Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Structural predictions of the binding site architecture for monoclonal antibody NC6.8 using computer-aided molecular modeling, ligand binding, and spectroscopy.

Monoclonal antibody NC6.8 binds the superpotent sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid with high affinity (Kd = 53 nM). Using computer-aided molecular modeling and several experimental techniques, such as competitive ligand binding, absorbance spectroscopy, and fluorescence spectroscopy, we have predicted the structure of the variable domain fragment (Fv) and identified the key residues in the combining site of the antibody. We have identified nine specific amino acids as being involved in ligand recognition and complexation. Most notable are H:33W, which is responsible for ligand-induced tryptophan fluorescence quenching, H:56R, which forms a salt bridge with the carboxylate moiety of the ligand, and L:34H, which, deep in the binding site, interacts with the cyanophenyl portion of the ligand. Two residues located deep in the putative binding pocket, H:35E and H:50E, provide the negatively charged potential for interaction with the protonated aryl nitrogen and the positive guanidinium group. These modeling predictions were made before the solution of high-resolution structures of the native Fab (2.6 A) and the Fab-ligand complex (2.2 A). Comparisons between the theoretical model and experimental native and liganded Fab structures are made.     

Measurement of cell-mediated inflammation in experimental murine autoimmune encephalomyelitis by radioisotopic labeling.

A radioisotopic index test was used to detect that time of onset and intensity of cell-mediated immune inflammation of experimental autoimmune encephalomyelitis (EAE) in mice. Mice were tested at various time intervals after an encephalitogenic immunization with mouse spinal cord to homogenate for delayed-type hypersensitivity (DTH) to myelin basic protein (MBP) by intradermal challenge with antigen in the ear pinna. After 25 hr, the intensity of DTH was measured by 125I-radiometry which depends upon the migration of 125I-UdR radiolabeled mononuclear cells into the antigen depot. Cells reactive to MBP were detected by the ear assay as early as 7 days after the initial encephalitogenic sensitization. The degree of cell-mediated immune inflammation in the brain and spinal cord during the evolution of EAE was also measured by a radioisotopic technique; increased 125I-UdR uptake could be detected in the brain 3 to 4 days before the onset of signs of EAE at days 11 to 12, whereas 125I-UdR in the spinal cord was detected only 1 day before, or concomitant with, the onset of signs of EAE. Both, or concomitant with, the onset of signs of EAE. Both the "ear" and "organ" radiometric index tests are useful in measuring the degree of cell-mediated inflammation in EAE, and supplement routine histopathological and observational assessments.     

Surface immunoglobulin on rabbit lymphoid cells. III. Double expression and separate endocytosis of surface immunoglobulin allotypes on heterozygous lymphocytes demonstrated by immunoelectron microscopic labeling.

The expression of immunoglobulin b locus (k chain) allotypes on the surface of rabbit peripheral blood lymphocytes (PBL's) is examined using an indirect double immunoelectron microscopic labeling technique. Ferritin and whelk hemocyanin individually conjugated to allotypically specific IgG are used as ultrastructurally identifiable molecular markers. These indicators are coupled to lymphocyte surface immunoglobulin (Ig) allotypic determinants by an antiallotype antibody linkage. Human red blood cells, conjugated with IgG of a specific allotype and used as test cells, demonstrate the absolute specificity and high efficiency of the ultrastructural labeling technique. Specific labeling on rabbit PBL's shows that 65–75% of the cells are positive for surface Ig. Lymphocytes from homozygous donors (b4b4 or b6b6) are labeled specifically with only the appropriate allotypic labeling system. Thirty-three percent of the PBL's from heterozygous donors (b4b6) express both allotypes (allelic inclusion) on the cell surface; the remaining proportion of Ig-bearing cells have only one detectable allotype present (allelic exclusion). We conclude that approximately 50% of the Ig-bearing PBL's demonstrate allelic inclusion for the b locus allotypes. On allelically included heterozygous lymphocytes, both allotypic determinants can undergo specific endocytosis. Endocytosis of one allotype on heterozygous cells can be induced by stimulation with antiallotypic serum without affecting the surface appearance of the other allelic marker (separate endocytosis).     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Rift Valley Fever Risk Map Model and Seroprevalence in Selected Wild Ungulates and Camels from Kenya

Since the first isolation of Rift Valley fever virus (RVFV) in the 1930s, there have been multiple epizootics and epidemics in animals and humans in sub-Saharan Africa. Prospective climate-based models have recently been developed that flag areas at risk of RVFV transmission in endemic regions based on key environmental indicators that precede Rift Valley fever (RVF) epizootics and epidemics. Although the timing and locations of human case data from the 2006–2007 RVF outbreak in Kenya have been compared to risk zones flagged by the model, seroprevalence of RVF antibodies in wildlife has not yet been analyzed in light of temporal and spatial predictions of RVF activity. Primarily wild ungulate serum samples from periods before, during, and after the 2006–2007 RVF epizootic were analyzed for the presence of RVFV IgM and/or IgG antibody. Results show an increase in RVF seropositivity from samples collected in 2007 (31.8%), compared to antibody prevalence observed from 2000–2006 (3.3%). After the epizootic, average RVF seropositivity diminished to 5% in samples collected from 2008–2009. Overlaying maps of modeled RVF risk assessments with sampling locations indicated positive RVF serology in several species of wild ungulate in or near areas flagged as being at risk for RVF. Our results establish the need to continue and expand sero-surveillance of wildlife species Kenya and elsewhere in the Horn of Africa to further calibrate and improve the RVF risk model, and better understand the dynamics of RVFV transmission.     

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.     

Ultrastructural effects of silicic acid on primary lung fibroblasts in tissue culture

Transmission and scanning electron microscopic examination of primary lung fibroblasts exposed in tissue culture to polymeric silicic acid (PSA) revealed profound cellular changes in the cell surface membranes, resulting in rapid endocytosis of affected membranes and formation of multivesicular bodies. Exposure to monomeric silicic acid did not appear to exhibit any immediate adverse effects. Appearance of numerous cytoplasmic vacuoles within 1 h of PSA exposure was easily visible by light microscopy. Electron microscopy revealed that PSA exposure caused formation of an 'osmiophilic' cell surface membrane. Numerous osmiophilic cytoplasmic blebs on the surface and subsequent endocytotic vesicles appeared to collapse and aggregate into multivesicular bodies. This study provides ultrastructural evidence of the direct interaction between lung fibroblasts and polymeric silicic acid, which has a dramatic effect the surface membrane, its subsequent internalization and cytoplasmic processing. This interaction could be one of the key steps in the damaging effects of silica containing dust.     

Combined use of regulatory elements within the cDNA to increase the production of a soluble mouse single-chain antibody, scFv, from tobacco cell suspension cultures

In order to facilitate production and secretion of a soluble form of a small, single-chain antibody ScFv (32 kDa) in tobacco cell suspension culture, several modifications were made simultaneously to the antibody cDNA that included elements that have been shown to regulate the expression of proteins in plants. The scFv cDNA was initially ligated into a binary vector under the control of the CaMV 35S promoter and the T7 terminator for expression in tobacco suspension culture. Subsequently, modifications were engineered into the cDNA for enhancement of scFv production. These included the following: (i) the signal peptide (SP) of the tobacco pathogenesis-related protein PR1a which was added in-frame to the N-terminal end of scFv cDNA; (ii) a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence), which was fused to the N-terminal end of the SP; and (iii) the endoplasmic reticulum retention signal peptide KDEL, which was added to the C-terminal end of the scFv protein. Using a modified disruption method involving pectinase, the highest expression of total scFv (344 ng scFv/g cell) occurred when the plant leader sequence, the TEV sequence, and the KDEL peptide were all present in the expression construct. Although the addition of the KDEL sequence significantly increased the total yield of protein 5.4-fold, it did not increase the overall amount of protein secreted. These studies indicate that while the SP is very important in promoting secretion of the scFv, it had little influence on increasing scFv secretion levels even when both the TEV and the KDEL sequences significantly increased overall protein levels.