomer of ouabain. Its structural investigation, however, has long been hindered by the paucity of sample material. Our recent purification of endogenous ouabain (3 μg) from bovine hypothalamus allowed the measurement of its1H-NMR. The obtained spectrum as well as reexamination of past microscale structural studies on endogenous ouabain led us to identify the purified material as ouabain in an unusual manner. It turned out that the structural analysis had been complicated by a facile ouabain–borate complexation in borosilicate glassware. In retrospect, it is not surprising that the polyhydroxylated ouabain molecule serves as a polydentate ligand to inorganic species. In its physiological environment, ouabain may exist as some unknown complex. The chemical species giving rise to the reported biological activities of hypothalamic inhibitory factor preparations remain to be clarified.
Matrix metalloproteinase deficiencies affect contact hypersensitivity: Stromelysin-1 deficiency prevents the response and gelatinase B deficiency prolongs the response
Authors: Wang Min , Qin Xuejin , Mudgett John S. , Ferguson Thomas A. , Senior Robert M. , Welgus Howard G.
Published: July 2002

Matrix metalloproteinases (MMPs) are expressed by T cells and macrophages, but there is a paucity of evidence for their role in immune responses. We have studied mice with deficiencies of stromelysin-1 (MMP-3) or gelatinase B (MMP-9) in a dinitrofluorobenzene (DNFB)-induced model of contact hypersensitivity (CHS). Stromelysin-1-deficient mice showed a markedly impaired CHS response to topical DNFB, although they responded normally to cutaneously applied phenol, an acute irritant. Lymphocytes from lymph nodes of DNFB-sensitized stromelysin-1-deficient mice did not proliferate in response to specific soluble antigen dinitrobenzenesulfonic acid, but did proliferate identically to lymph node lymphocytes from wild-type mice when presented with the mitogen Con A. An intradermal injection of stromelysin-1 immediately before DNFB sensitization rescued the impaired CHS response to DNFB in stromelysin-1-deficient mice. Unlike stromelysin-1-deficient mice, gelatinase B-deficient mice exhibited a CHS response comparable to wild-type controls at 1 day postchallenge, but the response persisted beyond 7 days in contrast to the complete resolution observed in wild-type mice by 7 days. However, gelatinase B-deficient mice had a normal rate of resolution of acute inflammation elicited by cutaneous phenol. Gelatinase B-deficient mice failed to show IL-10 production at the site of CHS, an essential feature of resolution in control mice. These results indicate that stromelysin-1 and gelatinase B serve important functions in CHS. Stromelysin-1 is required for initiation of the response, whereas gelatinase B plays a critical role in its resolution.
Antibodies to CD44 and integrin α4, but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment
Authors: Brocke Stefan , Piercy Christopher , Steinman Lawrence , Weissman Irving L. , Veromaa Timo
Published: July 2002

The role of various adhesion molecules in lymphocyte homing to the brain and in inflammatory autoimmune disease of the central nervous system (CNS) was examined in mice. Activated T cell lines and clones expressed CD44 and integrin α4, but not L-selectin, and entered the CNS independent of their antigen specificity. mAbs directed against CD44 and integrin α4prevented the transfer of experimental autoimmune encephalomyelitis (EAE) by myelin basic protein-specific T cells. T cells preincubated with anti-CD44 or antiintegrin α4were blocked only partially from entering the brain parenchyma. However, both antibodies efficiently prevented CNS inflammation and clinical expression of EAE when injectedin vivo. This effect lasted as long as antibodies were administered. Antibodies specific for L-selectin had no effect on homing of encephalitogenic T cells to the brain or development of EAE. Antiintegrin α4and anti-CD44 did not impair the activation and function of encephalitogenic T cellsin vitroand did not deplete integrin α4- or CD44-positive cellsin vivo. These data suggest that, in the absence of leukocyte recruitment, the entry of a reduced number of activated myelin basic protein-reactive T cells in the CNS is not sufficient for the development and expression of EAE. We propose that antibodies to integrin α4and CD44 prevent clinical disease by partially targeting the primary influx of encephalitogenic T cells and by preventing the secondary influx of leukocytes to lesions initiated by the transferred T cells.
P19
ARF
stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2
Authors: Tao Weikang , Levine Arnold J.
Published: July 2002

The
INK4a-ARF
locus encodes two distinct tumor suppressors, p16
INK4a
and p19
ARF
. Whereas p16
INK4a
restrains cell growth through preventing phosphorylation of the retinoblastoma protein, p19
ARF
acts by attenuating Mdm2-mediated degradation of p53, thereby stabilizing p53. Recent data indicate that Mdm2 shuttles between the nucleus and the cytoplasm and that nucleo-cytoplasmic shuttling of Mdm2 is essential for Mdm2’s ability to promote p53 degradation. Therefore, Mdm2 must export p53 from the nucleus to the cytoplasm where it targets p53 for degradation. We show here that coexpression of p19
ARF
blocks the nucleo-cytoplasmic shuttling of Mdm2. Moreover, subnuclear localization of Mdm2 changes from the nucleoplasm to the nucleolus in a shuttling time-dependent manner, whereas p19
ARF
is exclusively located in the nucleolus. In heterokaryons containing Mdm2 and p19
ARF
, the longer the Mdm2 shuttling is allowed, the more Mdm2 protein colocalizes with p19
ARF
in the nucleolus, implying that Mdm2 moves from the nucleoplasm to the nucleolus and then associates with p19
ARF
there. Furthermore, whether or not Mdm2 colocalizes with p19
ARF
in the nucleolus, p19
ARF
prevents Mdm2 shuttling. This observation suggests that Mdm2 might be exported through the nucleolus and p19
ARF
could inhibit the nuclear export of Mdm2 by tethering Mdm2 in the nucleolus. Taken together, p19
ARF
could stabilize p53 by inhibiting the nuclear export of Mdm2.
Purification of transcription cofactor complex CRSP
Authors: Ryu Soojin , Tjian Robert
Published: July 2002

Transcription of protein coding genes in metazoans involves the concerted action of enhancer binding proteins and the RNA polymerase II apparatus. The cross talk between these two classes of transcription factors is mediated by an elaborate set of cofactor complexes. For the activation of transcription by the promoter specificity protein 1 (Sp1), TATA binding protein-associated factors in the TFIID complex originally were identified as necessary coactivators, but the identity of additional cofactors required for activated transcription was unknown. Recently, we have reported the isolation and properties of a cofactor complex, CRSP (cofactor required for Sp1), which functions in conjunction with the TATA binding protein-associated factors to promote efficient activation of transcription by Sp1. CRSP contains unique subunits as well as polypeptides that are shared with other cofactor complexes. Here, we report a detailed purification protocol for the isolation of CRSP from human HeLa cells. Our purification strategy takes advantage of the ability of CRSP to bind Ni
2+
-nitrilotriacetic acid-agarose resin as well as other conventional chromatographic resins. We also describe a streamlined purification protocol that allows a more rapid and efficient means to isolate active CRSP.
Evidence that a plant virus switched hosts to infect a vertebrate and then recombined with a vertebrate-infecting virus
Authors: Gibbs Mark J. , Weiller Georg F.
Published: July 2002

There are several similarities between the small, circular, single-stranded-DNA genomes of circoviruses that infect vertebrates and the nanoviruses that infect plants. We analyzed circovirus and nanovirus replication initiator protein (Rep) sequences and confirmed that an N-terminal region in circovirus Reps is similar to an equivalent region in nanovirus Reps. However, we found that the remaining C-terminal region is related to an RNA-binding protein (protein 2C), encoded by picorna-like viruses, and we concluded that the sequence encoding this region of Rep was acquired from one of these single-stranded RNA viruses, probably a calicivirus, by recombination. This is clear evidence that a DNA virus has incorporated a gene from an RNA virus, and the fact that none of these viruses code for a reverse transcriptase suggests that another agent with this capacity was involved. Circoviruses were thought to be a sister-group of nanoviruses, but our phylogenetic analyses, which take account of the recombination, indicate that circoviruses evolved from a nanovirus. A nanovirus DNA was transferred from a plant to a vertebrate. This transferred DNA included the viral origin of replication; the sequence conservation clearly indicates that it maintained the ability to replicate. In view of these properties, we conclude that the transferred DNA was a kind of virus and the transfer was a host-switch. We speculate that this host-switch occurred when a vertebrate was exposed to sap from an infected plant. All characterized caliciviruses infect vertebrates, suggesting that the host-switch happened first and that the recombination took place in a vertebrate.