Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M(r) = 72,550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K(m) for sulphate was 6.9 microM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.     

Dissection of cross-reactivities using a panel of H-2Ld alloreactive T cell hybridomas

Using recombinant antibodies functionally expressed by secretion to the periplasm in Escherichia coli as a model system, we identified mutations located in turns of the protein which reduce the formation of aggregates during in vivo folding or which influence cell stability during expression. Unexpectedly, the two effects are based on different mutations and could be separated, but both mutations act synergistically in vivo. Neither mutation increases the thermodynamic stability in vitro. However, the in vivo folding mutation correlates with the yield of oxidative folding in vitro, which is limited by the side reaction of aggregation. The in vivo folding data also correlate with the rate and activation entropy of thermally induced aggregation. This analysis shows that it is possible to engineer improved frameworks for semi-synthetic antibody libraries which may be important in maintaining library diversity. Moreover, limitations in recombinant protein expression can be overcome by single amino acid substitutions.     

3-dimensional sonography in the diagnosis of meniscal lesions. An experimental and clinical study]

There is no consensus regarding the clinical significance of conventional two-dimensional ultrasound in the diagnosis of meniscal tears of the knee. Three-dimensional ultrasound spatially reconstructs a transparent image of subsequent ultrasound scans. In an experimental study of 96 menisci, radial and oblique tears were detected more often by three-dimensional ultrasound. In a clinical study of 60 menisci the two- and three-dimensional ultrasound reached a sensitivity of 92% and 100%, a specificity of 83% and 88%, a positive predictive value of 58% and 67%, and a negative predictive value of 98% and 100%, respectively. Altogether, there was no statistically significant difference between both methods. The high negative predictive value, however, shows that the three-dimensional ultrasound may be a clinically relevant examination for special questions in the diagnostics of meniscal tears.     

Renal handling and plasma elimination kinetics of carbonic anhydrase isoenzymes I, II and III in the rat

Using a radio-immunosorbent technique, the levels of the carbonic anhydrase (CA) isoenzymes CA I, II and III in plasma (1-3 micrograms ml-1), lymph (0.5-1.6 micrograms ml-1) and urine (0.03-0.06 micrograms ml-1), were determined in the rat. The renal clearances of CA I, II and III were 11 +/- 3, 42 +/- 11 and 35 +/- 4 nl min-1 (g kidney wet wt)-1 (n = 4-5), respectively. After a single i.v. injection of purified native or 125I-labelled isoenzymes, the elimination of CA I, and CA III from plasma followed a bi-exponential decline, with half-times of 7 and 9 min for the rapid phase and 112 min for the slow phase, respectively. Nephrectomy decreased the rapid phase and the build-up of catabolites. Therefore, the rapid phase of CA I and III elimination is probably explained by filtration of unbound isoenzyme at the glomeruli and subsequent degradation by the proximal tubules. The plasma elimination curve for CA II was different and followed a mono-exponential decline, with a half-time of 210 min both in normal and nephrectomized animals. This indicates that CA II is not filtered at the glomeruli. However, in acute renal failure, with leaking tubular cells, CA II was excreted into the urine. The slow elimination of the major part of the isoenzymes from plasma is explained by the binding of CA I, II and III to a plasma protein, immunochemically similar to transferrin, forming a macromolecular complex with a mol wt of 114 +/- 2 kDa.