Thermoneutral conditions correct the obese phenotype in female, but not male, Kiss1r knockout mice.

Kisspeptin, a neuropeptide that activates gonadotropin-releasing hormone (GnRH) neurons, has also been implicated as a regulator of energy balance. Kisspeptin receptor (Kiss1r) knockout (KO) mice display an obese phenotype in adulthood compared to wild-type (WT) controls due to reduced energy expenditure. Additionally, experimental evidence shows that the temperature of typical rodent housing conditions (22 °C) increases the metabolism of mice above basal levels. Female Kiss1r KO mice show reduced core temperature and impaired temperature adaptation to an acute cold challenge, suggesting their temperature homeostasis processes are altered. The present study examined the phenotype of gonadectomised Kiss1r KO mice at both sub-thermoneutral and thermoneutral temperature (22 °C and 30 °C). Our results confirmed the obese phenotype in Kiss1r KO mice at 22 °C, and revealed a sexually dimorphic effect of thermal neutrality on the phenotype. In female KO mice, the obesity observed at 22 °C was attenuated at 30 °C. Plasma leptin levels were higher in KO than WT female mice at 22 °C (P < 0.001) but not at 30 °C. Importantly, the expression of Ucp1 mRNA in brown adipose tissue was lower in KO mice compared to WT mice at 22 °C (P < 0.05), but not different from WT at 30 °C. In male KO mice, a metabolic phenotype was observed at 22 °C and 30 °C. These results provide further evidence for kisspeptin-mediated regulation of adiposity via altered energy expenditure. Moreover, thermoneutral housing alleviated the obese phenotype in female Kiss1r KO mice, compared to WT, indicating the impairment in these mice may relate to an inability to adapt to the chronic cold stress that is experienced at 22 °C.

Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification.

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

Acute milk yield response to frequent milking during early lactation is mediated by genes transiently regulated by milk removal.

Milking dairy cows four times daily (4×) instead of twice daily (2×) during early lactation stimulates an increase in milk yield that partly persists through late lactation; however, the mechanisms behind this response are unknown. We hypothesized that the acute mammary response to regular milkings would be transient and would involve different genes from those that may be specifically regulated in response to 4×. Nine multiparous cows were assigned at parturition to unilateral frequent milking (UFM; 2× of the left udder half, 4× of the right udder half). Mammary biopsies were obtained from both rear quarters at 5 days in milk (DIM), immediately after 4× glands had been milked (experiment 1, n = 4 cows), or 2.5 h after both udder halves had last been milked (experiment 2, n = 5 cows). Affymetrix GeneChip Bovine Genome Arrays were used to measure gene expression. We found 855 genes were differentially expressed in mammary tissue between 2× vs. 4× glands of cows in experiment 1 (false discovery rate ≤ 0.05), whereas none were differentially expressed in experiment 2 using the same criterion. We conclude that there is an acute transcriptional response to milk removal, but 4× milking did not elicit differential expression of unique genes. Therefore, there does not appear to be a sustained transcriptional response to 4× milking on day 5 of lactation. Using a differential expression plot of data from both experiments, as well as qRT-PCR, we identified at least two genes (chitinase 3-like-1 and low-density lipoprotein-related protein-2 that may be responsive to both milk removal and to 4× milking. Therefore, the milk yield response to 4× milking may be mediated by genes that are acutely regulated by removal of milk from the mammary gland.

Detailed computational study of p53 and p16: using evolutionary sequence analysis and disease-associated mutations to predict the functional consequences of allelic variants.

Deciding whether a missense allelic variant affects protein function is important in many contexts. We previously demonstrated that a detailed analysis of p53 intragenic conservation correlates with somatic mutation hotspots. Here we refine these evolutionary studies and expand them to the p16/Ink4a gene. We calculated that in order for 'absolute conservation' of a codon across multiple species to achieve P<0.05, the evolutionary substitution database must contain at least 3(M) variants, where M equals the number of codons in the gene. Codons in p53 were divided into high (73% of codons), intermediate (29% of codons), and low (0 codons) likelihood of being mutation hotspots. From a database of 263 somatic missense p16 mutations, we identified only four codons that are mutational hotspots at P<0.05 (8 mutations). However, data on function, structure, and disease association support the conclusion that 11 other codons with > or =5 somatic mutations also likely indicate functionally critical residues, even though P0.05. We calculated p16 evolution using amino acid substitution matrices and nucleotide substitution distances. We looked for evolutionary parameters at each codon that would predict whether missense mutations were disease associated or disrupted function. The current p16 evolutionary substitution database is too small to determine whether observations of 'absolute conservation' are statistically significant. Increasing the number of sequences from three to seven significantly improved the predictive value of evolutionary computations. The sensitivity and specificity for conservation scores in predicting disease association of p16 codons is 70-80%. Despite the small p16 sequence database, our calculations of high conservation correctly predicted loss of cell cycle arrest function in 75% of tested codons, and low conservation correctly predicted wild-type function in 80-90% of codons. These data validate our hypothesis that detailed evolutionary analyses help predict the consequences of missense amino-acid variants.

Response of Soybean Sudden Death Syndrome to Subsoil Tillage.

The soilborne pathogen Fusarium solani f. sp. glycines causes sudden death syndrome (SDS) of soybean. Previous research indicated that soil compaction related directly to disease foliar symptoms. Therefore, we hypothesized that decreasing soil compaction would increase soil porosity and provide a more aerated root zone that would hinder root infection by the fungus and decrease SDS foliar symptom severity. Two experimental areas (110 by 120 m) were established to evaluate the relationship between soil variables and SDS. Across the experimental area, strips (9.14 m wide) were subsoiled perpendicular to soybean rows to a depth of 40 to 45 cm, which alternated with strips that were not tilled. In both 1999 and 2000, subsoiling dramatically reduced foliar symptoms of SDS. Compared with no-till plots, subsoiled plots had lower soil bulk density, greater soil porosity, and less soil moisture. In areas where SDS occurs and soil compaction exists, the use of subsoiling can be used to reduce severity of foliar symptoms of SDS.

Expression and purification of the extracellular domain of human myelin protein zero.

Myelin protein zero (P0), an adhesion protein of the immunoglobulin superfamily, is the major protein of peripheral nervous system myelin in higher vertebrates. Protein zero is required for the formation and maintenance of myelin structure in the internode, likely through homophilic interactions at both the extracellular and the intracellular domains. Mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies of varying severity. Comparisons between the human and rat isoforms, whose three-dimensional structure has been determined by X-ray crystallography, suggest that these disease-associated genetic alterations lead to structural changes in the protein that alter P0-P0 interactions and hence affect myelin functionality. Knowing the crystal structures of native and altered human P0 isoforms could help to elucidate the structural changes in myelin membrane packing that underlie the altered functionality. Alterations of P0 extracellular domain (P0-ED) are of additional interest as previous X-ray diffraction studies on myelin membrane packing suggest that P0-ED molecules can assume distinct adhesive arrangements. Here, we describe an improved method to express and purify human P0-ED (hP0-ED) suitable for crystallographic analysis. A fusion protein consisting of maltose binding protein fused to hP0-ED was secreted to the periplasm of Escherichia coli to allow an appropriate folding pathway. The fusion protein was extracted via osmotic shock and purified by affinity chromatography. Factor Xa was used to cleave the fusion protein, and a combination of affinity and ion-exchange chromatography was used to further purify hP0-ED. We document several significant improvements to previous protocols, including bacterial growth to approximately 15 OD using orbital shakers and the use of diafiltration, which result in yields of approximately 150 mg highly pure protein per liter of medium.

TP53 mutations in breast cancer associated with BRCA1 or BRCA2 germ-line mutations: distinctive spectrum and structural distribution.

Several groups have studied the molecular pathology of inherited breast cancer. By combining several such studies, we show in this study that somatic TP53 abnormalities are more common in breast cancer associated with BRCA1 or BRCA2 germ-line mutations than in sporadic breast cancers (odds ratio, 2.8; P = 0.0003). Then, we compared the spectrum of TP53 mutations for breast cancers in the IARC TP53 mutation database with the 82 mutations reported in BRCA1/2-associated breast cancers. The spectrum differed significantly both in distribution (P < 1 x 10(-6)) and in base changes (P = 0.025). Mutations at A:T bp were more common in BRCA1/2-associated tumors and strand bias suggesting DNA repair abnormalities was found. Changes were common at TP53 codons that are not mutation hotspots. Structural modeling showed that most of these p53 non-hotspot amino acids characterized in breast tumors isolated from patients with deficient BRCA1/2 function are distributed in a region of the protein on the opposite side of the p53 DNA-binding surface. Our results suggest that BRCA1/2 mutations influence the type and distribution of TP53 mutations seen in breast cancer.

Proteobacterial histidine-biosynthetic pathways are paraphyletic.

In Lactococcus lactis there is a protein, HisZ, in the histidine-biosynthetic operon that exhibits significant sequence identity with histidyl-tRNA synthetase (HisRS) but does not aminoacylate tRNA. HisRS homologs that, like HisZ, cannot aminoacylate tRNA are represented in a highly divergent set of bacteria (including an aquificale, cyanobacteria, firmicutes, and proteobacteria), yet are missing from other bacteria, including mycrobacteria and certain proteobacteria. Phylogenetic analysis of the HisRS and HisRS-like family suggests that the HisZ proteins form a monophyletic group that attaches outside the predominant bacterial HisRS clade. These observations are consistent with a model in which the absences of HisZ from bacteria are due to its loss during evolution. It has recently been shown that HisZ from L. lactis binds to the ATP-PRPP transferase (HisG) and that both HisZ and HisG are required for catalyzing the first reaction in histidine biosynthesis. Phylogenetic analysis of HisG sequences shows conclusively that proteobacterial HisG and histidinol dehydrogenase (HisD) sequences are paraphyletic and that the partition of the Proteobacteria associated with the presence/absence of HisZ corresponds to that based on HisG and HisD paraphyly. Our results suggest that horizontal gene transfer played an important role in the evolution of the regulation of histidine biosynthesis.

Genotype-specific Trp53 mutational analysis in ultraviolet B radiation-induced skin cancers in Xpc and Xpc Trp53 mutant mice.

We have examined the mutational spectrum in the Trp53 gene from UVB radiation-induced skin cancers in Trp53+/+ and Trp53+/- mutant mice of all three possible Xpc genotypes. Mutations were detected in exons 2-10 of the Trp53 coding region in approximately 90% of >80 different skin cancers examined. In contrast to Trp53+/+ mice in which most mutations in the Trp53 gene were located in exons 5-8, the majority of the mutations in Trp53+/- mice were at other exons. We observed a high predilection for C-->T transition mutations at a unique CpG site in codon 122 (exon 4) of the Trp53 gene in Xpc-/- Trp53+/- mice. This site is not part of a pyrimidine dinucleotide. Mutations at this codon, as well as in codons 124 and 210, were observed exclusively in Xpc-/- or Xpc+/- mice. Mutations at the corresponding codons (127 and 213) in the human p53 gene have been reported in skin tumors from human patients with xeroderma pigmentosum. Hence, mutations at codons 122 (125), 124 (127), and 210 (213) may constitute signatures for defective or deficient nucleotide excision repair in mice (humans). In Xpc-/- mice, the majority of mutations were located at C residues in CpG sites, in which the C is presumably methylated. A similar bias can be deduced from studies in human XP individuals.

Defective nucleotide excision repair in xpc mutant mice and its association with cancer predisposition.

Mice that are genetically engineered are becoming increasingly more powerful tools for understanding the molecular pathology of many human hereditary diseases, especially those that confer an increased predisposition to cancer. We have generated mouse strains defective in the Xpc gene, which is required for nucleotide excision repair (NER) of DNA. Homozygous mutant mice are highly prone to skin cancer following exposure to UVB radiation, and to liver and lung cancer following exposure to the chemical carcinogen acetylaminofluorene (AAF). Skin cancer predisposition is significantly augmented when mice are additionally defective in Trp53 (p53) gene function. We also present the results of studies with mice that are heterozygous mutant in the Apex (Hap1, Ref-1) gene required for base excision repair and with mice that are defective in the mismatch repair gene Msh2. Double and triple mutant mice mutated in multiple DNA repair genes have revealed several interesting overlapping roles of DNA repair pathways in the prevention of mutation and cancer.

Distribution of plant-parasitic nematodes on sugarcane in louisiana and efficacy of nematicides.

A survey conducted from May 1995 through August 1998 revealed diverse nematode communities in Louisiana sugarcane fields. High populations of Mesocriconema, Paratrichodorus, Pratylenchus, and Tylenchorhynchus were widespread in nine sugarcane production parishes. Comparisons of plant cane and ratoon sugarcane crops indicated that nematode community levels increase significantly in successive ratoon crops. Nematicide trials evaluated the efficacy of aldicarb, ethoprop, and phorate against indigenous nematode populations. Aldicarb consistently increased the number of millable stalks, cane tonnage, and yield of sucrose in soils with a high sand content. Yield increases were concomitant with reductions in the density of the nematode community shortly after planting and at harvest. In soils with a higher clay content, the chemicals were less effective in controlling nematode populations and, as a result, yield increases were minimal.

Fabs specific for 8-oxoguanine: control of DNA binding.

Free radicals produce a broad spectrum of DNA base modifications including 7,8-dihydro-8-oxoguanine (8-oxoG). Since free radicals have been implicated in many pathologies and in aging, 8-oxoG has become a benchmark for factors that influence free radical production. Fab g37 is a monoclonal antibody that was isolated by phage display in an effort to create a reagent for detecting 8-oxoG in DNA. Although this antibody exhibited a high degree of specificity for the 8-oxoG base, it did not appear to recognize 8-oxoG when present in DNA. Fab g37 was modified using HCDR1 and HCDR2 segment shuffling and light chain shuffling. Fab 166 and Fab 366 which bound to 8-oxoG in single-stranded DNA were isolated. Fab 166 binds more selectively to single-stranded oligonucleotides containing 8-oxoG versus control oligonucleotides than does Fab 366 which binds DNA with reduced dependency on 8-oxoG. Numerous other clones were also isolated and characterized that contained a spectrum of specificities for 8-oxoG and for DNA. Analysis of the primary sequences of these clones and comparison with their binding properties suggested the importance of different complementarity determining regions and residues in determining the observed binding phenotypes. Subsequent chain shuffling experiments demonstrated that mutation of SerH53 to ArgH53 in the Fab g37 heavy chain slightly decreased the Fab's affinity for 8-oxoG but significantly improved its binding to DNA in an 8-oxoG-dependent manner. The light chain shuffling experiments also demonstrated that numerous promiscuous light chains could enhance DNA binding when paired with either the Fab g37 or Fab 166 heavy chains; however, only the Fab 166 light chain did so in an additive manner when combined with the Fab 166 heavy chain that contains ArgH53. A three-point model for Fab 166 binding to oligonucleotides containing 8-oxoG is proposed. We describe a successful attempt to generate a desired antibody specificity, which was not present in the animal's original immune response.

Streptococcus parasanguis pepO encodes an endopeptidase with structure and activity similar to those of enzymes that modulate peptide receptor signaling in eukaryotic cells.

Studies in our laboratory have identified two fimbria-associated adhesins, FimA and Fap1, of Streptococcus parasanguis FW213. In this study, we isolated and sequenced DNA fragments linked to fimA to determine if they contained additional factors associated with adherence, virulence, or survival in the host. An open reading frame just upstream and divergently transcribed from the fimA operon was identified and named pepO. Northern hybridization indicated that pepO is transcribed as a monocistronic message. pepO encodes a predicted 631-amino-acid protein with a molecular mass of approximately 70.6 kDa. PepO contains the essential motif HEXXH, typical of many zinc-dependent metalloproteases and metallopeptidases. PepO has significant sequence identity to mammalian metallopeptidases, including endothelin-converting enzyme, which converts a potent vasoconstrictor into its active form, and neutral endopeptidase (NEP), which is involved in terminating the activity of opioid peptides. The opioid peptide metenkephalin is a natural substrate of NEP. Cell extracts of FW213 cleaved metenkephalin at the same site as does NEP, while an extract from an insertionally inactivated pepO mutant did not. These results indicate that FW213 pepO encodes an enzyme with activity similar to that of known mammalian endopeptidases. Phylogenetic analysis of PepO and its homologues suggests lateral genetic exchange between bacteria and eukaryotes.

Evolutionary conservation and somatic mutation hotspot maps of p53: correlation with p53 protein structural and functional features.

Missense mutations in p53 frequently occur at 'hotspot' amino acids which are highly conserved and represent regions of structural or functional importance. Using the p53 mutation database and the p53 DNA sequences for 11 species, we more precisely defined the relationships among conservation, mutation frequency and protein structure. We aligned the p53 sequences codon-by-codon and determined the degree of substitution among them. As a whole, p53 is evolving at an average rate for a mammalian protein-coding gene. As expected, the DNA binding domain is evolving more slowly than the carboxy and amino termini. A detailed map of evolutionary conservation shows that within the DNA binding domain there are repeating peaks and valleys of higher and lower evolutionary constraint. Mutation hotspots were identified by comparing the observed distribution of mutations to the pattern expected from a random multinomial distribution. Seventy-three hotspots were identified; these 19% of codons account for 88% of all reported p53 mutations. Both high evolutionary constraint and mutation hotspots are noted at amino acids close to the protein-DNA interface and at others more distant from DNA, often buried within the core of the folded protein but sometimes on its surface. The results indicate that targeting highly conserved regions for mutational and functional analysis may be efficient strategies for the study of cancer-related genes.

Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine.

A major stable oxidation product of DNA cytosine is uracil glycol (Ug). Because of the potential of Ug to be a strong premutagenic lesion, it is important to assess whether it is a blocking lesion to DNA polymerase as is its structural counterpart, thymine glycol (Tg), and to evaluate its pairing properties. Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA polymerase I Klenow fragment (exo-). During translesion DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence contexts examined. Furthermore, only dAMP was incorporated opposite template Ug and Tg and the kinetic parameters of incorporation showed that dAMP was inserted opposite Ug more efficiently than opposite Tg. Ug opposite G and A was also recognized and removed in vitro by the E. coli DNA repair glycosylases, endonuclease III (endo III), endonuclease VIII (endo VIII), and formamidopyrimidine DNA glycosylase. The steady state kinetic parameters indicated that Ug was a better substrate for endo III and formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII, however, Tg was a better substrate.

Uracil glycol deoxynucleoside triphosphate is a better substrate for DNA polymerase I Klenow fragment than thymine glycol deoxynucleoside triphosphate.

A major stable oxidation product of DNA cytosine is 5,6-dihydroxy-5, 6-dihydrouracil (Ug). Ug can be formed directly in DNA or in the cellular nucleotide pools by deamination of the unstable primary product, cytosine glycol. Here, we synthesized dUgTP and showed that dUgTP was incorporated in place of dTTP and was a much better substrate for the model enzyme DNA polymerase I Klenow fragment lacking proofreading activity, Kf (exo-), than deoxythymidine glycol triphosphate (dTgTP). The relative efficiency for dUgTP insertion opposite A was 10 times higher than for dTgTP; however, the extension of a primer with 3' dUg was about 100 times more efficient than the extension of a primer with 3' dTg. At the insertion step, the differences in Vmax appeared to be responsible since the apparent Kms for dUgTP and dTgTP were about the same. In contrast, both the apparent Km and Vmax for elongation of dUg were markedly different from those of dTg. Molecular modeling was performed with both Tg and Ug and provides a rational structural explanation for these observations.

First Report of Erwinia carotovora subsp. carotovora Causing Bacterial Root Rot of Sweetpotato (Ipomoea batatas) in Louisiana.

Bacterial root and stem rot of sweetpotato (Ipomoea batatas (L.) Lam.) was first fully characterized in the U.S. in 1977 (2). It was thought to be caused exclusively by Erwinia chrysanthemi. Although a previous report described sweetpotato as a host for E. carotovora subsp. carotovora, based on artificial inoculations, others have reported that neither E. carotovora subsp. carotovora nor E. carotovora subsp. atroseptica decay sweetpotato storage roots (1). In October 1995, storage roots of sweetpotato cv. Beauregard were received from St. Landry Parish, LA, that displayed typical bacterial root rot. Isolations from these roots yielded bacteria that showed a similarity of 0.945 to E. carotovora subsp. carotovora with the Biolog GN Bacterial Identification System (version 3.50). This isolate (Ecc-LH) also differed from isolates of E. chrysanthemi (Ech) from sweetpotato and other hosts in that it was insensitive to erythromycin, did not produce phosphatase or lecithinase, and did not produce gas from glucose. Ecc-LH differed from known strains of E. carotovora subsp. atroseptica in that it did not produce reducing substances from sucrose or acid from palatinose. When Beauregard storage roots were inoculated by inserting micropipette tips containing 50 µl of 1.0 × 108 CFU/ml, both Ecc-LH and Ech-48 produced typical bacterial root rot symptoms. However, when they were compared by infectivity titrations at 28 to 32°C, Ecc-LH was less virulent than Ech-48. Ecc-LH had an ED50 of approximately 1.0 × 106 CFU/ml and did not cause appreciable disease below inoculum concentrations of 1.0 × 105, whereas Ech-48 had an ED50 of approximately 1.0 × 108 and caused soft rot at the lowest concentration tested, 1.0 × 103. Similar disease incidence was observed in infectivity titrations at 22 to 24°C, but Ech-48 caused less severe soft rot. E. carotovora subsp. carotovora was reisolated from inoculated storage roots and its identity was reconfirmed by Biolog. When terminal vine cuttings of Beauregard were dipped in 1.0 × 108 CFU/ml and planted in a greenhouse, bacterial stem rot symptoms developed on plants inoculated with Ech-48 at about 4 weeks postinoculation, or when new growth began. However, no symptoms developed on plants inoculated with Ecc-LH. This is the first report of natural occurrence of E. carotovora subsp. carotovora causing bacterial root rot of sweetpotato in Louisiana. E. chrysanthemi remains the most important pathogen causing bacterial soft rot in sweetpotato since it is widely associated with sweetpotato, is more virulent on storage roots and also causes a stem rot. E. carotovora subsp. carotovora can cause root rot, but has been isolated in only one location to date, is less virulent on storage roots, and apparently does not cause stem rot on the predominant cultivar in U.S. sweetpotato production, Beauregard. References: (1) C. A. Clark and J. W. Moyer. 1988. Compendium of Sweet Potato Diseases. American Phytopathological Society, St. Paul, MN. (2) N. W. Schaad and D. Brenner. Phytopathology 67:302, 1977.

Spinal cord myelin is vulnerable to decompression.

Spinal cord white matter is the major site of tissue damage resulting from decompression sickness (DCS or "the bends"). Damage is thought to result from bubble nucleation within the tissue. Why DCS occurs predominantly in the spinal cord and not in the brain is not known; neither is the exact pathological mechanism by which the spinal cord is damaged, nor how multiple sclerosis (MS)-like symptoms may ensue. To investigate the molecular basis of white matter damage, we subjected myelinated mouse tissues to varying durations of decompression, and then after recompression to one atmosphere, examined them for changes in myelin structure and composition. X-ray diffraction showed that the myelin period in spinal cord decreased by 4%, whereas those of optic and sciatic nerves were stable. The change in period was accompanied by a change in membrane bilayer profile--i.e., relative to control, the width of the bilayer decreased by approximately 6 A, whereas the interbilayer spaces each increased by approximately 3 A. The changes in electron density levels suggested a redistribution of matter from the interbilayer spaces into the lipid headgroup layers. By contrast with these structural changes, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance thin layer chromatography (HPTLC) revealed no noticeable change in myelin composition--i.e., there was no release of myelin-specific proteins or lipids. Our findings indicate that spinal cord myelin has an inherent structural vulnerability that may facilitate the targeting of this tissue during pressure changes.

Thermodynamics of the interactions of lac repressor with variants of the symmetric lac operator: effects of converting a consensus site to a non-specific site.

What are the thermodynamic consequences of the stepwise conversion of a highly specific (consensus) protein-DNA interface to one that is nonspecific? How do the magnitudes of key favorable contributions to complex stability (burial of hydrophobic surfaces and reduction of DNA phosphate charge density) change as the DNA sequence of the specific site is detuned? To address these questions we investigated the binding of lac repressor (LacI) to a series of 40 bp fragments carrying symmetric (consensus) and variant operator sequences over a range of temperatures and salt concentrations. Variant DNA sites contained symmetrical single and double base-pair substitutions at positions 4 and/or 5 [sequence: see text] in each 10 bp half site of the symmetric lac operator (Osym). Non-specific interactions were examined using a 40 bp non-operator DNA fragment. Disruption of the consensus interface by a single symmetrical substitution reduces the observed equilibrium association constant (K(obs)) for Osym by three to four orders of magnitude; double symmetrical substitutions approach the six orders in magnitude difference between specific and non-specific binding to a 40 bp fragment. At these adjacent positions in the consensus site, the free energy effects of multiple substitutions are non-additive: the first reduces /deltaG(obs)o/ by 3 to 5 kcal mol(-1), approximately halfway to the non-specific level, whereas the second is less deleterious, reducing /deltaG(obs)o/ by less than 3 kcal mol(-1). Variant-specific dependences of K(obs) on temperature and salt concentration characterize these LacI-operator interactions. In general, binding constants and standard free energies of binding both exhibit characteristic extrema near 290 K. As a consequence, both the enthalpic and entropic contributions to stability of Osym and variant complexes change from positive (i.e. entropy driven) at lower temperatures to negative (i.e. enthalpy driven) at higher temperatures, indicating that the heat capacity change upon binding, deltaC(obs)o, is large and negative. In general, /deltaC(obs)o/ decreases as the specificity and stability of the variant complex decreases. Stabilities of complexes of LacI with Osym and all variant operators are strongly [salt]-dependent. Binding constants for the variant complexes exhibit a power-dependence on [salt] that is larger in magnitude (i.e. more negative) than for Osym, but no obvious trend relates changes in contributions from the polyelectrolyte effect and the observed reductions in stability (delta deltaG(obs)o). These variant-specific thermodynamic signatures provide novel insights into the consequences of converting a consensus interface to a less specific one; such insights are not obtained from comparisons at the level of delta deltaG(obs)o. We propose that this variant-specific behavior arises from a strong effect of operator sequence on the extent of induced conformational changes in the protein (and possibly also in the DNA site) which accompany binding.

Concurrent factitious disorder and factitious disorder by proxy. Double jeopardy.

Two forms of medical dissimulation-factitious disorder and factitious disorder by proxy-present enormous challenges to clinicians accustomed to receiving valid symptom reports from their patients. The consequences of such "disease forgery" are heightened when a patient simultaneously engages in both forms of deception. We discuss a 34-year-old nurse who simulated or induced a panoply of physical and psychological ailments in both herself and her daughter. The staff's insistence on access to outside information sources proved indispensible in establishing both diagnoses, facilitating ongoing treatment for the patient and ensuring appropriate protection of the child.