The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the ITIS gene, which is predicted to encode a 348-kDa protein named huntingtin. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cell lines from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.Huntington disease (HD) is an inherited neurodegenerative disorder characterized by progressive motor, psychiatric, and cognitive disturbances. The neuropathology of HD includes selective loss of neurons that is most severe in the caudate and putamen but also affects other brain regions. It has been hypothesized that neuronal death in HD is due to a metabolic defect that leads to excitotoxicity (1, 2). The genetic mutation, however, has not yet been directly linked to neuronal metabolism.HD has been associated with the abnormal expansion of a polymorphic trinucleotide (CAG) repeat sequence occurring in the coding region of a gene (IT1S) located on chromosome 4 (3). In HD, the length of this repeat is substantially increased, ranging from 40 to over 100 copies (3-5). Juvenile-onset HD cases are associated with the highest numbers of repeats (3, 6). The IT1S gene encodes a large unknown protein (-343 kDa) that has been termed "huntingtin" (3). Its mRNA is normally distributed in diverse tissues in human and rat (3, 7) and is expressed predominantly in neurons in brain (7,8). In HD heterozygotes, both normal and mutant mRNA are present, suggesting that the trinucleotide expansion does not prevent transcription (9). Thus, the pathophysiology of HD likely depends on the effect of the mutant allele at the protein level. Understanding huntingtin is therefore crucial for determining how the genetic mutation could be linked to the pathophysiology of HD and for developing treatments based on the molecular defect. We have developed polyclonal and monoclonal antibodies specific to huntingtin to enable its identifica...

Huntington's disease (HD) chromosomes contain an expanded unstable (CAG)n repeat in chromosome 4p16.3. We have examined nine families with potential de novo expression of the disease. With one exception, all of the affected individuals had 42 or more repeat units, well above the normal range. In four families, elderly unaffected relatives inherited the same chromosome as that containing the expanded repeat in the proband, but had repeat lengths of 34-38 units, spanning the gap between the normal and HD distributions. Thus, mutation to HD is usually associated with an expansion from an already large repeat.

ABSTRACT:1 H NMR chemical shifts have been obtained in the solvents deuterochloroform and dimethyl sulfoxide. The difference in the chemical shifts of an OH or NH group in these two solvents, Δδ = δ(DMSO) − δ(CDCl 3 ), can be converted into the hydrogen bond acidity, A, of the group using the equation A = 0.0065 + 0.133Δδ. The NMR A value, A NMR , can be used as a quantitative assessment of intramolecular hydrogen bonding. We list values of Δδ and A NMR for 55 compounds containing an OH group and 60 compounds with an NH group. For the hydroxy compounds, if A > 0.5 then the OH group is not part of an intramolecular hydrogen bond, but if A < 0.1 then the OH group forms part of an intramolecular hydrogen bond. For NH compounds, if A > 0.16 the NH group is not part of an intramolecular hydrogen bond, and if A < 0.05 the NH group is part of an intramolecular hydrogen bond. No comparison compounds are needed, and the method is extremely simple. We further show how it is possible to relate intramolecular hydrogen bonding to the actual effect on values of a number of physicochemical, environmental, and biochemical properties. ■ INTRODUCTION Shalaeva et al.1 have recently shown that the presence of an intramolecular hydrogen bond (intraHB) in a molecule can considerably alter the properties of a molecule. These include properties relevant to drug design such as solubility, permeability, and partition. It is therefore important to be able to identify molecules that possess intraHBs and, if possible, to assess the effect of an intraHB on the molecular properties. Testa and co-workers 2−5 were the first to show that the effect of intraHBs could be observed in water−solvent partition coefficients (as log P) and particularly in differences between partition coefficients in water−octanol and water−aprotic solvent systems. They set out differences in log P for water− octanol and water−heptane partitions (eq 1) and showed that intraHBs greatly reduce the value of Δ(log P) oct−hept . They also observed similar effects due to intraHBs in other water−octanol and water−solvent systems.4,5 Leo 6 used octanol and chloroform as the two solvent systems in order to calculate the hydrogen bond acidity of a solute, and Feng et al. 7 used dibutyl ether and cyclohexane as the solvent systems to calculate solute hydrogen bond acidity.

The membrane structure of the naturally occurring gramicidins A, B, and C was investigated using circular dichroism (CD) spectroscopy and single-channel recording techniques. All three gramicidins form channels with fairly similar properties (Bamberg, E., K. Noda, E. Gross, and P. Läuger. 1976. Biochim. Biophys. Acta. 419:223-228.). When incorporated into lysophosphatidylcholine micelles, however, the CD spectrum of gramicidin B is different from that of gramicidin A or C (cf. Prasad, K. U., T. L. Trapane, D. Busath, G. Szabo, and D. W. Urry. 1983. Int. J. Pept. Protein Res. 22:341-347.). The structural identity of the channels formed by gramicidin B has, therefore, been uncertain. We find that when gramicidins A and B are incorporated into dipalmitoylphosphatidylcholine vesicles, their CD spectra are fairly similar, suggesting that the two channel structures could be similar. In planar bilayers, gramicidins A, B, and C all form hybrid channels with each other. The properties of the hybrid channels are intermediate to those of the symmetric channels, and the appearance rates of the hybrid channels (relative to the symmetric channels) corresponds to what would be predicted if all three gramicidin molecules were to form structurally equivalent channels. These results allow us to interpret the different behavior of channels formed by the three gramicidins solely on the basis of the amino acid substitution at position 11.