bluuebunny/crossref_metadata_2025 huggingface dataset:
Cysteine Protease Inhibitors Cure an Experimental Trypanosoma cruzi Infection
Authors: Engel Juan C. , Doyle Patricia S. , Hsieh Ivy , McKerrow James H.
Published: July 2002

Trypanosoma cruzi is the causative agent of Chagas' disease. The major protease, cruzain, is a target for the development of new chemotherapy. We report the first successful treatment of an animal model of Chagas' disease with inhibitors designed to inactivate cruzain. Treatment with fluoromethyl ketone–derivatized pseudopeptides rescued mice from lethal infection. The optimal pseudopeptide scaffold was phenylalanine-homophenylalanine. To achieve cure of infection, this pseudopeptide scaffold was incorporated in a less toxic vinyl sulfone derivative. N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl also rescued mice from a lethal infection. Six of the treated mice survived over nine months, three without further treatment. Three mice that had entered the chronic stage of infection were retreated with a 20-d regimen. At the conclusion of the experiments, five of the six mice had repeated negative hemacultures, indicative of parasitological cure. Studies of the effect of inhibitors on the intracellular amastigote form suggest that the life cycle is interrupted because of inhibitor arrest of normal autoproteolytic cruzain processing at the level of the Golgi complex. Parasites recovered from the hearts of treated mice showed the same abnormalities as those treated in vitro. No abnormalities were noted in the Golgi complex of host cells. This study provides proof of concept that cysteine protease inhibitors can be given at therapeutic doses to animals to selectively arrest a parasitic infection.
Molecular Requirements for T Cell Recognition by a Major Histocompatibility Complex Class II–restricted T Cell Receptor: The Involvement of the Fourth Hypervariable Loop of the Vα Domain
Authors: Thatte Jayant , Qadri Ayub , Radu Caius , Ward E. Sally
Published: July 2002

The role of two central residues (K68, E69) of the fourth hypervariable loop of the Vα domain (HV4α) in antigen recognition by an MHC class II–restricted T cell receptor (TCR) has been analyzed. The TCR recognizes the NH2-terminal peptide of myelin basic protein (Ac1-11, acetylated at NH2 terminus) associated with the class II MHC molecule I-Au. Lysine 68 (K68) and glutamic acid 69 (E69) of HV4α have been mutated both individually and simultaneously to alanine (K68A, E69A). The responsiveness of transfectants bearing wild-type and mutated TCRs to Ac1-11–I-Au complexes has been analyzed in the presence and absence of expression of the coreceptor CD4. The data demonstrate that in the absence of CD4 expression, K68 plays a central role in antigen responsiveness. In contrast, the effect of mutating E69 to alanine is less marked. CD4 coexpression can partially compensate for the loss of activity of the K68A mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants, are highly sensitive to anti-CD4 antibody blockade. The observations support models of T cell activation in which both the affinity of the TCR for cognate ligand and the involvement of coreceptors determine the outcome of the T cell–antigen-presenting cell interaction.
Crucial Role of the Interleukin 1 Receptor Family Member T1/St2 in T Helper Cell Type 2–Mediated Lung Mucosal Immune Responses
Authors: Coyle Anthony J. , Lloyd Clare , Tian Jane , Nguyen Trang , Erikkson Christina , Wang Lin , Ottoson Par , Persson Per , Delaney Tracy , Lehar Sophie , Lin Steve , Poisson Louis , Meisel Christian , Kamradt Thomas , Bjerke Torbjorn , Levinson Douglas , Gutierrez-Ramos Jose Carlos
Published: July 2002

T1/ST2 is an orphan receptor of unknown function that is expressed on the surface of murine T helper cell type 2 (Th2), but not Th1 effector cells. In vitro blockade of T1/ST2 signaling with an immunoglobulin (Ig) fusion protein suppresses both differentiation to and activation of Th2, but not Th1 effector populations. In a nascent Th2-dominated response, anti-T1/ST2 monoclonal antibody (mAb) inhibited eosinophil infiltration, interleukin 5 secretion, and IgE production. To determine if these effects were mediated by a direct effect on Th2 cells, we next used a murine adoptive transfer model of Th1- and Th2-mediated lung mucosal immune responses. Administration of either T1/ST2 mAb or T1/ST2-Ig abrogated Th2 cytokine production in vivo and the induction of an eosinophilic inflammatory response, but failed to modify Th1-mediated inflammation. Taken together, our data demonstrate an important role of T1/ST2 in Th2-mediated inflammatory responses and suggest that T1/ST2 may prove to be a novel target for the selective suppression of Th2 immune responses.
Src-like Adaptor Protein (Slap) Is a Negative Regulator of T Cell Receptor Signaling
Authors: Sosinowski Tomasz , Pandey Akhilesh , Dixit Vishva M. , Weiss Arthur
Published: July 2002

Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)–, and interleukin 2–dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3ζ, ZAP-70, SH2 domain–containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.
Interferon γ Induction of Pulmonary Emphysema in the Adult Murine Lung
Authors: Wang Zhongde , Zheng Tao , Zhu Zhou , Homer Robert J. , Riese Richard J. , Chapman Harold A. , Shapiro Steven D. , Elias Jack A.
Published: July 2002

Chronic inflammation containing CD8+ lymphocytes, neutrophils, and macrophages, and pulmonary emphysema coexist in lungs from patients with chronic obstructive pulmonary disease. Although this inflammatory response is believed to cause the remodeling that is seen in these tissues, the mechanism(s) by which inflammation causes emphysema have not been defined. Here we demonstrate that interferon γ (IFN-γ), a prominent product of CD8+ cells, causes emphysema with alveolar enlargement, enhanced lung volumes, enhanced pulmonary compliance, and macrophage- and neutrophil-rich inflammation when inducibly targeted, in a transgenic fashion, to the adult murine lung. Prominent protease and antiprotease alterations were also noted in these mice. They included the induction and activation of matrix metalloproteinase (MMP)-12 and cathepsins B, H, D, S, and L, the elaboration of MMP-9, and the selective inhibition of secretory leukocyte proteinase inhibitor. IFN-γ causes emphysema and alterations in pulmonary protease/antiprotease balance when expressed in pulmonary tissues.
The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV
Authors: Richard Eva , Arredondo-Vega Francisco X. , Santisteban Ines , Kelly Susan J. , Patel Dhavalkumar D. , Hershfield Michael S.
Published: July 2002

Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans.
The Production of a New MAGE-3 Peptide Presented to Cytolytic T Lymphocytes by HLA-B40 Requires the Immunoproteasome
Authors: Schultz Erwin S. , Chapiro Jacques , Lurquin Christophe , Claverol Stéphane , Burlet-Schiltz Odile , Warnier Guy , Russo Vincenzo , Morel Sandra , Lévy Frédéric , Boon Thierry , Van den Eynde Benoît J. , van der Bruggen Pierre
Published: July 2002

By stimulating human CD8+ T lymphocytes with autologous dendritic cells infected with an adenovirus encoding MAGE-3, we obtained a cytotoxic T lymphocyte (CTL) clone that recognized a new MAGE-3 antigenic peptide, AELVHFLLL, which is presented by HLA-B40. This peptide is also encoded by MAGE-12. The CTL clone recognized MAGE-3–expressing tumor cells only when they were first treated with IFN-γ. Since this treatment is known to induce the exchange of the three catalytic subunits of the proteasome to form the immunoproteasome, this result suggested that the processing of this MAGE-3 peptide required the immunoproteasome. Transfection experiments showed that the substitution of β5i (LMP7) for β5 is necessary and sufficient for producing the peptide, whereas a mutated form of β5i (LMP7) lacking the catalytically active site was ineffective. Mass spectrometric analyses of in vitro digestions of a long precursor peptide with either proteasome type showed that the immunoproteasome produced the antigenic peptide more efficiently, whereas the standard proteasome more efficiently introduced cleavages destroying the antigenic peptide. This is the first example of a tumor-specific antigen exclusively presented by tumor cells expressing the immunoproteasome.
Fast Inactivation of Delayed Rectifier K Conductance in Squid Giant Axon and Its Cell Bodies
Authors: Mathes Chris , Rosenthal Joshua J.C. , Armstrong Clay M. , Gilly William F.
Published: July 2002

Inactivation of delayed rectifier K conductance (gK) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to VK (∼−10 mV in 50–70 mM external K) for a variable time and then assaying available gK with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as with long depolarizations. Under our experimental conditions (12–18°C, 4 mM internal MgATP) a large fraction of gK inactivates within 250 ms at −10 mV in both cell bodies and axons, although inactivation tends to be more complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is more temperature-sensitive and becomes very prominent above 12°C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of gK, suggesting a strong coupling of this inactivation path to the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating IK but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that do not closely resemble those associated with either C-type or N-type inactivation of cloned Kv1 channels studied in heterologous expression systems.
On the Structural Basis for Size-selective Permeation of Organic Cations through the Voltage-gated Sodium Channel
Authors: Sun Ye-Ming , Favre Isabelle , Schild Laurent , Moczydlowski Edward
Published: July 2002

Recent evidence indicates that ionic selectivity in voltage-gated Na+ channels is mediated by a small number of residues in P-region segments that link transmembrane elements S5 and S6 in each of four homologous domains denoted I, II, III, and IV. Important determinants for this function appear to be a set of conserved charged residues in the first three homologous domains, Asp(I), Glu(II), and Lys(III), located in a region of the pore called the DEKA locus. In this study, we examined several Ala-substitution mutations of these residues for alterations in ionic selectivity, inhibition of macroscopic current by external Ca2+ and H+, and molecular sieving behavior using a series of organic cations ranging in size from ammonium to tetraethylammonium. Whole-cell recording of wild-type and mutant channels of the rat muscle μ1 Na+ channel stably expressed in HEK293 cells was used to compare macroscopic current–voltage behavior in the presence of various external cations and an intracellular reference solution containing Cs+ and very low Ca2+. In particular, we tested the hypothesis that the Lys residue in domain III of the DEKA locus is responsible for restricting the permeation of large organic cations. Mutation of Lys(III) to Ala largely eliminated selectivity among the group IA monovalent alkali cations (Li+, Na+, K+, Rb+, Cs+) and permitted inward current of group IIA divalent cations (Mg2+, Ca2+, Sr2+, Ba2+). This same mutation also resulted in the acquisition of permeability to many large organic cations such as methylammonium, tetramethylammonium, and tetraethylammonium, all of which are impermeant in the native channel. The results lead to the conclusion that charged residues of the DEKA locus play an important role in molecular sieving behavior of the Na+ channel pore, a function that has been previously attributed to a hypothetical region of the channel called the “selectivity filter.” A detailed examination of individual contributions of the Asp(I), Glu(II), and Lys(III) residues and the dependence on molecular size suggests that relative permeability of organic cations is a complex function of the size, charge, and polarity of these residues and cation substrates. As judged by effects on macroscopic conductance, charged residues of the DEKA locus also appear to play a role in the mechanisms of block by external Ca2+ and H+, but are not essential for the positive shift in activation voltage that is produced by these ions.
Rapid and Direct Effects of pH on Connexins Revealed by the Connexin46 Hemichannel Preparation
Authors: Brady Trexler E. , Bukauskas Feliksas F. , Bennett Michael V.L. , Bargiello Thaddeus A. , Verselis Vytas K.
Published: July 2002

pH is a potent modulator of gap junction (GJ) mediated cell–cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell–cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting (∼10−7 M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.
Kvβ1.2 Subunit Coexpression in HEK293 Cells Confers O2 Sensitivity to Kv4.2 but not to Shaker Channels
Authors: Pérez-García María Teresa , López-López José Ramón , González Constancio
Published: July 2002

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane α subunits and cytoplasmic modulatory β subunits. The differential expression and association of α and β subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvβ1.2 coexpression on the properties of Shaker and Kv4.2 KV channel α subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvβ1.2 functionally associates with these two α subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvβ1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvβ1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2′-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+β channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+β currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that β subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4.2+β channels to respond to hypoxia.
Molecular Coupling of S4 to a K+ Channel's Slow Inactivation Gate
Authors: Loots Eli , Isacoff Ehud Y.
Published: July 2002

The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K+ channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4–pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.
Altered State Dependence of C-Type Inactivation in the Long and Short Forms of Human Kv1.5
Authors: Kurata Harley T. , Soon Gordon S. , Fedida David
Published: July 2002

Evidence from both human and murine cardiomyocytes suggests that truncated isoforms of Kv1.5 can be expressed in vivo. Using whole-cell patch-clamp recordings, we have characterized the activation and inactivation properties of Kv1.5ΔN209, a naturally occurring short form of human Kv1.5 that lacks roughly 75% of the T1 domain. When expressed in HEK 293 cells, this truncated channel exhibited a V1/2 of −19.5 ± 0.9 mV for activation and −35.7 ± 0.7 mV for inactivation, compared with a V1/2 of −11.2 ± 0.3 mV for activation and −0.9 ± 1.6 mV for inactivation in full-length Kv.15. Kv1.5ΔN209 channels exhibited several features rarely observed in voltage-gated K+ channels and absent in full-length Kv1.5, including a U-shaped voltage dependence of inactivation and “excessive cumulative inactivation,” in which a train of repetitive depolarizations resulted in greater inactivation than a continuous pulse. Kv1.5ΔN209 also exhibited a stronger voltage dependence to recovery from inactivation, with the time to half-recovery changing e-fold over 30 mV compared with 66 mV in full-length Kv1.5. During trains of human action potential voltage clamps, Kv1.5ΔN209 showed 30–35% greater accumulated inactivation than full-length Kv1.5. These results can be explained with a model based on an allosteric model of inactivation in Kv2.1 (Klemic, K.G., C.-C. Shieh, G.E. Kirsch, and S.W. Jones. 1998. Biophys. J. 74:1779–1789) in which an absence of the NH2 terminus results in accelerated inactivation from closed states relative to full-length Kv1.5. We suggest that differential expression of isoforms of Kv1.5 may contribute to K+ current diversity in human heart and many other tissues.
An expression of spectral radius via Aluthge transformation
Authors: Yamazaki Takeaki
Published: July 2002

For an operator

T
∈
B
(
H
)

T\in B(H)

, the Aluthge transformation of

T
T

is defined by

T
~

=

|

T

|

1
2

U

|

T

|

1
2

\widetilde {T}=|T|^{\frac {1}{2}}U|T|^{\frac {1}{2}}

. And also for a natural number

n
n

, the

n
n

-th Aluthge transformation of

T
T

is defined by

T

n

~

=

(

T

n
−
1

~

)

~

\widetilde {T_{n}}=\widetilde {(\widetilde {T_{n-1}})}

and

T

1

~

=

T
~

\widetilde {T_{1}}=\widetilde {T}

. In this paper, we shall show

lim

n
→
∞

‖

T

n

~

‖
=
r
(
T
)
,

\begin{equation*} \lim _{n\to \infty }\|\widetilde {T_{n}}\|=r(T),\end{equation*}

where

r
(
T
)

r(T)

is the spectral radius.
Automatic realizability of Galois groups of order 16
Authors: Grundman Helen , Smith Tara
Published: July 2002

This article examines the realizability of small groups of order

2

k

,
k
≤
4

2^{k}, k \leq 4

, as Galois groups over arbitrary fields of characteristic not 2. In particular we consider automatic realizability of certain groups given the realizability of others.
Tables of linear congruential generators of different sizes and good lattice structure
Authors: L’Ecuyer Pierre
Published: July 2002

We provide sets of parameters for multiplicative linear congruential generators (MLCGs) of different sizes and good performance with respect to the spectral test. For

ℓ
=
8
,
9
,
…
,
64
,
127
,
128

\ell = 8, 9, \dots , 64, 127, 128

, we take as a modulus

m
m

the largest prime smaller than

2
ℓ

2^\ell

, and provide a list of multipliers

a
a

such that the MLCG with modulus

m
m

and multiplier

a
a

has a good lattice structure in dimensions 2 to 32. We provide similar lists for power-of-two moduli

m
=

2

ℓ

m = 2^{\ell }

, for multiplicative and non-multiplicative LCGs.
Two-primary algebraic 𝐾-theory of rings of integers in number fields
Authors: Rognes J. , Weibel C. , M. Kolster appendix by
Published: July 2002

We relate the algebraic

K
K

-theory of the ring of integers in a number field

F
F

to its étale cohomology. We also relate it to the zeta-function of

F
F

when

F
F

is totally real and Abelian. This establishes the

2
2

-primary part of the “Lichtenbaum conjectures.” To do this we compute the

2
2

-primary

K
K

-groups of

F
F

and of its ring of integers, using recent results of Voevodsky and the Bloch–Lichtenbaum spectral sequence, modified for finite coefficients in an appendix. A second appendix, by M. Kolster, explains the connection to the zeta-function and Iwasawa theory.
A uniqueness theorem for harmonic functions on the upper-half plane
Authors: Ou Biao
Published: July 2002

Consider harmonic functions on the upper-half planeR+2={(x,y)|y>0}R^{2}_{+}\! = \{(x,y)|\;y > 0 \}satisfying the boundary conditionuy=−exp⁡(u)u_{y}=-\exp (u)and the constraint∫R+2exp⁡(2u)>∞\int _{R^{2}_{+}}\exp (2u) > \infty. We prove that all such functions are of form (1.2) below.
TOPAL 2.0: improved detection of mosaic sequences within
multiple alignments
Authors: McGuire Gráinne , Wright Frank
Published: July 2002

Motivation: The Dss statistic was proposed by (Mol. Biol. Evol., 14, 1125–1131, McGuire et al. (1997)) for scanning data sets for the presence of recombination, an important step in some phylogenetic analyses. The statistic, however, could not distinguish well between among-site rate variation and recombination, and had no statistical test for significant values. This paper addresses these shortfalls.

Results: A modification to the Dss statistic is proposed which accounts for rate variation to a large extent. A statistical test, based on parametric bootstrapping, is also suggested.

Availability: The TOPAL package (version 2) may be accessed from http://www.bioss.sari.ac.uk/~frank/Geneticsand by anonymous ftp from ftp://ftp.bioss.sari.ac.ukin the directory pub/phylogeny/topal.

Contact: frank@bioss.sari.ac.uk
Apoptosis is promoted by the dsRNA‐activated factor (DRAF1) during viral infection independent of the action of interferon or p53
Authors: WEAVER BRIAN K. , ANDO OSAMU , KUMAR K. PRASANNA , REICH NANCY C.
Published: July 2002

An apoptotic cellular defense mechanism is triggered in response to viral dsRNA generated during the course of infection by many DNA and RNA viruses. We demonstrate that apoptosis induced by dsRNA or a paramyxovirus is independent of the action of interferon as it can proceed in a variety of cell lines and primary cells deficient in an interferon response. Initiation of apoptosis appears to be triggered by activation of a cellular transcription factor, the dsRNA‐activated factor (DRAF1). DRAF1 is composed of interferon regulatory factor 3 (IRF‐3) and the transcriptional coactivators CREB binding protein (CBP) or p300. We find that activation of IRF‐3 in the absence of viral infection stimulates apoptosis. In addition, a negative interfering mutant blocks both target gene induction and apoptosis, demonstrating a requirement for gene expression by IRF‐3/DRAF1 to promote apoptosis. IRF‐3/DRAF1 target gene expression is also induced in response to a distinct apoptotic stimulus, the DNA damaging agent etoposide. The activity of the p53 tumor suppressor does not appear to be required for IRF‐3/DRAF1‐mediated apoptosis.—Weaver, B. K., Ando, O., Kumar, K. P., Reich, N. C Apoptosis is promoted by the dsRNA‐activated factor (DRAF1) during viral infection independent of the action of interferon or p53. FASEB J. 15, 501‐514 (2001)
Cardiac contractile impairment associated with increased phosphorylation of troponin I in endotoxemic rats
Authors: Tavernier Benoît , Li Jian‐Mei , El‐Omar Magdi M. , Lanone Sophie , Yang Zhao‐Kang , Trayer Ian P , Mebazaa Alexandre , Shah Ajay M.
Published: July 2002

The subcellular mechanisms underlying intrinsic myocardial depression during sepsis remain poorly defined, in particular the relative roles of altered intracellular Ca2+ transients versus changes in myofilament properties. We studied contractile function of cardiac myocytes isolated 12 h after induction of endotoxemia (5 mg/kg intravenous E. coli lipopolysaccharide [LPS]) in conscious rats. Cardiomyocytes from LPS‐injected rats had depressed twitch shortening compared with control cells (4.1±0.2% versus 7.8±0.3%; P<0.001; 0.5‐Hz stimulation), but similar Ca2+ transients (peak indo‐1 ratio 1.13±0.02 versus 1.12±0.02; P = NS). Contractile depression was unaffected by inhibitors of nitric oxide synthase. Steady‐state myofilament response to Ca2+, assessed by tetanization of intact cells over a range of [Ca2+], was reduced significantly in the LPS group (P<0.001). Intracellular pH (SNARF fluorescence) was similar in both groups, but the amount of phosphorylated troponin I present in endotoxemic myocardium was approximatel2y+ twofold greater than in control myocardium. In addition, steady‐state myofilament response to Ca2+ was unaffected by isoproterenol (3 nmol/L) in endotoxemic cells, whereas there was a rightward shift in control cells. A reduction in myofilament response to Ca2+ is the major determinant of intrinsic cardiac depression in systemic endotoxemia. This condition appears to be related to an increase in myocardial troponin I phosphorylation.
Subcellular localization of presenilins during mouse preimplantation development
Authors: Jeong Sung‐Jin , Kim Hye‐Sun , Chang Keun‐A , Geum Dong‐Ho , Park Cheol Hyoung , Seo Ji‐Heui , Rah Jong‐Cheol , Lee Jun Ho , Choi Se Hoon , Lee Sang Goo , Kim Kyungjin , Suh Yoo‐Hun
Published: July 2002

The genes defective in familial Alzheimer's disease encode the proteins presenilin 1 and 2 (PS1 and 2). Expression of presenilins (PSs) and their proteolytic processing are regulated during neuronal development. Even though these proteins are detected and regulated mainly in Golgi and endoplasmic reticulum, their subcellular distribution during the development is not known. The present study aimed to investigate the localization of PSs and their role during early developmental stage using mouse embryo model. At preimplantation stage, PSs were detected not only in cytoplasm, but also in the nucleus from oocyte to 2.5 dpc (day postcoitum), then disappeared in the nucleus at blastocyst stage (3.5 dpc). Antisense against PS1 and PS2 decreased the transition to blastocyst stage, whereas each antisense alone had no effect. Treatment with lactacystin (26S proteosome inhibitor), which arrest cell cycle at M phase, redistributed PSs into centrosome‐kinetochore microtubule. PS2 overexpression in HEK 293 cell arrested cell cycle at S phase. These data suggest that PSs play key roles in cell division and differentiation during early development.—Jeong, S.‐J., Kim, H.‐S., Chang, K.‐A., Geum, D.‐H., Park, C. H., Seo, J.‐H., Rah, J.‐C., Lee, J. H., Choi, S. H., Lee, S. G., Kim, K., Suh, Y.‐H. Subcellular localization of presenilins during mouse preimplantation development. FASEB J. 14, 2171–2176 (2000)
The past, the future and the biology of memory storage
Authors: Kandel Eric R. , Pittenger Christopher
Published: July 2002

We here briefly review a century of accomplishments in studying memory storage and delineate the two major questions that have dominated thinking in this area: the systems question of memory, which concerns where in the brain storage occurs; and the molecular question of memory, which concerns the mechanisms whereby memories are stored and maintained. We go on to consider the themes that memory research may be able to address in the 21st century. Finally, we reflect on the clinical and societal import of our increasing understanding of the mechanisms of memory, discussing possible therapeutic approaches to diseases that manifest with disruptions of learning and possible ethical implications of the ability, which is on the horizon, to ameliorate or even enhance human memory.
Environmental factors which may have led to the appearance of colour vision
Authors: Maximov Vadim V.
Published: July 2002

It is hypothesized that colour vision and opponent processing of colour signals in the visual system evolved as a means of overcoming the extremely unfavourable lighting conditions in the natural environment of early vertebrates. The significant flicker of illumination inherent in the shallow–water environment complicated the visual process in the achromatic case, in particular preventing early detection of enemies. The presence of two spectral classes of photoreceptors and opponent interaction of their signals at a subsequent retinal level allowed elimination of the flicker from the retinal image. This new visual function provided certain advantages concerning reaction times and favoured survival. This assumption explains why the building blocks for colour vision arose so early, i.e. just after the active predatory lifestyle was mastered. The principal functions of colour vision inherent in extant animals required a more complex neural machinery for colour processing and evolved later as the result of a change in visual function favouring colour vision.
Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae
Authors: Sharp Judith A. , Franco Alexa A. , Osley Mary Ann , Kaufman Paul D.
Published: July 2002

Budding yeast centromeres are comprised of ∼125-bp DNA sequences that direct formation of the kinetochore, a specialized chromatin structure that mediates spindle attachment to chromosomes. We report here a novel role for the histone deposition complex chromatin assembly factor I (CAF-I) in building centromeric chromatin. The contribution of CAF-I to kinetochore function overlaps that of the Hir proteins, which have also been implicated in nucleosome formation and heterochromatic gene silencing. cacΔ hirΔ double mutant cells lacking both CAF-I and Hir proteins are delayed in anaphase entry in a spindle assembly checkpoint-dependent manner. Further, cacΔ andhirΔ deletions together cause increased rates of chromosome missegregation, genetic synergies with mutations in kinetochore protein genes, and alterations in centromeric chromatin structure. Finally, CAF-I subunits and Hir1 are enriched at centromeres, indicating that these proteins make a direct contribution to centromeric chromatin structures.
Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore
Authors: Measday Vivien , Hailey Dale W. , Pot Isabelle , Givan Scott A. , Hyland Katherine M. , Cagney Gerard , Fields Stan , Davis Trisha N. , Hieter Philip
Published: July 2002

The budding yeast kinetochore is composed of an inner and outer protein complex, which binds to centromere (CEN) DNA and attaches to microtubules. We performed a genetic synthetic dosage lethality screen to identify novel kinetochore proteins in a collection of chromosome transmission fidelity mutants. Our screen identified several new kinetochore-related proteins including YLR381Wp/Ctf3p, which is a member of a conserved family of centromere-binding proteins. Ctf3p interacts with Mcm22p, Mcm16p, and the outer kinetochore protein Ctf19p. We used chromatin immunoprecipitation to demonstrate that Ctf3p, Mcm22p, and Mcm16p bind to CEN DNA in a Ctf19p-dependent manner. In addition, Ctf3p, Mcm22p, and Mcm16p have a localization pattern similar to other kinetochore proteins. The fission yeast Ctf3p homolog, Mis6, is required for loading of a CENP-A centromere specific histone, Cnp1, onto centromere DNA. We find however that Ctf3p is not required for loading of the budding yeast CENP-A homolog, Cse4p, ontoCEN DNA. In contrast, Ctf3p and Ctf19p fail to bind properly to the centromere in a cse4-1 mutant strain. We conclude that the requirements for CENP-A loading onto centromere DNA differ in fission versus budding yeast.
Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation
Authors: Nishioka Kenichi , Chuikov Sergei , Sarma Kavitha , Erdjument-Bromage Hediye , Allis C. David , Tempst Paul , Reinberg Danny
Published: July 2002

A novel histone methyltransferase, termed Set9, was isolated from human cells. Set9 contains a SET domain, but lacks the pre- and post-SET domains. Set9 methylates specifically lysine 4 (K4) of histone H3 (H3-K4) and potentiates transcription activation. The histone H3 tail interacts specifically with the histone deacetylase NuRD complex. Methylation of histone H3-K4 by Set9 precludes the association of NuRD with the H3 tail. Moreover, methylation of H3-K4 impairs Suv39h1-mediated methylation at K9 of H3 (H3-K9). The interplay between the Set9 and Suv39h1 histone methyltransferases is specific, as the methylation of H3-K9 by the histone methyltransferase G9a was not affected by Set9 methylation of H3-K4. Our studies suggest that Set9-mediated methylation of H3-K4 functions in transcription activation by competing with histone deacetylases and by precluding H3-K9 methylation by Suv39h1. Our results suggest that the methylation of histone tails can have distinct effects on transcription, depending on its chromosomal location, the combination of posttranslational modifications, and the enzyme (or protein complex) involved in the particular modification.
The structure of apo protein‐tyrosine phosphatase 1B C215S mutant: More than just an S → O change
Authors: Scapin Giovanna , Patel Sangita , Patel Vira , Kennedy Brian , Asante‐Appiah Ernest
Published: July 2002

Protein‐tyrosine phosphatases catalyze the hydrolysis of phosphate monoesters via a two‐step mechanism involving a covalent phospho‐enzyme intermediate. Biochemical and site‐directed mutagenesis experiments show that the invariant Cys residue present in the PTPase signature motif (H/V)CX5R(S/T) (i.e., C215 in PTP1B) is absolutely required for activity. Mutation of the invariant Cys to Ser results in a catalytically inactive enzyme, which still is capable of binding substrates and inhibitors. Although it often is assumed that substrate‐trapping mutants such as the C215S retain, in solution, the structural and binding properties of wild‐type PTPases, significant differences have been found in the few studies that have addressed this issue, suggesting that the mutation may lead to structural/conformational alterations in or near the PTP1B binding site. Several crystal structures of apo‐WT PTP1B, and of WT‐ and C215S‐mutant PTP1B in complex with different ligands are available, but no structure of the apo‐PTP1B C215S has ever been reported. In all previously reported structures, residues of the PTPase signature motif have an identical conformation, while residues of the WPD loop (a surface loop which includes the catalytic Asp) assume a different conformation in the presence or absence of ligand. These observations led to the hypothesis that the different spectroscopic and thermodynamic properties of the mutant protein may be the result of a different conformation for the WPD loop. We report here the structure of the apo‐PTP1B C215S mutant, which reveals that, while the WPD loop is in the open conformation observed in the apo WT enzyme crystal structure, the residues of the PTPases signature motif are in a dramatically different conformation. These results provide a structural basis for the differences in spectroscopic properties and thermodynamic parameters in inhibitor binding observed for the wild‐type and mutant enzymes.
A radiobiological basis for bioeffect planning
Authors: Wigg David R.
Published: July 2002

The main purpose of this thesis is to encourage the development of bioeffect planning as an experimental tool by which means bioeffect plans may be compared with standard isodose plans. The limitations of isodose planning become apparent in many common clinical circumstances when such comparisons can be made. Most of this thesis addresses the fundamental problems of the derivation of useful biological models for clinical application and the description of tumor and normal tissue parameter values and their variability. Particular emphasis has been placed on comparing the predictive value of the models and parameters against clinical results of fractionated and continuous irradiation. A bioeffect planning system has been developed using an IGE Target computer and this permits testing the sensitivity of the models to changes in parameter values as applied to an actual patient with or without CT based plans. As a clinician the author is painfully aware of the tantalizing challenge of attempting to perceive the operation of biological systems as a whole when so many variables need to be considered simultaneously. Bioeffect planning provides a means of demonstrating some of these complicated interactions. There are many reasons why bioeffect planning has not been adequately developed. These include concerns about the clinical applications of theoretical models, the uncertainty of normal tissue and tumor parameter values and the nonavailability of suitable computer systems capable of bioeffect planning. These concerns are fully justified and isodose planning must remain the gold standard for clinical treatment. Yet these hazards must be judged against the certainties that isodose planning, in which the only variable usually considered is the total dose, can be substantially misleading.
Quantitative assessment of diffuse coronary artery disease using digital angiography
Authors: Zhou Yifang
Published: July 2002

Diffuse coronary artery disease (DCAD), a common form of atherosclerosis, may be responsible for the failure of improved coronary flow reserve after successful PTCA (percutaneous transluminal coronary angioplasty). However, DCAD is especially difficult to diagnose because the lumen cross‐sectional area is diffusely diseased along multiple vessels. In this study, the normal coronary arterial tree was defined from a supply and demand perspective through the establishment of a hemodynamic model. The definition is embodied by the following correlations between different morphometric and hemodynamic parameters. For any supplying segment in a normal coronary artery tree, the coronary blood flow is proportional to its dependent distal cumulative arterial length. Furthermore, the cross‐sectional area of any supplying segment and its cumulative distal arterial volume relate to its distal cumulative arterial length in forms of fractal power law. The power law indices were found to observe certain constraints. The proposed correlations were validated using flow simulation on a complete set of anatomical data of a normal pig's coronary arterial tree. Validations of morphometric and hemodynamic parameters were also performed on angiographic data from a swine animal model using techniques of 3‐D reconstruction of the arterial tree and videodensitometry. The detectability of DCAD was evaluated by simulating a moderate level of DCAD and propagation of measurement error for morphometric and hemodynamic parameters. In conclusion, the proposed methodology can be used for detection and quantification of DCAD.
Detector line spread functions determined analytically by transport of Compton recoil electrons
Authors: van't Veld A. A. , van Luijk P. , Praamstra F. , van der Hulst P. C.
Published: July 2002

To achieve the maximum benefit of conformal radiation therapy it is necessary to obtain accurate knowledge of radiation beam penumbras based on high‐resolution relative dosimetry of beam profiles. For this purpose there is a need to perform high‐resolution dosimetry with well‐established routine dosimeters, such as ionization chambers or diodes. Profiles measured with these detectors must be corrected for the dosimeter's nonideal response, caused by finite dimensions and, in the case of an ionization chamber, the alteration of electron transport and a contribution of electrons recoiled in the chamber wall and the central electrode. For this purpose the line spread function (LSF) of the detector is needed. The experimental determination of LSFs is cumbersome and restricted to the specific detector and beam energy spectrum used. Therefore, a previously reported analytical model [Med. Phys. 27, 923–934 (2000)] has been extended to determine response profiles of routine dosimeters: shielded diodes and, in particular, ionization chambers, in primary dose slit beams. The model combines Compton scattering of incident photons, the transport of recoiled electrons by Fermi–Eyges small‐angle multiple scattering theory, and functions to limit electron transport. It yields the traveling direction and the energy of electrons upon incidence on the detector surface. In the case of ionization chambers, geometrical considerations are then sufficient to calculate the relative amount of ionization in chamber air, i.e., the detector response, as a function of the detector location in the slit beam. In combination with the previously reported slit beam dose profiles, the LSF can then readily be derived by reconstruction techniques. Since the spectral contributions are preserved, the LSF of a dosimeter is defined for any beam for which the effective spectrum is known. The detector response profiles calculated in this study have been verified in a telescopic slit beam geometry, and were found to correspond to experimental profiles within 0.2 and 0.3 mm (full width at half‐maximum) for a Wellhoefer IC15 chamber in a 6 and 25 MV‐X x‐ray beam, respectively. For a shielded diode these figures were found to be 0.2 and 0.1 mm, respectively. It is shown that a shielded diode in a primary beam needs only a small size‐based correction of measured profiles. The effect of the LSF of an IC15 chamber on penumbra width has been determined for a set of model penumbras. The LSFs calculated by the application of the analytical model yield a broadening by 2 mm of a 3 mm wide penumbra (20%–80%). This is 0.5 mm (6 MV‐X) to 1 mm (25 MV‐X) smaller than found with the experimental LSFs. With a spatial correction based on the LSFs that were determined in this study, this broadening of up to 2 mm is eliminated, so that ionization chambers like the IC15 can be used for high‐resolution relative dosimetry on a routine basis.
A generalized approach to parallel magnetic resonance imaging
Authors: Sodickson Daniel K. , McKenzie Charles A.
Published: July 2002

Parallel magnetic resonance (MR) imaging uses spatial encoding from multiple radiofrequency detector coils to supplement the encoding supplied by magnetic field gradients, and thereby to accelerate MR image acquisitions beyond previous limits. A generalized formulation for parallel MR imaging is derived, demonstrating the relationship between existing techniques such as SMASH and SENSE, and suggesting new algorithms with improved performance. Hybrid approaches combining features of both SMASH‐like and SENSE‐like image reconstructions are constructed, and numerical conditioning techniques are described which can improve the practical robustness of parallel image reconstructions. Incorporation of numerical conditioning directly into parallel reconstructions using the generalized approach also removes a cumbersome and potentially error‐prone sensitivity calibration step involving division of two distinct in vivo reference images. Hybrid approaches in combination with numerical conditioning are shown to extend the range of accelerations over which high‐quality parallel images may be obtained.