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EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

REFERENCES



Accelerated apoptosis is one mechanism proposed
for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type
1 (HIV-1 ) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of
gp...

in vitro calmodulin binding to a peptide corresponding to
the C-terminal calmodulin-binding domain of gp160. Stable Tet-off
Jurkat cell lines were developed that inducibly express wild type gp160
or gp160A835W. Increasing expression of wild type gp16...

INTRODUCTION

TOP

ABSTRACT

INTRODUCTION

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The importance of apoptosis in AIDS is controversial although there is
abundant in vitro evidence supporting a role for apoptosis in the pathogenesis of HIV-1 ( 12 ), and although mechanisms regulating this apoptosis in vivo are not clear, current ev...



The coat glycoprotein of HIV-1, gp160, is post-translationally cleaved
to an extracellular subunit, gp120, and a transmembrane subunit, gp41.
The subunits are non-covalently associated, and both are required for
viral entry into the cell. Two calmodu...



We demonstrate here that gp160 expression enhances FAS-mediated
apoptosis in Jurkat cells by increasing calmodulin expression and
accelerating caspase 3activation and that these effects that require
calmodulin binding to gp160 are blocked by a single...

EXPERIMENTAL PROCEDURES

TOP

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

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 Peptide Synthesis--

Peptides corresponding to the C-terminal
calmodulin-binding site of gp160 (residues 826-843) were
synthesized at the University of Alabama at Birmingham Comprehensive
Cancer Center Peptide Synthesis and Analysis Shared Facility and
purified by high p...



 TdT Apoptosis Staining--

In situ apoptosis
staining was performed using terminal deoxynucleotide transferase
(Roche Molecular Biochemicals). Briefly, cells were collected after
treatments and cytospun onto microscope slides and fixed in 10%
formalin in PBS. Slides were then ...

cobalt chloride). After a brief wash, slides were blocked in 1% bovine
serum albumin, 0.1% gelatin in PBS for 15min at room temperature.
Slides were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody for 1h at room temperature, ...



 Western Blot for Caspase 3and Calmodulin--

Jurkat cells were
treated as indicated and lysed in buffer containing 50m M

HEPES, pH 7.4,150m M NaCl, 10% glycerol, 1% Triton X-100, 1m M sodium orthovanadate, 10m M EDTA,
10m M EGTA, 1m M ammonium molybdate, 50m M NaF, 0.5µ M okadaic acid, 5m M

benzamidine, and 50µg/ml pepstatin. Equivalent amounts of protein
(100µg) were separated on 12.5% SDS-PAGE and transferred to
Immobilon P membrane (Millipore, Bedford, MA). Membranes were fixed in
0.2% glutaraldehyde in Tris-buffered saline (TBS ) fo...



 Dansyl-Calmodulin Binding--

The binding of calmodulin to
peptides corresponding to the C-terminal calmodulin-binding domain of
gp160 or the A835W mutant was determined fluorimetrically with
dansyl-calmodulin (Ocean Biologics, Corvalis, OR) as reported
previously ( 21 ). Briefly...

KCl, and 1m M CaCl 2 with or without 5 m M EGTA. Emission spectra were obtained with concentrations
of peptides ranging from 0to 1µ M, and mellitin was used
as a positive control for binding.

RESULTS

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Earlier work has shown that expression of HIV-1 envelope glycoprotein,
gp160, overcomes a block in the FAS pathway in Molt4 cells ( 19 ), a
FAS-resistant human T-cell line deficient in hematopoietic stem cell
phosphatase. The results presented here s...







The long cytoplasmic tail of gp160 is a feature conserved among HIV-1,
HIV-2, and simian immunodeficiency virus. No specific function has been
attributed to the cytoplasmic region, and despite considerable
variability in the overall sequence, all kno...







To determine whether the mechanism of gp160-dependent
enhanced apoptosis occurs at the level of the FAS receptor, we
investigated whether transfection of gp160 enhanced
ceramide-mediated apoptosis. Ceramide is a proposed mediator of
apoptosis produce...







Caspase 3,or caspase 3-like proteases, are critical effectors that
play an important role in most types of apoptosis ( 8 ). During
activation, caspase 3is first cleaved by an upstream caspase (caspase
8in the FAS pathway) and then undergoes a second ...

block gp160-enhanced FAS- and ceramide-induced apoptosis,
suggesting a calmodulin-dependent mechanism. The molecular site of action of calmodulin antagonists in gp160-enhanced apoptosis is
currently under investigation. However, the data presented he...







To confirm the effect of calmodulin binding by gp160 on FAS-mediated
apoptosis, we created a point mutation in the C-terminal CaM-binding
domain based on comparisons with the calmodulin-binding domain of MLCK

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Brain Activity and Tinnitus



Exposure to short periods of very loud noise can cause tinnitusÑa persistent ringing or buzzing in the ears that cannot be blocked out. Tinnitus may affect around 10%Ð15% of the population; severe tinnitus is very debilitating (1%Ð2% of the populatio...

The human ear is essentially a very sensitive vibration sensor, one that is able to receive the minute longitudinal vibrations in air that make up sound waves. It can detect sounds from 20 Hertz (Hz) (very low pitch) to 20,000 Hz (very high pitch) bu...

Some studies in both animals and humans have suggested that tinnitus and hearing loss may be related. These studies have found that neurons in regions of the auditory cortex that have been deprived of stimuli because of hearing loss change their rece...

One of the key research targets in tinnitus has been investigation of cortical activity, especially in animal models of tinnitus, but studies in humans have been rare. Previous studies have identified temporal and frontal temporal changes in individu...

In this month's PLoS Medicine, Nathan Weisz and colleagues studied 17 patients with chronic tinnitus and hearing loss and 16 control individuals with normal hearing. Patients were asked to fill in a questionnaire about the impact of tinnitus on their...

The team's methods differed from previous work in that the team chose to examine the power spectrum of neuromagnetic oscillatory activity during rest, whereas previous studies had focused on measuring neurophysiological responses following sounds.

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The fact that the end-tidal CO 2 was not influenced by
hypoxia virtually excludes the distorting influence of changes in
P CO 2 on hypoxia-induced cerebral vasodilation
in the present study ( 28 ). Normally, the hypoxic
ventilatory response consists ...







In contrast to normoxia, L -NMMA induced a small increase in
mean arterial pressure of 6% during hypoxia. This is probably caused
by inhibition of hypoxia-induced nitric oxide-mediated systemic
vasodilation by L -NMMA. The fact that L -NMMA
did not i...







An unexpected finding of this study is that basal cerebral blood flow
is significantly different between the normoxic and hypoxic conditions.
Because the study is performed in a randomized single-blind fashion,
this finding must be accidental. This i...

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  For the nuclear localization experiments NIH3T3 cells were plated on glass coverslips, at 3000 cells in a 30-µl drop of DMEM/10% donor calf serum. Following attachment, the cells were cultured in 0.5 ml of the same medium and left for 24 h. They we...

  For the KRAB domain repression assays COS-7 cells were plated at a density of 1.4 x 10 6 cells in 10-cm dishes and transfected 48 h later, using a total of 2.6 µg of DNA per transfection, as described above. Cell lysates were collected 48 h later, ...

  For RNA extraction, primary cultures of astrocytes, oligodendrocytes, endothelial cells, pericytes, and neurons were a gift of Dr. Krause-Finkeldey. All cultures were prepared from newborn or perinatal rats as described previously ( 51 ). They were...

  For immunocytochemistry, promoter analysis, and to study the effect of LIF, Schwann cells were prepared from newborn or postnatal day (P) 3 or P4 rat sciatic nerve and serum-purified using cytosine arabinoside, followed in some experiments by antib...

  In transfection experiments with the 1.1-kb P 0 and 9.0-kb MBP promoters P3-purified Schwann cells were expanded by passaging three times in DMEM, 10% FCS, and 2 µ M forskolin (Merck Biosciences) and crude glial growth factor prepared from bovine p...

   Immunocytochemistry— Sciatic nerves from embryonic and newborn mice were teased on to gelatin coated microscope slides as described previously ( 57 ). Slides were air-dried for 30 min, fixed in ice-cold methanol for 10 min at 4 °C, then equilibrat...

        RESULTS       TOP   ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES   RESULTS   DISCUSSION   REFERENCES     Identification of ZFP-57, a Novel Zinc Finger Protein Expressed in the Schwann Cell Lineage— To isolate Schwann cell genes containin...

   

          View larger version (43K):  [in this window]  [in a new window]  F IG. 1. Outline of the strategy used to clone genes expressing zinc finger motif genes. A, diagram showing that zinc finger ( ZF ) motifs arranged in tandem arrays in a hypot...

       Data base analyses revealed that one sequence obtained was more than 95% identical to the murine transcription factor ZFP-57 previously identified by Okazaki et al. ( 61 ) in F9 teratocarcinoma cells. We cloned and sequenced over 11 kb of mous...

   Expression of Zfp-57 during Embryonic and Postnatal Development of the Sciatic Nerve— The expression of the Zfp-57 gene during the early and postnatal development of the mouse sciatic nerve was compared with the expression of the zinc finger trans...

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          View larger version (52K):  [in this window]  [in a new window]  F IG. 9. Expression of Zfp-57 and Krox-20 in Swiss 3T3 cells in the presence of FGF-2. Semiquantitative RT-PCR analysis of cultured Swiss 3T3 cells at different time points af...

             DISCUSSION       TOP   ABSTRACT   INTRODUCTION   EXPERIMENTAL PROCEDURES   RESULTS   DISCUSSION   REFERENCES    We have isolated the rat Zfp-57 gene using a fast and simple approach to search for novel zinc finger transcription factors e...

  ZFP-57 is a relative of the Xenopus laevis transcription factor IIIA type gene family of zinc finger transcription factors ( 66 ). In addition to the C2H2 zinc finger motif ( 67 ) it also contains a KRAB-DNA binding motif, a characteristic shared b...

  In other zinc finger proteins the KRAB motif is functionally associated with transcriptional repression ( 62, 71 ). Consistent with this, we find that the KRAB domain in ZFP-57 acts as a powerful repressor of activated transcription when linked to ...

  The KRAB domain is also necessary for the normal localization of the protein to speckles of centromeric heterochromatin within the nucleus of NIH3T3 cells, another characteristic typical of proteins containing this domain ( 36, 62, 71 ). Neverthele...

  Consistent with the cloning of Zfp-57 from E12 sciatic nerve, mRNA expression was relatively high during early development of peripheral nerves. We also found relatively high levels of expression in spinal cord and sensory ganglia at E12, with high...

   

  In peripheral nerve, Zfp-57 mRNA is substantially down-regulated after a peak of expression between E15 and E17 and by P28 has dropped to low levels, though the protein is still readily detectable in adult nerve. The onset of down-regulation overla...

  In line with this, we tested the hypothesis that ZFP-57 function might be associated with cell proliferation using a variety of conditions. In Swiss 3T3 cells the number of cells synthesizing DNA is not altered by enforced expression of ZFP-57 (see...

  Despite the fact that ZFP-57 can repress myelin gene promoters when constitutively expressed in Schwann cells, it does not inhibit induction of Krox-20 or the myelin proteins periaxin or P 0 in a cAMP-based assay of myelin-related differentiation (...

  In conclusion, this is the first report of a zinc finger protein containing a KRAB domain with a demonstrated function as a transcriptional repressor in Schwann cells of the peripheral nervous system. Zfp-57 is under strong developmental regulation...

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   GDP·AlF[4] ^−complexes, Gln^204 must sever its hydrogen bond with
   Ser^206 and W^nuc and rotate ≈120° about χ[1] and ≈90° about χ[2] and
   χ[3] (to gauche+ and gauche−, respectively) such that its carbamoyl
   group donates a hydrogen bond to the equatorial oxygen of the
   pentacoordinate γ-phosphoryl group and accepts a hydrogen bond from
   W^nuc. Such would incur a substantial penalty in catalytic efficiency
   and perhaps account, at least in part, for the low catalytic rate of
   GTP hydrolysis in G[α] and perhaps in other G proteins.

   We propose that the ground state G[iα1]·GTP complex is “auto-inhibited”
   with Gln^cat locked into an unproductive conformation. Active site
   residues in the EF-Tu·GppNHp complex also assumes anti-catalytic
   positions; in this case His^cat, the residue corresponding to Gln^cat,
   cannot interact with the substrates because of steric interference by
   other active site residues ([119]41). In G[iα1], catalysis could occur
   only if the bonds that hold Gln^cat in this position are broken, and
   the side chain freed to interact with the pentacoordinate transition
   state. This model predicts that changes that disrupt the ground state
   conformation of Gln^204, while not otherwise compromising the active
   site, would increasek [cat].

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   most similar homologs. This conserved site is proposed to be a binding
   or catalytic site for a ligand or substrate that is common to YabJ and
   other members of the YER057c/YjgF/UK114 family of proteins.

   Purine biosynthesis in Bacillus subtilis is regulated by the purine
   operon repressor gene, purR. The purR operon consists of purR and yabJ,
   an ORF of unknown function. The repressor PurR binds to control regions
   upstream of the transcription start sites and regulates transcription
   of purEKBC(orf)QLFMNHD in the pur operon, of purR and yabJ in the purR
   operon, and of purA ([30]1). PurR binding to control-site DNA is
   blocked by 5-phosphoribosyl-1-pyrophosphate (PRPP), a central
   nucleotide metabolite that acts as an inducer of pur operon and purA
   transcription. 5-phosphoribosyl-1-pyrophosphate is the starting
   material for purine biosynthesis. No other metabolite, nucleotide,
   nucleoside, or nucleotide base is known to affect PurR DNA binding in
   vitro ([31]2).

   Recently, yabJ, the distal coding sequence in the purR operon, was
   shown to affect PurR function in vivo ([32]3). Regulation of
   transcription of purine biosynthetic genes is sensitive to levels of
   some nutrients and depends on the interrelated pools of

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   has been identified, purified, and shown to catalyze a Claisen-type
   condensation between long chain acyl-CoA substrates such as
   myristoyl-CoA (C[14]) and malonyl-ACP. This enzyme, presumed to play a
   key role in initiating meromycolic acid biosynthesis, was crystallized,
   and its structure was determined at 2.1-Ã… resolution. The mtFabH
   homodimer is closely similar in topology and active-site structure to
   Escherichia coli FabH (ecFabH), with a CoA/malonyl-ACP-binding channel
   leading from the enzyme surface to the buried active-site cysteine
   residue. Unlike ecFabH, mtFabH contains a second hydrophobic channel
   leading from the active site. In the ecFabH structure, this channel is
   blocked by a phenylalanine residue, which constrains specificity to
   acetyl-CoA, whereas in mtFabH, this residue is a threonine, which
   permits binding of longer acyl chains. This same channel in mtFabH is
   capped by an α-helix formed adjacent to a 4-amino acid sequence
   insertion, which limits bound acyl chain length to 16 carbons. These
   observations offer a molecular basis for understanding the unusual
   substrate specificity of mtFabH and its probable role in regulating the
   biosynthesis of the two different length acyl chains required for
   generation of mycolic acids. This mtFabH presents a new target for
   structure-based design of novel antimycobacterial agents.

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   molecules. Two features of this site can explain the distinct substrate
   specificities of mtFabH and ecFabH. The presence of Phe^87B (where B is
   monomer B) in this fatty acyl-binding site of ecFabH obstructs binding
   of straight fatty acid chains longer than ∼4 carbons, thereby
   accounting for the selectivity of the E. coli enzyme for acetyl over
   longer chain substrates ([120]6). In mtFabH, residue 87B is a
   threonine, whose smaller size permits binding of longer chain fatty
   acids (Fig.[121]8). The -OγH of the Thr^87Bside chain is
   hydrogen-bonded to a bound solvent, thereby orienting the side chain
   methyl group toward the position of the acyl substrate and contributing
   to the hydrophobicity of its environment. This channel is also blocked
   in ecFabH by Arg^196B and Leu^191A(where A is monomer A), which in
   mtFabH are isoleucine and glutamine, respectively, and by Ile^203A and
   Leu^205A, which are displaced in mtFabH by the changes around the
   insertion at position 202.
   [122]Figure 8
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   Thr^87B;F87B, Phe^87B.

   The end of the putative acyl-binding channel (channel 2) distal to the
   active-site Cys^112 in mtFabH is capped by the α-helix at positions
   194–202 induced just before the 4-residue insertion (Fig.[126]9). The
   Arg^2024A side chain, which is hydrogen-bonded to the peptide carbonyl
   oxygen of Pro^144A, blocks the end of this substrate channel, as do, to
   a lesser extent, the side chains of Gln^191A, Ile^196B, Phe^198A,
   Ala^199B, and Gln^200B. In the ecFabH structure, this area is open to
   solvent, but the inner part of the channel proximal to the active site
   is blocked by other residues as described above, preventing binding of
   longer chains.
   [127]Figure 9
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   Figure 9

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   GDPáAlF[4] ^−complexes, Gln^204 must sever its hydrogen bond with
   Ser^206 and W^nuc and rotate Å120¡ about χ[1] and Å90¡ about χ[2] and
   χ[3] (to gauche+ and gauche−, respectively) such that its carbamoyl
   group donates a hydrogen bond to the equatorial oxygen of the
   pentacoordinate γ-phosphoryl group and accepts a hydrogen bond from
   W^nuc. Such would incur a substantial penalty in catalytic efficiency
   and perhaps account, at least in part, for the low catalytic rate of
   GTP hydrolysis in G[α] and perhaps in other G proteins.

   We propose that the ground state G[iα1]áGTP complex is Òauto-inhibitedÓ
   with Gln^cat locked into an unproductive conformation. Active site
   residues in the EF-TuáGppNHp complex also assumes anti-catalytic
   positions; in this case His^cat, the residue corresponding to Gln^cat,
   cannot interact with the substrates because of steric interference by
   other active site residues ([119]41). In G[iα1], catalysis could occur
   only if the bonds that hold Gln^cat in this position are broken, and
   the side chain freed to interact with the pentacoordinate transition
   state. This model predicts that changes that disrupt the ground state
   conformation of Gln^204, while not otherwise compromising the active
   site, would increasek [cat].