OBJECTIVE: Anti-DNA antibodies are believed to be important in the pathogenesis of systemic lupus erythematosus (SLE). Antibodies that bind specifically and with high affinity to dsDNA are most closely involved in tissue damage. Analysis of the sequences of the variable regions of human monoclonal anti-DNA antibodies is useful in defining the structural features that give rise to these binding properties. This article systematically reviews the evidence derived from such sequences. METHOD: Previous reviews of this subject have been hampered by incomplete knowledge of the human immunoglobulin variable region repertoire. In this article, the original sequence data from reports of over 50 human monoclonal antibodies (mAb) are reinterpreted by alignment to the most similar alleles of the most similar germline genes. This allows accurate estimation of the site and nature of somatic mutations. RESULTS: Human IgG monoclonal anti-DNA antibodies generally carry more mutations than IgM. In many cases these have been selected by an antigen-driven process. In many of the more specific, higher affinity dsDNA binders, there is an accumulation of basic residues in the complementarity determining regions. However, many exceptions to this rule exist, particularly among IgM mAb. CONCLUSIONS: Unlike murine anti-DNA antibodies, these human mAb show little evidence for preferential use of particular V(H), V(K) and V(lambda) genes or families to encode antibodies of this specificity.

Antibodies, Antinuclear↗

PubMed1998

The role of in vitro expression systems in the investigation of antibodies to DNA.

OBJECTIVES: Antibodies to DNA are believed to be important in the development of tissue inflammation and clinical activity in systemic lupus erythematosus (SLE). Sequence analysis of monoclonal murine and human anti-DNA antibodies suggests that somatic mutations and basic residues are important features at the DNA-binding site. To test this hypothesis, it is possible to alter these residues by site-directed mutagenesis of cloned variable region cDNA. The mutagenized cDNA sequence is then expressed in the form of a protein molecule whose properties can be tested in assays of binding or pathogenicity. The purpose of this article is to provide a systematic review of the evidence derived by such methods in the study of anti-DNA antibodies. METHODS: Various different expression systems are available. Experiments using bacterial and eukaryotic expression systems are considered in turn. The advantages and disadvantages of the systems are described and the results obtained are compared. RESULTS AND CONCLUSIONS: High yields of antibody fragments such as scFv and Fab can be achieved by expression in bacteria. Such studies tend to confirm that reversion of somatic mutations or removal of basic residues at the antigen binding site reduce affinity for DNA. Tests of pathogenicity can only be performed by expressing whole antibodies in eukaryotic cells. The limited data available from expression of mutagenized cDNA in such systems argue against a simple relationship between changes in DNA binding affinity and changes in pathogenic potential. Further studies are therefore required to analyze the sequence requirements for pathogenicity.

Animals↗

PubMed1998

Antibacterial steroidal alkaloids from Sarcococca saligna.

Two new pregnane-type steroidal alkaloids, saligcinnamide [(20S,2'E)-20-(N,N-dimethylamino)-3beta-(3'-phenyl-2'-propenyl-N-meth ylamido)pregnane](1) and N(a)-methyl epipachysamine-D [(20S)-20-(N,N-dimethylamino)-3beta-(N-methylbenzamido)pregnane](2 ), along with a known base, epipachysamine D [(20S)-20-(N,N-dimethylamino)-3beta-(benzamido)pregnane] (3), were isolated from the EtOH extracts of the roots and stems of Sarcococca saligna. The new bases exhibited antibacterial activity against several human pathogenic bacteria. Two derivatives of 1, dihydrosaligcinnarnide [(20S)-20-(N,N-dimethylamino)-3beta-(3'-phenylpropionoyl-N-meth ylamido)pregnane](4) and dihydrosaligcinnamine [(20S)-20-(N,N-dimethylamino)-3beta-N-(3'-phenylpropyl-N-methylamino)pre gnane](5), and a derivative of 2, N(a)-methyl epipachysamine [(20S)-20-(N,N-dimethylamino)-3beta-(N-benzyl,N-methylamino)pregnane](6) were prepared and their antibacterial activity determined.

Alkaloids↗

PubMed1998

Permeation of unfolded basic fibroblast growth factor (bFGF) across rabbit buccal mucosa--does unfolding of bFGF enhance transport?

PURPOSE: To investigate whether recombinant human basic fibroblast growth factor (rhbFGF) would permeate freshly-excised rabbit buccal mucosa. In addition, the effect of a permeation enhancer (Na+ glycocholate) and the possibility of reversibly unfolding the globular protein to a more linear conformation to increase the permeability of the test protein was evaluated. METHODS: The in vitro flux of bFGF through freshly-excised rabbit buccal mucosa was determined using side-by-side diffusion systems. Detection of bFGF was performed using gradient elution, reversed-phase high-pressure liquid chromatography (RP-HPLC). Fluorescence spectroscopy and heparin affinity chromatography were used to assess the tertiary structure of bFGF. RESULTS: Preliminary in vitro results have demonstrated that the bFGF flux increased from 1.4 +/- 0.13 ng min-1 cm-2 to 3.2 +/- 0.38 ng min-1 cm-2 with the addition of 15 mM Na+ glycocholate (NaG) to the donor solution. Subsequent addition of guanidine HCl (GnHCl) to the donor solution (3 M) was not followed by a further increase in the flux of bFGF (2.9 +/- 0.26 ng min-1 cm-2). However, when the order of addition of the additives was reversed (GnHCl first followed by NaG), the flux of bFGF across rabbit buccal mucosa was increased. Upon addition of GnHCl, there was a significant (p < .05) increase in bFGF flux from 1.2 +/- 0.15 ng min-1 cm-2 to 5.0 +/- 0.58 ng min-1 cm-2. Addition of NaG further increased the flux to 8.5 +/- 1.1 ng min-1 cm-2 which was approximately 3- to 3.5-fold greater than that determined with the protein alone in the absence of any donor phase additives. The percent of parent bFGF remaining following a 3-hr exposure of a bFGF solution to either the mucosal, serosal, or both sides of rabbit buccal mucosa were 54.3 +/- 5.7%, 71.8 +/- 6.3%, and 36.2 +/- 5.4%, respectively with the majority of parent bFGF lost during the first 15 minutes. A model endopeptidase (endoproteinase Arg-C from mouse submaxillary gland) was shown in vitro to contribute to the loss in parent bFGF. CONCLUSIONS: The permeation of bFGF across rabbit buccal mucosa may be significantly increased by initially unfolding the protein with GnHCl and then treating the tissue with the permeation enhancer, NaG. Refolding and possible reactivation of bFGF's bioactivity may occur following membrane transport and subsequent dilution into an infinite sink.

Animals↗

PubMed1998

Effect of thyroid hormone on plasma apolipoproteins and apoA- and apoB-containing lipoprotein particles.

METHODS: Apolipoprotein and apoB- and apoA-containing lipoprotein particle concentrations were determined in 10 athyreotic patients 4 weeks after withdrawal of Synthroid replacement therapy [T4, 0.96 +/- 0.66 microgram mL-1; thyroid-stimulating hormone (TSH), 62.7 +/- 22.8 muIU mL-1] and again 4 weeks after reinstitution of treatment. RESULTS: Thyroid hormone replacement was associated with significant decreases in plasma cholesterol (TC), triglyceride (TG), apolipoprotein B and apolipoprotein C-III (P < 0.01). Both the cholesterol ester-rich LP-B particles and triglyceride-rich LP-Bc particles declined significantly in response to thyroid hormone (LP-B withdrawal 81.6 +/- 24.0 vs. replacement 65.1 +/- 22.0; LP-Bc withdrawal 14.3 +/- 6.0 vs. replacement 10.9 +/- 4.8 mg%, P < 0.01). ApoC-III also decreased in high-density lipoprotein (HDL) (apoC-III-HS), and in very low-density lipoprotein (VLDL) + low-density lipoprotein (LDL) (apoC-III-HP), but this reduction was proportionate so that the apo-C-III-HS/apoC-III-HP ratio, an indirect estimate of the efficiency of lipoprotein lipase (LPL), was unchanged. Apolipoprotein A-I concentrations also decreased significantly (withdrawal 140.7 +/- 27.0 vs. replacement 127.1 +/- 30.0 mg%, P < 0.01) in parallel with the changes in LP-A-I and LP-A-I:A-II particles (LP-A-I withdrawal 35.8 +/- 7.7 vs. replacement 31.5 +/- 6.3; LP-A-I:AII withdrawal 104.9 +/- 20.0 vs. replacement 95.5 +/- 26.0; P < 0.05). CONCLUSION: These findings indicate that thyroid hormone influences the transport not only of both TG-rich and cholesterol-rich apoB-containing lipoprotein particles but also of those that contain apoAI.

Apolipoproteins A↗

PubMed1998

Bronchial epithelial-myoepithelial carcinoma.

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Aged↗

PubMed1998

Characterization of SU1 isoamylase, a determinant of storage starch structure in maize.

Function of the maize (Zea mays) gene sugary1 (su1) is required for normal starch biosynthesis in endosperm. Homozygous su1- mutant endosperms accumulate a highly branched polysaccharide, phytoglycogen, at the expense of the normal branched component of starch, amylopectin. These data suggest that both branched polysaccharides share a common precursor, and that the product of the su1 gene, designated SU1, participates in kernel starch biosynthesis. SU1 is similar in sequence to alpha-(1-->6) glucan hydrolases (starch-debranching enzymes [DBEs]). Specific antibodies were produced and used to demonstrate that SU1 is a 79-kD protein that accumulates in endosperm coincident with the time of starch biosynthesis. Nearly full-length SU1 was expressed in Escherichia coli and purified to apparent homogeneity. Two biochemical assays confirmed that SU1 hydrolyzes alpha-(1-->6) linkages in branched polysaccharides. Determination of the specific activity of SU1 toward various substrates enabled its classification as an isoamylase. Previous studies had shown, however, that su1- mutant endosperms are deficient in a different type of DBE, a pullulanase (or R enzyme). Immunoblot analyses revealed that both SU1 and a protein detected by antibodies specific for the rice (Oryza sativa) R enzyme are missing from su1- mutant kernels. These data support the hypothesis that DBEs are directly involved in starch biosynthesis.

Isoamylase↗

PubMed1998

Developing rehabilitative behavioral interventions for long-term care: technology transfer, acceptance, and maintenance issues.

Rehabilitative behavioral interventions that are documented in clinical trials to improve nursing home resident outcomes and are recommended by practice guidelines are often not adapted for daily use in nursing homes and other long-term care (LTC) facilities. Failure to evaluate issues other than clinical efficacy when developing interventions contributes to this gap between efficacy and effectiveness in practice. A potential solution is a research model that supplements traditional clinical intervention research with methodology designed specifically to evaluate the ability of LTC facilities to implement the interventions. This paper discusses several critical issues of intervention and implementation that should be addressed, including targeting interventions, advocacy, cost-effectiveness, training, and quality control. We also describe how clinical trials could be designed and staged to increase the probability that effective interventions will be implemented in the day-to-day care of frail older patients in LTC facilities.

Aged↗

PubMed1998

What brought the adaptive immune system to vertebrates?--The jaw hypothesis and the seahorse.

A hypothesis is discussed that the adaptive immune system of vertebrates evolved in the gastrointestinal regions of primitive jawed fish (placoderms) due to increased localized injuries and infections which were inadvertently brought about by the novel jaw structures and the predatory life style. The question whether the modern jawless fish, cyclostomes, have adaptive immunity or not is briefly but critically reviewed. The discovery that the gut-associated immune tissues in mammals constitute the primary immune tissues for the local T cells and that some epithelial gamma delta T cells have a unique propensity is summarized and discussed in relation to the jaw hypothesis. Initial study of the seahorse (Hippocampus) indicates that the gut-associated immune tissues may be absent in this teleost species, suggesting an evolutionary link between the adaptive immune system and the jaw structure or eating habit.

Adaptation, Physiological↗