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In an earlier work, the authors developed a new method, the mobile block Hessian (MBH) approach, to accurately calculate vibrational modes for partially optimized molecular structures [J. Chem. Phys.2007, 126(22), 224102.]. It is based on the introduction of blocks, consisting of groups of atoms, that can move as rigid bodies. The internal geometry of the blocks need not correspond to an overall optimization state of the total molecular structure. The standard MBH approach considers free blocks with six degrees of freedom. In the extended MBH approach introduced herein, the blocks can be connected by one or two adjoining atoms, which further reduces the number of degrees of freedom. The new approach paves the way for the normal-mode analysis of biomolecules such as proteins. It rests on the hypothesis that low-frequency modes of proteins can be described as pure rigid-body motions of blocks of consecutive amino acid residues. The method is validated for a series of small molecules and further applied to alanine dipeptide as a prototype to describe vibrational interactions between two peptide units; to crambin, a small protein with 46 amino acid residues; and to ICE/caspase-1, which contains 518 amino acid residues.

Patients with ALS commonly exhibit pseudobulbar affect.

The reaction path for the rearrangement of chorismate to prephenate in B.subtilis has been determined in a QM/MM study including the entire protein environment while treating the reaction with ab initio quantum chemistry. In addition to the reactant, chorismate, the side-chains of glu78 and arg90 are included in the quantum region to explore whether the strong ionic hydrogen bonding of the side chains to the substrate has a catalytic effect. The hydrogen bonds from glu78 and arg90 induce electronic effects that activate the substrate. The energetic residue analysis finds that the binding from arg7, arg63, and arg90 are all catalytic due to a differential stabilization along the reaction path of the transition state with respect to the reactant by the local environment. A global QM/MM optimization including the entire protein environment shows only slight changes in the protein environment around the active site along the reaction path. The rearrangement reaction occurs with almost a complete break in the C−O ether bond in chorismate before the C−C bond forms to create prephenate. In this study, the reacting complex forms a hydrogen bond to arg63 that stabilizes the region near the protein surface where the substrate may enter the active site.

Physical work capacity in 35 ALS patients and 6 untrained controls was evaluated during progressive bicycle ergometery. In the ALS patients, maximum oxygen consumption (VO2max) and work capacity (Wmax) drop in relation to the decrease in ALS functional score. However, the oxygen cost (ml O2/kpm) of submaximal exercise was significantly increased. Prolonged submaximal exercise tests in six ALS patients and six matched untrained controls indicated that the exercise-induced increase in plasma free fatty acids, beta-hydroxybuty-rate, esterified carnitine, and muscle esterified carnitine was significantly retarded in ALS patients.

A model for the three-dimensional structure of the repeating Asn-Pro-Asn-Ala tetrapeptide of the immunodominant circumsporozoite protein of Plasmodium falciparum has been developed. A trial structure in the form of a type I beta turn with asparagine side chains hydrogen-bonded to the backbone peptide linkages was used as a starting point. A repeating oligomer of this trial structure was modeled using energy minimization and molecular dynamics computer simulations in conjunction with image boundary conditions. The most stable structure generated is a right-handed 12(38) helix, which is unlike any previously identified protein secondary structure. The helix has 12 residues per turn, corresponding to an angle of twist of 120 degrees per tetrapeptide unit, and a pitch of 4.95 A, corresponding to a rise of 1.65 A per tetrapeptide unit. It is highly stabilized by extensive hydrogen bonding, with each tetrapeptide unit acting as an acceptor for five hydrogen bonds and as a donor for five hydrogen bonds to residues in adjacent turns as well as having four weak internal hydrogen bonds. A number of nearly isoenergetic variations on the most stable structure that still retained the basic 12(38) helical motif were also discovered. The implications of these structures for vaccine development are discussed.

To characterize the functionally important anharmonic motions of proteins, simulations of carboxymyoglobin (MbCO) dynamics have been performed during which dihedral transitions were prohibited. Comparison of torsionally restrained and unrestrained protein dynamics simulated at three levels of hydration and at temperatures ranging from 100 to 400 K suggests that hydration "catalyzes" protein mobility by facilitating collective anharmonic motions that do not require dihedral transitions. When dihedral transitions were prohibited, dehydrated MbCO, to a good approximation, exhibited only harmonic fluctuations, whereas hydrated MbCO exhibited both harmonic and anharmonic motions. The fluctuation of helix centers of mass also remained highly anharmonic in the torsionally restrained hydrated system. Atomic mean-square fluctuation at 300 K was reduced upon prohibition of dihedral transitions by only 28% and 10% for MbCO hydrated by 350 and 3830 water molecules, respectively.

A restrained molecular dynamics simulation approach that explicitly includes the effect of the surrounding solvent molecules is applied to the NMR determination of the conformations of the B-loop fragments of human transforming growth factor alpha and epidermal growth factor. Backbone interproton distance restraints are obtained by using two-dimensional rotating frame nuclear Overhauser effect spectroscopy (ROESY). The simulations are carried out both in "vacuum" and in "water." The results are discussed in terms of the energetics, agreement with the NMR distances, and the flexibility of the peptides.

ts1 is a murine leukemia virus that causes rapidly evolving hindlimb paralysis in susceptible strains of mice. Following perinatal infection, three physical forms of unintegrated viral DNA were detected in the spinal cord by Southern blot hybridization. Linear and supercoiled closed-circle viral double-stranded DNAs were detected in both the central nervous system and non-central nervous system tissues. An elevated level of a novel minus-sense single-stranded form of viral DNA, which had a very high mobility in agarose gels, was correlated with the onset of symptoms of paralysis. As the severity of paralysis progressed, the level of this single-stranded form increased rapidly, with the highest level in the spinal cords of moribund mice. Since the virulence of a number of cytopathic retroviruses has been associated with the presence of increased amounts of unintegrated viral DNA in the tissues of the infected hosts, this novel form of highly mobile unintegrated single-stranded DNA may have a role in the neuropathogenesis of ts1.

Familial amyotrophic lateral sclerosis (FALS) constitutes 5 to 10 of cases of ALS and, in most families, its inheritance is consistent with an autosomal dominant trait with age-dependent penetrance. The biochemical abnormality underlying the disorder is unknown. We analyzed DNA from 131 Members of 6 multigenerational ALS families, which included 13 affected Members, for genetic linkage to 39 expressed and DNA markers, using the techniques of 2-point linkage analysis, multilocus linkage analysis, and exclusion mapping. We identified FALS families with structures suitable for linkage, by computer simulation techniques. A DNA bank established to provide optimum use of available FALS families provided DNA from immortalized lymphoblast cell lines and frozen postmortem tissue. We could not link FALS to any of the markers studied, but excluded chromosome regions unlikely to be a locus of the FALS gene. With the help of this exclusion data, we will concentrate on regions of the human genome that remain unexcluded.

we studied copolymer 1 (Copaxone) in a multicenter (11-university) phase III trial of patients with relapsing-remitting multiple sclerosis (MS). Two hundred fifty-one patients were randomized to receive copolymer 1 (n = 125) or placebo (n = 126) at a dosage of 20 mg by daily subcutaneous injection for 2 years. The primary end point was a difference in the MS relapse rate. The final 2-year relapse rate was 1.19 ± 0.13 for patients receiving copolymer 1 and 1.68 ± 0.13 for those receiving placebo, a 29 reduction in favor of copolymer 1 (p= 0.007) (annualized rates = 0.59 for copolymer 1 and 0.84 for placebo). Trends in the proportion of relapse-free patients and median time to first relapse favored copolymer 1. Disability was measured by the Expanded Disability Status Scale (EDSS), using a two-neurologist (examining and treating) protocol. When the proportion of patients who improved, were unchanged, or worsened by ≥1 EDSS step from baseline to conclusion (2 years) was evaluated, significantly more patients receiving copolymer 1 were found to have improved and more receiving placebo worsened (p= 0.037). Patient withdrawals were 19 (15.2) from the copolymer 1 group and 17 (13.5) from the placebo group at approximately the same intervals. The treatment was well tolerated. The most common adverse experience was an injection-site reaction. Rarely, a transient self-limited systemic reaction followed the injection in 15.2 of those receiving copolymer 1 and 3.2 of those receiving placebo. This reaction was characterized by flushing or chest tightness with palpitations, anxiety, or dyspnea and commonly lasted for 30 seconds to 30 minutes. This rigorous study confirmed the findings of a previous pilot trial and demonstrated that copolymer 1 treatment can significantly and beneficially alter the course of relapsing-remitting MS in a well-tolerated fashion.

Molecular dynamics (MD) simulation covering a wide range of hydration indicate that myoglobin is fully hydrated by 350 water molecules, in agreement with experiment. These waters, originally placed uniformly about the protein, form clusters that hydrate every charged group throughout the entire simulation. Some atoms in charged groups are hydrated by two water layers while 37% of the protein surface remains uncovered. The locations of the 350 waters are consistent with those of crystallographic waters resolved by x-ray and neutron diffraction. Hydration by 350 waters at 300 K stabilizes the conformation of carboxymyoglobin measured by x-ray diffraction throughout the entire protein, halves the rate of torsional transitions, and promotes alternative conformations for surface atoms. The glass transition observed experimentally in hydrated myoglobin near 220 K is also seen in the simulations and correlates with an increase in the number of dihedral angles undergoing transitions. The anharmonic protein motion above 220 K is enhanced by protein hydration.

ts1 is a highly neuropathogenic and lymphocytopathic mutant of Moloney murine leukemia virus TB (MoMuLV-TB). We previously reported that the primary neuropathogenic determinant of ts1 maps to a single amino acid substitution, Val-25-->Ile, in precursor envelope protein gPr80env. This Val-25-->Ile substitution apparently renders gPr80env inefficient for transport from the endoplasmic reticulum to the Golgi apparatus. These findings suggest that the cytopathic effect of ts1 in neural cells might be due to the accumulation of gPr80env in the endoplasmic reticulum. Since endothelial and glial cells are targets of ts1 infection in the central nervous system, we established primary endothelial and astrocyte cultures to investigate the mechanism of cell killing caused by ts1. A continuous cell line, TB, was used as a control. Our results showed that both ts1 and MoMuLV-TB replicated and induced a cytopathic effect in astrocyte cultures, albeit to different degrees; ts1 appeared to be more lethal than MoMuLV-TB. On the other hand, ts1 and MoMuLV-TB infections of endothelial or TB cells were not cytopathic. The cytopathic effect in infected astrocytes correlated with the inefficiency of gPr80env transport and the intracellular accumulation of gPr80env as well as aberrant virus particles.

When 251 relapsing-remitting patients with multiple sclerosis were randomized to receive daily subcutaneous injections of glatiramer acetate, previously called copolymer 1 (Copaxone; n = 125) or placebo (n = 126) for 24 months, there were no laboratory abnormalities associated with glatiramer acetate treatment and it was well tolerated with few side effects. Patients receiving glatiramer acetate had significantly fewer relapses and were more likely to be neurologically improved, whereas those receiving placebo were more likely to worsen. This study was extended for 1 to 11 months (mean of 5.2 months for the glatiramer acetate group and 5.9 months for the placebo group). The blinding and study conditions used during the core 24-month study were unchanged throughout the extension. The results of this extension study confirm the excellent tolerance and safety profile of glatiramer acetate for injection. The clinical benefit of glatiramer acetate for both the relapse rate and for neurologic disability was sustained at the end of the extension trial.

We conducted this study to better inform amyotrophic lateral sclerosis (ALS) patients about home ventilation and to assist them in decision-making. We gathered data on the prevalence of ALS patients on home ventilation in northern Illinois and the percentage who chose it, and we asked identified subjects, their families, and physicians for their attitudes toward home ventilation. Fewer than 10 of ALS patients had chosen home ventilation, and fewer than 5 were still on it. Seventeen patients (90) were glad to have chosen home ventilation and would choose it again. Family caregivers reported major burdens, and only half would choose it for themselves. The mean yearly cost of home ventilation was 153,252. Home ventilation is effective for ALS patients and desired by the small number who undergo it, but it imposes significant burdens on families.

The relationship between speech intelligibility on a single-word identification test and the average second-formant (F2) slope of selected test words was examined for a group of 25 men and ten women with amyotrophic lateral sclerosis (ALS). A Spearman rank-order correlation coefficient greater than 0.80 was obtained for both the male and female subjects. This moderately high correlation indicates that the F2 slope index is a useful acoustic measure of speech proficiency in ALS. F2 slope indices are reported for normal control populations of geriatric men and women. In addition, progressive deterioration of the F2 slope index is illustrated in a case study of one woman with ALS.

ts1 is a temperature-sensitive mutant of Moloney murine leukemia virus that causes hind-limb paralysis in mice. In tissues of the central nervous systems of paralyzed moribund FVB/N mice, a major component of the unintegrated viral DNA of ts1 consists of highly mobile physical forms of viral-specific DNA (HM DNA). Previous studies with ecotropic virus-specific polarity probes showed that the gp70-coding region of the env gene in the HM DNA was minus-sense single-stranded DNA. The physical forms of the HM DNA have now been characterized in more detail with additional ecotropic virus-specific probes that hybridized to the p15E-coding region of the env gene and two locations within the U3 region of the long terminal repeat. Two major classes of HM DNA were found: class I molecules consist of short minus-sense single-stranded DNA; class II molecules are partial DNA duplexes that are longer than the class I molecules. The two classes of HM DNA molecules are intermediate products of reverse transcription of the viral RNA of ts1. Since tissues that are infected with cytopathic retroviruses may contain high levels of unintegrated viral DNA, the HM DNA may have a role in inducing neurodegeneration in the central nervous systems of mice that are infected with ts1.

The ts1 mutant of Moloney murine leukemia virus TB causes degenerative neurologic and immunologic disease in mice, characterized by development of spongiform encephalomyelopathy resulting in hindlimb paralysis, marked thymic atrophy associated with immunodeficiency, and generalized body wasting. To investigate the pathogenesis of the thymic atrophy caused by ts1, we constructed a chimeric virus, ts1-Cas(NS), in which a major portion of the U3 region of the long terminal repeat of ts1, a T-lymphotropic and neurovirulent murine leukemia virus, was replaced by the corresponding U3 region of Cas-Br-E, a B-lymphotropic and neurovirulent murine leukemia virus. In FVB/N mice, ts1-Cas(NS) induced paralytic and wasting disease with incidence, severity, and latency similar to that induced by ts1, but it failed to cause thymic atrophy as severe as that observed in ts1-infected mice. Furthermore, thymocytes cultured from ts1-Cas(NS)-infected mice died at a much slower rate than those of ts1-infected mice. The U3 substitution in ts1-Cas(NS) specifically diminished the ability of the virus to replicate in the thymus, whereas viral replication in the spinal cord was not significantly affected; thus, neurovirulence was not changed. The correlation of reduced thymic atrophy with decreased thymic viral titers and the decreased ability of ts1-Cas(NS) to cause thymocyte death in mice suggest strongly that the marked thymic atrophy in ts1-infected mice is not an indirect effect occurring secondary to neurodegenerative and wasting disease but is a direct cytopathic effect of high-level viral replication in the thymus.

A 10-fold reduction in the incubation period of murine neurotropic retrovirus spongiform polioencephalomyelopathy was effected by a 1,000-fold concentration of the cloned virus inoculum.

Facial, trigeminal, and hypoglossal motoneuron involvement was quantified in 25 amyotrophic lateral sclerosis patients and in normal controls. Measures included (1) maximum voluntary contraction of the lower lip, mandible, and tongue using custom-designed force transducers, (2) clinical functions of each muscle group, and in some patients (3) orofacial mobility using videofluoroscopy. All measures indicated that the tongue muscles were most severely affected, even in patients who initially had symptoms in the extremities.

The ALS Patient Care Database was created to improve the quality of care for patients with ALS by 1) providing neurologists with data to evaluate and improve their practices, 2) publishing data on temporal trends in the care of patients with ALS, and 3) developing hypotheses to be tested during formal clinical trials.