Anti-α-fodrin antibodies do not add much to the diagnosis of Sjögren's syndrome

The presence of anti-α-fodrin autoantibodies has been reported to be a highly specific and sensitive test for the diagnosis of Sjögren's syndrome (SjS). We looked (in Nijmegen) for anti-α-fodrin, anti-Ro60, and anti-La autoantibodies in a cohort of 51 patients with rheumatic diseases (primary SjS [21], secondary SjS [6], rheumatoid arthritis [RA] [12], systemic lupus erythematosus [SLE] [6], and scleroderma [6]) and in 28 healthy subjects, using ELISA, immunoblotting, and immunoprecipitation. The same samples were analyzed with an alternative anti-α-fodrin ELISA in Hanover. The Nijmegen ELISA of the sera from primary SjS showed sensitivities of 43% and 48% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The Hanover ELISA showed sensitivities of 38% and 10% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The ELISAs for α-fodrin showed six (Nijmegen) and four (Hanover) anti-α-fodrin-positive RA sera. IgA and IgG anti-fodrin antibodies were also present in four patients with secondary SjS. The sensitivities of Ro60 and La-antibodies in the Nijmegen ELISA were 67% and 62%, respectively. Unlike anti-α-fodrin antibodies, all anti-Ro60 and anti-La positive sera could be confirmed by immunoblotting or RNA immunoprecipitation. Thus, anti-Ro and anti-La autoantibodies were more sensitive than anti-α-fodrin autoantibodies in ELISA and were more frequently confirmed by other techniques. Anti-La antibodies appear to be more disease-specific than anti-α-fodrin antibodies, which are also found in RA sera. Therefore, the measurement of anti-α-fodrin autoantibodies does not add much to the diagnosis of Sjögren's syndrome.     

Plasmodium falciparum: effect of anti-malarial drugs on the production and secretion characteristics of histidine-rich protein II

Plasmodium falciparum histidine-rich protein II (HRP2) is one of the best documented malaria proteins. However, little is known about the development of HRP2 concentrations under the influence of anti-malarial drugs. HRP2 levels were determined in cell medium mixture, cellular compartment, and in culture supernatant using a double-site sandwich ELISA specific for HRP2. Characteristic increases in the overall HRP2 levels were found during the later ring and the trophozoite stages. Throughout the later schizont development, rupture, and reinvasion, however, the HRP2 levels remained comparatively stable. When the cultures were exposed to serial dilutions of anti-malarial drugs, a distinct inhibition of HRP2 production was seen with increasing concentrations of drugs, resulting in sigmoid dose-response curves, similar to those obtained from conventional drug sensitivity assays. HRP2 therefore allows for a very accurate estimation of parasite development and its inhibition and may therefore be ideally suited for use in drug sensitivity or bioassays.     

Active site control of myosin cross-bridge zeta potential

The electrical properties of contractile proteins contribute to muscle structure and perhaps function but have not been characterized adequately. Electrophoretic mobility, mu(e), is sensitive to the net electric charge and hydrodynamic size of a molecule in solution. Zeta potential, zeta, particle charge, Q(e), and particle charge-to-mass ratio are proportional to mu(e). We measured mu(e) for nucleotide complexes of skeletal muscle heavy meromyosin (HMM) and subfragment 1 (S1). The results indicate that mu(e) for HMM changes depending on the ligand bound in the active site. The changes in electric charge appear to occur mainly on the S1 moieties. For HMM(MgATPgammaS)(2) and HMM(MgADP.P(i))(2) the values of mu(e) are -0.077 and -0.17 (microm/s)/(V/cm), respectively. For these complexes, mu(e) is independent of [ATP], [ADP], and [P(i)]. When P(i) dissociates from HMM(MgADP.P(i))(2) to form HMM(MgADP)(2), mu(e) decreases to -0.61 (microm/s)/(V/cm). This large decrease in mu(e) is independent of free [ADP] or [ATP]. Increasing [P(i)], on the other hand, increases mu(e) for HMM(MgADP)(2) to values near those observed for the steady-state intermediate. For HMM, mu(e) = -0.34 and is independent of P(i). MgADP binding to HMM decreases mu(e) to -0.57 (microm/s)/(V/cm), and the dissociation constant is 9 microM. Taken together, these data indicate that mu(e) and, thus, zeta are controlled by ligand binding to the active site. The magnitudes of the particle charge-to-mass ratios for the HMM complexes are all in a range that falls within published values determined for a variety of other proteins. Possible roles that the observed nucleotide-dependent changes in cross-bridge electric charge might have in the contractile cycle in muscle are considered.     

Intratumoral low-volume jet-injection for efficient nonviral gene transfer

Jet-injection has become an applicable technology among other established nonviral delivery systems, such as particle bombardment or in vivo electroporation. The low-volume jet injector employed in this study uses compressed air to inject solutions of 1.5–10 μL containing naked DNA into the desired tissue. The novel design of this prototype makes multiple jet-injections possible. Therefore, repeated jet-injections into one target tissue can be performed easily. This jet-injector hand-held system was used for the direct in vivo gene transfer of plasmid DNA into tumors to achieve efficient expression of reporter genes (β-galactosidase, green fluorescent protein [GFP]) and of therapeutic genes (TNF-α) in different tumor models. The study presented here revealed the key parameters of efficient in vivo jet-injection (jet-injection volume, pressure, jet penetration, DNA stability) to define the optimal conditions for a jet-injection-aided nonviral gene therapy.     

ELECTRON MICROSCOPY OF DNA MOLECULES "STAINED" WITH HEAVY METAL SALTS

Single DNA molecules can be rendered visible in the electron microscope by "staining" with water-soluble salts of heavy metals. The best results were obtained with lanthanum nitrate, uranyl acetate, and lead perchlorate. The molecules appear as filaments approximately 20 A wide. Their length was not determined, but it could be shown that it varied with the molecular weight of the DNA used. The same heavy metal salts will preferentially "stain" the nucleic acid in a protein-DNA complex. Evidence is provided for the possibility of a partial separation of a double-stranded molecule into single strands on adsorption to the supporting film.     

Molecular characterisation of soybean polysaccharides: an approach by size exclusion chromatography, dynamic and static light scattering methods

Water soluble polysaccharides from soybean (SSPS) have a pectin-like structure and are used as stabilisers in acidified beverages. Physicochemical properties such as structure, molecular weight and shape or conformation are primary factors controlling their functional properties. Two soybean polysaccharides, a native SSPS and a modified SSPS treated with beta-(1-->4)-D-galactosidase (GPase/SSPS) were studied by dynamic and static light scattering (DLS, SLS) and size exclusion chromatography (SEC). Consecutive filtrations using a range of membrane pore size removed a small fraction of macromolecular aggregates from dilute polysaccharide solutions with relatively little effect on the major component molecules as monitored by DLS and SEC measurements. Access to aggregate-free dilute solutions of SSPS and GPase/SSPS allowed the direct measurement of molecular characteristics. SLS results showed that SSPS had a weight average molecular weight of (645+/-11)x 10(3)g/mol and a radius of gyration, Rg, of (23.5+/-2.8)nm. By comparing R(g) with the hydrodynamic radius, Rh (21.1+/-0.5 nm) obtained from DLS, the structural parameter rho (Rg/Rh) was found to be 1.1, suggesting that SSPS has an overall globular shape due to a highly branched structure. The modified SSPS had a significantly lower molecular weight (287+/-18)x 10(3)g/mol but a similar radius of gyration (23.2+/-1.7 nm). The structure parameter rho of GPase/SSPS was higher (rho=1.3) because of a smaller hydrodynamic radius (17.7+/-1.8 nm). This suggests that GPase/SSPS has a much less branched structure yet still differs significantly from a linear random coil conformation (rho=1.7-2.0). The results derived from SLS and DLS are in agreement with the conclusions obtained from a chemical analysis where the reduction of molecular weight of GPase/SSPS was caused by the cleavage of galactan side chains.     

Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome

Birt-Hogg-Dubé syndrome (BHD), a genodermatosis characterized by multiple hamartomas of the hair follicle (fibrofolliculoma), predisposes individuals to an increased risk of developing renal neoplasms and spontaneous pneumothorax. Previously, we localized the BHD locus (also known as FLCN) to chromosome 17p11.2 by linkage analysis and subsequently identified germline mutations in a novel gene in probands from eight of the nine families with BHD in our screening panel. Affected members of five of the families inherited an insertion/deletion of a cytosine in a C8 tract in exon 11. This mutation was also identified by exon 11 screening in probands from 22 of 52 additional families with BHD and therefore represents a hypermutable "hotspot" for mutation in BHD. Here, we screened the remaining 30 families from this large BHD cohort by direct sequence analysis and identified germline BHD mutations in 84% (51/61) of all families with BHD recruited to our study. Mutations were located along the entire length of the coding region, including 16 insertion/deletion, 3 nonsense, and 3 splice-site mutations. The majority of BHD mutations were predicted to truncate the BHD protein, folliculin. Among patients with a mutation in the exon 11 hotspot, significantly fewer renal tumors were observed in patients with the C-deletion than those with the C-insertion mutation. Coding-sequence mutations were not found, however, in probands from two large families with BHD whose affected members shared their family's BHD-affected haplotype. Of the 53 families with BHD whose members inherited either a germline mutation or the affected haplotype, 24 (45%) had at least one member with renal neoplasms. Three families classified with familial renal oncocytoma were identified with BHD mutations, which represents the first disease gene associated with this rare form of renal neoplasm. This study expands the BHD-mutation spectrum and evaluates genotype-phenotype correlations among families with BHD.     

Concurrent targeted exchange of three bases in mammalian hprt by oligonucleotides

The repair of point mutations in hprt gene by single-stranded oligonucleotides represents a model to test targeted nucleotide exchange. We studied the concurrent nucleotide exchange of two or three nucleotides in the hprt deficient hamster cell line V79-151. The used oligonucleotides resulted in mismatches at two (151, 159) or three (151, 144, and 159) hprt positions. The hprt point mutation at position 151 was repaired in about 2/10(6) cells as shown by hprt sequencing in clones surviving HAT selection. The second nucleotide exchange at hprt position 159 was found in 7% of these HAT selected clones. Using oligonucleotides resulting in three mismatches, 29% of the clones showed nucleotide exchanges at the two hprt positions (151, 144) and about 4% at three positions (151, 144, and 159). These results indicate that single-stranded oligonucleotides can generate two or three nucleotide exchanges in a mammalian chromosomal gene.