E Haber

A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.

Proceedings of the National Academy of Sciences (PNAS), a peer reviewed journal of the National Academy of Sciences (NAS) - an authoritative source of high-impact, original research that broadly spans the biological, physical, and social sciences.

Plasma homocysteine levels are elevated in 20-30% of all patients with premature atherosclerosis. Although elevated homocysteine levels have been recognized as an independent risk factor for myocardial infarction and stroke, the mechanism by which these elevated levels cause atherosclerosis is unknown. To understand the role of homocysteine in the pathogenesis of atherosclerosis, we examined the effect of homocysteine on the growth of both vascular smooth muscle cells and endothelial cells at concentrations similar to those observed in clinical studies. As little as 0.1 mM homocysteine caused a 25% increase in DNA synthesis, and homocysteine at 1 mM increased DNA synthesis by 4.5-fold in rat aortic smooth muscle cells (RASMC). In contrast, homocysteine caused a dose-dependent decrease in DNA synthesis in human umbilical vein endothelial cells. Homocysteine increased mRNA levels of cyclin D1 and cyclin A in RASMC by 3- and 15-fold, respectively, indicating that homocysteine induced the mRNA of cyclins important for the reentry of quiescent RASMC into the cell cycle. Furthermore, homocysteine promoted proliferation of quiescent RASMC, an effect markedly amplified by 2% serum. The growth-promoting effect of homocysteine on vascular smooth muscle cells, together with its inhibitory effect on endothelial cell growth, represents an important mechanism to explain homocysteine-induced atherosclerosis.

One hundred fifty patients with potentially life-threatening digitalis toxicity were treated with digoxin-specific antibody fragments (Fab) purified from immunoglobulin G produced in sheep. The dose of Fab fragments was equal to the amount of digoxin or digitoxin in the patient's body as estimated from medical histories or determinations of serum digoxin or digitoxin concentrations. The youngest patient received Fab fragments within several hours of birth, and the oldest patient was 94 years old. Seventy-five patients (50%) were receiving long-term digitalis therapy, 15 (10%) had taken a large overdose of digitalis accidentally, and 59 (39%) had ingested an overdose of digitalis with suicidal intent. The clinical response to Fab was unspecified in two cases, leaving 148 patients who could be evaluated. One hundred nineteen patients (80%) had resolution of all signs and symptoms of digitalis toxicity, 14 (10%) improved, and 15 (10%) showed no response. After termination of the Fab infusion, the median time to initial response was 19 minutes, and 75% of the patients had some evidence of a response by 60 minutes. There were only 14 patients with adverse events considered to possibly or probably have been caused by Fab; the most common events were rapid development of hypokalemia and exacerbation of congestive heart failure. No allergic reactions were identified in response to Fab treatment. Of patients who experienced cardiac arrest as a manifestation of digitalis toxicity, 54% survived hospitalization.(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated surface areas on proteins that would be accessible to contacts with large (1-nm radius) spherical probes. Such spheres are comparable in size to antibody domains that contain antigen-combining sites. We found that all the reported antigenic sites correspond to segments particularly accessible to a large sphere. The antigenic sites were also evident as the most prominently exposed regions (hills and ridges) in contour maps of the solvent-accessible (small-probe) surface. In myoglobin and cytochrome c, virtually all of the van der Waals surface is accessible to the large probe and therefore potentially antigenic; in myohemerythrin, distinct large-probe-inaccessible, and nonantigenic, surface regions are apparent. The correlation between large-sphere-accessibility and antigenicity in myoglobin, lysozyme, and cytochrome c appears to be better than that reported to exist between antigenicity and segmental flexibility; that is, surface regions that are rigid often constitute antigenic epitopes, whereas some of the flexible parts of the molecules do not appear antigenic. We propose that the primary reason why certain polypeptide-chain segments are antigenic is their exceptional surface exposure, making them readily available for contacts with antigen-combining sites. Exposure of these segments frequently results in high mobility and, in consequence, to the reported correlation between antigenicity and segmental flexibility.