Chromosome duplications and deletions and their mechanisms of origin.

Duplications and deletions of the same gene loci or chromosome regions are known to produce different clinical manifestations and are significant factors in human morbidity and mortality. Extensive cytogenetic and molecular cytogenetic studies with cosmid and YAC probes in two patients with unique mosaicism for reciprocal duplication-deletion allowed us to further understand the origin of these abnormalities. The first patient's mosaic karyotype was 46,XX, inv dup(11) (q23q13)/46,XX,del(11)(q13q23). The second patient had a 46,XY,dup(7)(p11.2p13)/46,XY,del(7)(p11.2p13)/46,XY karyotype. Fluorescence in situ hybridization studies on the first patient placed the two breakpoints near the folate-sensitive fragile sites FRA11A and FRA11B. The presence of repeated sequences responsible for these fragile sites may have been involved in the patient's duplication-deletion. Our investigation leads us to conclude that, in addition to known mechanisms (such as unequal crossovers between homologs, unequal sister chromatid exchanges, excision of intrachromatid loops, and meiotic recombination within a single chromatid), duplication-deletion can also arise by the formation of an overlying loop followed by an uneven crossover at the level of the DNA strand.     

No fever and leucocytosis in response to a lipopolysaccharide challenge in an insectivorous bat

Bat immune systems may allow them to respond to zoonotic agents more efficiently than other mammals. As the first line of defence, the taxonomically conserved acute phase immune reaction of leucocytosis and fever is crucial for coping with infections, but it is unknown if this response is a key constituent to bat immunological success. We investigated the acute phase reaction to a standard lipopolysaccharide (LPS) challenge in Pallas''s mastiff bats (Molossus molossus). Challenged bats lost mass, but in contrast to other mammals showed no leucocytosis or fever. There also was no influence on body temperature reduction during torpor. When compared to recent genome-wide assays for constituent immune genes, this lack of a conserved fever response to LPS contributes to a clearer understanding of the innate immune system in bat species and of the coevolution of bats with a wide diversity of pathogens.     

Single- and double-stranded RNA measurements by flow cytometry in solid neoplasms

We investigated the ability of single- and double-stranded RNA measurements to discriminate between neoplastic and non-neoplastic solid tissues. Sixty-one solid nonhematopoietic neoplasms, 10 reactive non-neoplastic lesions, and 26 normal tissue samples were the test materials. Single-stranded ribonucleic acid (s-RNA) levels and double-stranded ribonucleic acid (ds-RNA) excess in these specimens were defined in relationship to normal human lymphocytes. The mean s-RNA index in normal, reactive, benign, and diploid and aneuploid malignant tissue samples was 0.90, 1.54, 1.9, 1.2, and 2.2, respectively. For ds-RNA, the mean excess level for normal, reactive, benign, and diploid and aneuploid malignant specimens was 8.5%, 18.5%, 51.0%, 36.0%, and 41.3%, respectively. No statistical differences in s-RNA level were found between non-neoplastic and neoplastic tissue samples. A significant difference in ds-RNA excess was found between non-neoplastic and benign, and diploid and aneuploid malignant neoplastic tissue (P less than 0.001). The specificity of s-RNA level and ds-RNA excess was 94.4% and 100%, and the sensitivity was 29.5% and 67.2%, respectively. Notably, ds-RNA determinations identified 70.0% of the diploid neoplastic samples, in contrast to 20% by s-RNA. Our preliminary data suggest that ds-RNA may be a useful parameter and may complement DNA ploidy in identification of solid neoplasms, especially if the yield of intact cells is improved.     

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB₂ by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB₂ in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB₂.