Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene

Multiple-lentigines (ML)/LEOPARD (multiple lentigines, electrocardiographic-conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome is an autosomal dominant condition--characterized by lentigines and café au lait spots, facial anomalies, cardiac defects--that shares several clinical features with Noonan syndrome (NS). We screened nine patients with ML/LEOPARD syndrome (including a mother-daughter pair) and two children with NS who had multiple café au lait spots, for mutations in the NS gene, PTPN11, and found, in 10 of 11 patients, one of two new missense mutations, in exon 7 or exon 12. Both mutations affect the PTPN11 phosphotyrosine phosphatase domain, which is involved in <30% of the NS PTPN11 mutations. The study demonstrates that ML/LEOPARD syndrome and NS are allelic disorders. The detected mutations suggest that distinct molecular and pathogenetic mechanisms cause the peculiar cutaneous manifestations of the ML/LEOPARD-syndrome subtype of NS.     

Comparative biology and pathology of oxidative stress in Alzheimer and other neurodegenerative diseases: beyond damage and response

In this review, we consider comparative aspects of the biology and pathology of oxygen radicals in neurodegenerative disease and how these findings have influenced our concept of oxidative stress. The common definition of oxidative stress is a breach of antioxidant defenses by oxygen radicals leading to damage to critical molecules and disrupted physiology. Inherent in this definition is that oxidative stress is an unstable situation, for if there is net damage, viability of the system decreases with time, leading to disequilibria and death. While this circumstance defines acute conditions, such as stroke and head trauma which result in dysfunction and death, it does not fit physiological situations or chronic diseases closely aligned to normal physiology. Therefore, we propose that oxidative modifications in Alzheimer disease may actually serve as a homeostatic response to stress resulting in a shift of neuronal priority from normal function to basic survival. This phenomenon is comparable to normal physiological conditions of metabolic decrease, such as those seen in hibernation and estivation. Thus, Alzheimer disease could be seen as part of normal aging that includes additional pathology due to inadequate homeostatic response.     

Assessing insulin resistance: application of a fasting glucose to insulin ratio in growth hormone-treated children

BACKGROUND: Insulin resistance (IR) is an important risk factor for cardiovascular disease and type-2 diabetes mellitus. Therefore simple measures of IR have been proposed to screen the at-risk patient. A fasting serum glucose (mg/dl) to plasma insulin (microU/ml) ratio (FGIR) of < 7 was recently suggested as a screening tool for IR in certain pediatric patients. METHODS: To determine the utility of simple indicators of IR, the FGIR of < 7 was applied to a group of patients with established risk for IR. The study group was comprised of non-growth hormone (GH)-deficient patients with Turner syndrome (TS, n = 92) and idiopathic short stature (ISS, n = 73) receiving GH. The occurrence of a FGIR of < 7 in these cohorts was compared to data from previous publications. RESULTS/CONCLUSIONS: The application of a FGIR of < 7 confirmed a rise in IR with GH therapy in both groups as well as a higher occurrence in the TS group, rising from 22 to 48% between 12 and 24 months of GH therapy. We conclude that simple measures of IR such as the FGIR may be useful in screening and following patients at risk for IR.     

DEFOG: a practical scheme for deciphering families of genes

We developed a novel efficient scheme, DEFOG (for "deciphering families of genes"), for determining sequences of numerous genes from a family of interest. The scheme provides a powerful means to obtain a gene family composition in species for which high-throughput genomic sequencing data are not available. DEFOG uses two key procedures. The first is a novel algorithm for designing highly degenerate primers based on a set of known genes from the family of interest. These primers are used in PCR reactions to amplify the members of the gene family. The second combines oligofingerprinting of the cloned PCR products with clustering of the clones based on their fingerprints. By selecting members from each cluster, a low-redundancy clone subset is chosen for sequencing. We applied the scheme to the human olfactory receptor (OR) genes. OR genes constitute the largest gene superfamily in the human genome, as well as in the genomes of other vertebrate species. DEFOG almost tripled the size of the initial repertoire of human ORs in a single experiment, and only 7% of the PCR clones had to be sequenced. Extremely high degeneracies, reaching over a billion combinations of distinct PCR primer pairs, proved to be very effective and yielded only 0.4% nonspecific products.     

Flexible linkers leash the substrate binding domain of SspB to a peptide module that stabilizes delivery complexes with the AAA+ ClpXP protease

SspB dimers bind proteins bearing the ssrA-degradation tag and stimulate their degradation by the ClpXP protease. Here, E. coli SspB is shown to contain a dimeric substrate binding domain of 110-120 N-terminal residues, which binds ssrA-tagged substrates but does not stimulate their degradation. The C-terminal 40-50 residues of SspB are unstructured but are required for SspB to form substrate-delivery complexes with ClpXP. A synthetic peptide containing the 10 C-terminal residues of SspB binds ClpX, stimulates its ATPase activity, and prevents SspB-mediated delivery of GFP-ssrA for ClpXP degradation. This tripartite structure--an ssrA-tag binding and dimerization domain, a flexible linker, and a short peptide module that docks with ClpX--allows SspB to deliver tagged substrates to ClpXP without interfering with their denaturation or degradation.     

Developing Inflorescences of Male and Female <Emphasis Type="Italic">Rumex acetosa</Emphasis> L. Show Differences in Gibberellin Content

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

Expression of N-formylated proteins in Escherichia coli

In bacteria, protein expression initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. In addition, because deformylation is a prerequisite for removal of the initiating methionine, this post-translational processing step is also prevented by actinonin, and the N-formyl methionine residue is retained by proteins from which it is normally removed. We have demonstrated the applicability of this system for obtaining N-modified forms of several different proteins and use one of these modified molecules to show that the N-terminal amino group is not required for ClpXP degradation of proteins bearing an N-terminal recognition signal.     

Acute cold exposure,leptin, and somatostatin analog (octreotide) modulate thyroid 5'-deiodinase activity

We investigated the effect of acute cold exposure, leptin, and the somatostatin analog octreotide (OCT) on thyroid type I (D1) and II (D2) deiodinase activities. Microsomal D1 and D2 activities were measured by the release of (125)I from (125)I-reverse triiodothyronine (rT(3)) under different assay conditions. Rats exposed to 4 degrees C (15, 30, 60, and 120 min) showed progressive reduction in thyroidal D1 and D2, reaching approximately 40% at 2 h (P < 0.05) despite increased circulating TSH (P < 0,05) associated with the higher thyroid D1 and D2 in hypothyroid rats. A single injection of leptin (8 microg/100 g body wt sc) induced increased thyroid and liver D1 (P < 0.05), but not thyroid D2, activities at 30 and 120 min, independently of the serum TSH rise shown only at 2 h. OCT (1 microg/kg body wt sc) increased D1 and D2 activity significantly 24 h after a single injection, with no changes in serum TSH. Therefore, leptin and somatostatin are potential physiological upregulators of thyroid deiodinases, and their low secretion during acute cold exposure may be a potential mechanism contributing to cold-induced reduction in thyroid deiodinase activity.