Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP\u3csup\u3e+\u3c/sup\u3e

A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ of 4 x 1.3 x 0.7 mm (3.6 mm3) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 A resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 A resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR–folate–NADP+ ternary-complex structure in order to examine the protonation state, protein dynamics and solvent structure of the complex, furthering understanding of the catalytic mechanism

Comments on information loss and remnants

The information loss and remnant proposals for resolving the black hole information paradox are reconsidered. It is argued that in typical cases information loss implies energy loss, and thus can be thought of in terms of coupling to a spectrum of ``fictitious'' remnants. This suggests proposals for information loss that do not imply planckian energy fluctuations in the low energy world. However, if consistency of gravity prevents energy non-conservation, these remnants must then be considered to be real. In either case, the catastrophe corresponding to infinite pair production remains a potential problem. Using Reissner-Nordstrom black holes as a paradigm for a theory of remnants, it is argued that couplings in such a theory may give finite production despite an infinite spectrum. Evidence for this is found in analyzing the instanton for Schwinger production; fluctuations from the infinite number of states lead to a divergent stress tensor, spoiling the instanton calculation. Therefore naive arguements for infinite production fail.Comment: 30 pages (harvmac l mode) UCSBTH-93-35 (minor reference and typo corrections

Supersymmetry and Positive Energy in Classical and Quantum Two-Dimensional Dilaton Gravity

An $N = 1$ supersymmetric version of two dimensional dilaton gravity coupled to matter is considered. It is shown that the linear dilaton vacuum spontaneously breaks half the supersymmetries, leaving broken a linear combination of left and right supersymmetries which squares to time translations. Supersymmetry suggests a spinorial expression for the ADM energy $M$, as found by Witten in four-dimensional general relativity. Using this expression it is proven that ${M}$ is non-negative for smooth initial data asymptotic (in both directions) to the linear dilaton vacuum, provided that the (not necessarily supersymmetric) matter stress tensor obeys the dominant energy condition. A {\it quantum} positive energy theorem is also proven for the semiclassical large-$N$ equations, despite the indefiniteness of the quantum stress tensor. For black hole spacetimes, it is shown that $M$ is bounded from below by $e^{- 2 \phi_H}$, where $\phi_H$ is the value of the dilaton at the apparent horizon, provided only that the stress tensor is positive outside the apparent horizon. This is the two-dimensional analogue of an unproven conjecture due to Penrose. Finally, supersymmetry is used to prove positive energy theorems for a large class of generalizations of dilaton gravity which arise in consideration of the quantum theory.Comment: 21 page

Hawking Radiation and Unitary evolution

We find a family of exact solutions to the semi-classical equations (including back-reaction) of two-dimensional dilaton gravity, describing infalling null matter that becomes outgoing and returns to infinity without forming a black hole. When a black hole almost forms, the radiation reaching infinity in advance of the original outgoing null matter has the properties of Hawking radiation. The radiation reaching infinity after the null matter consists of a brief burst of negative energy that preserves unitarity and transfers information faster than the theoretical bound for positive energy.Comment: LaTex file + uuencoded ps version including 4 figure

Molecular basis for passive immunotherapy of Alzheimer's disease

Amyloid aggregates of the amyloid-{beta} (A{beta}) peptide are implicated in the pathology of Alzheimer's disease. Anti-A{beta} monoclonal antibodies (mAbs) have been shown to reduce amyloid plaques in vitro and in animal studies. Consequently, passive immunization is being considered for treating Alzheimer's, and anti-A{beta} mAbs are now in phase II trials. We report the isolation of two mAbs (PFA1 and PFA2) that recognize A{beta} monomers, protofibrils, and fibrils and the structures of their antigen binding fragments (Fabs) in complex with the A{beta}(1–8) peptide DAEFRHDS. The immunodominant EFRHD sequence forms salt bridges, hydrogen bonds, and hydrophobic contacts, including interactions with a striking WWDDD motif of the antigen binding fragments. We also show that a similar sequence (AKFRHD) derived from the human protein GRIP1 is able to cross-react with both PFA1 and PFA2 and, when cocrystallized with PFA1, binds in an identical conformation to A{beta}(1–8). Because such cross-reactivity has implications for potential side effects of immunotherapy, our structures provide a template for designing derivative mAbs that target A{beta} with improved specificity and higher affinity

Predictability and Semiclassical Approximation at the onset of Black Hole formation

We combine analytical and numerical techniques to study the collapse of conformally coupled massless scalar fields in semiclassical 2D dilaton gravity, with emphasis on solutions just below criticality when a black hole almost forms. We study classical information and quantum correlations. We show explicitly how recovery of information encoded in the classical initial data from the outgoing classical radiation becomes more difficult as criticality is approached. The outgoing quantum radiation consists of a positive-energy flux, which is essentially the standard Hawking radiation, followed by a negative-energy flux which ensures energy conservation and guarantees unitary evolution through strong correlations with the positive-energy Hawking radiation. As one reaches the critical solution there is a breakdown of unitarity. We show that this breakdown of predictability is intimately related to a breakdown of the semiclassical approximation.Comment: 26 pages RevTex + 8 figures in a separate postscript fil

Sulfotyrosine Recognition as Marker for Druggable Sites in the Extracellular Space

Chemokine signaling is a well-known agent of autoimmune disease, HIV infection, and cancer. Drug discovery efforts for these signaling molecules have focused on developing inhibitors targeting their associated G protein-coupled receptors. Recently, we used a structure-based approach directed at the sulfotyrosine-binding pocket of the chemokine CXCL12, and thereby demonstrated that small molecule inhibitors acting upon the chemokine ligand form an alternative therapeutic avenue. Although the 50 members of the chemokine family share varying degrees of sequence homology (some as little as 20%), all members retain the canonical chemokine fold. Here we show that an equivalent sulfotyrosine-binding pocket appears to be conserved across the chemokine superfamily. We monitored sulfotyrosine binding to four representative chemokines by NMR. The results suggest that most chemokines harbor a sulfotyrosine recognition site analogous to the cleft on CXCL12 that binds sulfotyrosine 21 of the receptor CXCR4. Rational drug discovery efforts targeting these sites may be useful in the development of specific as well as broad-spectrum chemokine inhibitors

Blushift of a tachyon in the charged 2D black hole

We study the propagation of string fields (metric $G_{\mu\nu}$, Mawxell gauge potential $A_{\mu}$, dilaton $\Phi$, and tachyon $T$) in a two-dimensional (2D) charged black hole. It is shown that the tachyon is a propagating field both inside and outside the black hole. This becomes infinitely blueshifted at the inner horizon. We confirm that the inner horizon is unstable, whereas the outer horizon is stable.Comment: 15 pages 3 figures, RevTeX, to be published Phys. Rev. D52 (15 November,1995), to obtain the figures contact Author ([email protected]

CXCL12 and [N33A]CXCL12 in 5637 and HeLa Cells: Regulating HER1 Phosphorylation via Calmodulin/Calcineurin

In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals via both G-protein-dependent and β-arrestin-dependent pathways responsible for inducing a rapid and a late wave, respectively, of ERK1/2 phosphorylation. In contrast, the structural variant [N33A]CXCL12 triggered no β-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1) Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2) Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3) In cells knockdown of β-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4) HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate β-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1

A chronic fatigue syndrome – related proteome in human cerebrospinal fluid

BACKGROUND: Chronic Fatigue Syndrome (CFS), Persian Gulf War Illness (PGI), and fibromyalgia are overlapping symptom complexes without objective markers or known pathophysiology. Neurological dysfunction is common. We assessed cerebrospinal fluid to find proteins that were differentially expressed in this CFS-spectrum of illnesses compared to control subjects. METHODS: Cerebrospinal fluid specimens from 10 CFS, 10 PGI, and 10 control subjects (50 μl/subject) were pooled into one sample per group (cohort 1). Cohort 2 of 12 control and 9 CFS subjects had their fluids (200 μl/subject) assessed individually. After trypsin digestion, peptides were analyzed by capillary chromatography, quadrupole-time-of-flight mass spectrometry, peptide sequencing, bioinformatic protein identification, and statistical analysis. RESULTS: Pooled CFS and PGI samples shared 20 proteins that were not detectable in the pooled control sample (cohort 1 CFS-related proteome). Multilogistic regression analysis (GLM) of cohort 2 detected 10 proteins that were shared by CFS individuals and the cohort 1 CFS-related proteome, but were not detected in control samples. Detection of ≥1 of a select set of 5 CFS-related proteins predicted CFS status with 80% concordance (logistic model). The proteins were α-1-macroglobulin, amyloid precursor-like protein 1, keratin 16, orosomucoid 2 and pigment epithelium-derived factor. Overall, 62 of 115 proteins were newly described. CONCLUSION: This pilot study detected an identical set of central nervous system, innate immune and amyloidogenic proteins in cerebrospinal fluids from two independent cohorts of subjects with overlapping CFS, PGI and fibromyalgia. Although syndrome names and definitions were different, the proteome and presumed pathological mechanism(s) may be shared