Significant reduction of factor D and immunosuppressive complement fragment Ba by hemofiltration.

Because of their effect on the immune response, especially in patients with chronic or acute renal failure, factor D (FD) and the immunosuppressive complement fragment Ba are substances which may be important for the immunological status. Since they cannot be eliminated by conventional Cuprophan hemodialysis because of their high molecular weight (24,000 and 33,000 D, respectively), the effect of hemofiltration (HF) on the plasma concentration of both components was tested. It was shown that plasma levels of FD can be lowered by 43.5% during an HF treatment and the plasma concentration of Ba by 30.6%. Moreover, the two substances could be detected in the hemofiltrate. Up to 75 mg FD and up to 37 mg Ba could be eliminated per treatment, depending on the plasma concentrations and the filtration volume. A convective method such as chronic HF is therefore clearly superior to diffusive methods of blood purification when substances with such a high molecular weight have to be eliminated. It has still to be established whether the elimination of FD and Ba by chronic intermittent HF results in a sustained improvement in the immunological status of patients treated in this way.

Promoter recognition in archaea is mediated by transcription factors: identification of transcription factor aTFB from Methanococcus thermolithotrophicus as archaeal TATA-binding protein.

At least two transcription factors, aTFB and aTFA, are required for accurate and faithful in vitro transcription of homologous templates in cell-free extracts from the methanogenic Archaeon Methanococcus thermolithotrophicus. We have recently shown that the function of aTFB can be replaced by eucaryal TATA-binding proteins. Here we demonstrate using template commitment experiments that promoter recognition in an Archaeon is mediated by transcription factors. The archaeal TATA box was identified as recognition site for binding of aTFB by gel shift analyses. aTFB binds also to the TATA box of adenovirus 2 major late promoter suggesting homology of eucaryal and archaeal TATA boxes. Our analyses provide evidence for a common molecular mechanism of transcription initiation by eucaryal RNA polymerases and archaeal RNA polymerase. They indicate also an evolutionary homology for aTFB and TBP.

Functional interaction of yeast and human TATA-binding proteins with an archaeal RNA polymerase and promoter.

TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show that eucaryal TBP is stably sequestered at the archaeal promoter and that this interaction is further stabilized in combination with aTFA. Binding studies revealed that recognition of an archaeal promoter by TBP involves specific binding to the TATA box. These findings demonstrate a common function of TBP and aTFB and imply a common evolutionary origin of eucaryal and archaeal transcriptional machinery.

Surface modifying additives for improved device-blood compatibility.

Copolymers composed of polar and nonpolar blocks, when blended with a base polymer in low concentrations, migrate to the base polymer surface during and after fabrication. Migration of these surface modifying additives (SMAs) dramatically changes the outermost surface molecular layers that comprise the region that determines biocompatibility. The blood compatibility of cardiopulmonary bypass and hemodialysis components have been improved by using SMA blended polymers or SMA coated surfaces. The particular SMAs used were a series of triblock copolymers with a general formulation of polycaprolactone-polydimethylsiloxane-polycaprolactone. X-ray fluorescence (XRF), fourier transform infrared (FTIR), refractive increments (RI), and gel permeation chromatography (GPC) were used to characterize the molecular weight of SMA and the bulk concentration of SMA after blending. Electron spectroscopy for chemical analysis (ESCA) proved that the surface of blended polymers was highly saturated with SMA. Results of in vitro experiments with human blood demonstrated that SMA blended polymers delay contact activation (kallikrein-like activity), reduce coagulation activity (thrombin-antithrombin [TAT] generation), and do not adversely affect complement activation (terminal complement complex [TCC] generation) or mononuclear cells activation (IL-1 beta production). Ex vivo canine AV shunt studies showed improvement of platelet compatibility of SMA blended polymers. Reduction of cellular and protein system activation by using components fabricated with SMA blood contacting surfaces can potentially result in reduced morbidity associated with extracorporeal circulation.

Studies of biocompatibility of different dialyzer membranes: role of complement system, intracellular calcium and inositol-triphosphate.

PMNLs are activated during extracorporeal circulation. The aim of this cross-over biocompatibility study was to investigate the role of complement system, intracellular calcium [Ca2+]i and inositol-triphosphate (IP3) on PMNL degranulation during hemodialysis (HD) with following membranes: polyamide, hemophane and cuprophane. In a second study the effect of complement system, intracellular calcium and IP3 on lactoferrin release during HD with polysulfone and polymethylmethacrylate (PMMA) was also investigated. HD with cuprophane leads to the highest formation of terminal complement component (TCC) followed by PMMA and hemophane. There was a strong correlation between maximal arterial TCC formation and procentual increase of plasma lactoferrin during hemodialysis treatment with all membranes. Both HD with PMMA and hemophane leads to a significant increase of resting [Ca2+]i after 30 minutes of HD. Lowest TCC formation and lowest rise in [Ca2+]i were observed with polysulfone and polyamide. Procentual and absolute increase of [Ca2+]i did also correlate with maximal TCC formation during HD using PMMA, hemophane, polyamide and polysulfone. Since cuprophane induces an initial drop of PMNLs, these cells could not be isolated during HD with cuprophane membranes. Resting PMNL IP3 values were similar before and 30 minutes after begin of hemodialysis and comparable with all membranes used. These data indicate that TCC and intracellular calcium are important signals for PMNL degranulation during HD with cuprophane, PMMA and hemophane. However, mild degranulation of specific PMNL granules can also occur in the absence of significant change in TCC, [Ca2+]i or IP3 levels during HD with polyamide or polysulfone.

Bacteria- and endotoxin-free dialysis fluid for use in chronic hemodialysis.

As the quality of water in the dialysis fluid varies considerably, dialysis fluid is contaminated with a high percentage of bacteria and endotoxins. The bacterial populations contained in the dialysis fluid are as heterogeneous as the chemical structure of the endotoxins that result. The latter can pass through the dialysis membrane whereby high-flux membranes permit a larger number of retransportable molecules than low-flux membranes. A central aim toward a future, safe dialysis process should, therefore, be the production of a dialysate that is free of bacteria and endotoxins. As we were able to demonstrate in various examinations, this goal is most likely to be achieved with the aid of sterile filtration using hollow fiber modules of polyamid. To avoid disinfection of the polyamid membrane, as this would only reach bacteria but not endotoxins, the filter was changed after at most 10 h. The achieved dialysis fluid was free of bacteria and endotoxins. We were also able to show that the release of interleukin-1 was reduced. In addition, side-effects, such as a drop in blood pressure, headaches, muscular cramps, and nausea, were reduced.

Role of proteinase/antiproteinase inhibitor disequilibrium in the bioincompatibility induced by artificial surfaces.

As one aspect of bioincompatibility, the importance of activation of proteolytic systems as a result of an imbalance between protease and antiprotease activity has been increasingly recognized. This principle is illustrated by selected studies in our laboratory. These concern (i) generation of kinins on membranes with negative surface charge, (ii) activation of the complement system as a function of binding to the membrane of the regulatory protein H, (iii) generation of thrombin-antithrombin complexes (TAT), and (iv) generation of plasmin/antiplasmin complexes with an interesting discrepancy between in vivo and in vitro.

Cell-free transcription of the nifH1 gene of Methanococcus thermolithotrophicus indicates that promoters of archaeal nif genes share basic features with the methanogen consensus promoter.

The nifH1 gene of Methanococcus thermolithotrophicus, which encodes the putative dinitrogenase reductase of an archaeon, was accurately transcribed in a homologous cell-free transcription system. Extracts of cells grown with N2 or ammonia as nitrogen source initiated transcription at the nifH1 promoter with similar efficiencies. We confirmed that cells grown under non-N2-fixing conditions do not contain significant amounts of nifH1-specific mRNA. The levels of cell-free transcription initiation at the nifH1 promoter were similar to those observed at a tRNA promoter. The DNA sequence from -40 to +5 relative to the initiator nucleotide of nifH1 mRNA contained all the information required for promoter activity. A mutational analysis of this section of DNA demonstrated that a TATA box at -25 and the TTGT motif (initiator element) at the transcription start site are essential for cell-free transcription. These elements are similar to the structural determinants of a known tRNA promoter of Methanococcus. Mutation of a sequence, showing homology to the bacterial NifA site, which overlaps the transcription start site, did not affect promoter activity. Hence, cell-free transcription of the Methanococcus nifH1 gene is independent of upstream activator elements and does not require alternate cis-acting sequences that differ from the methanogen consensus promoter. These findings suggest that the activation of nif promoters is brought about by fundamentally different mechanisms in Archaea and bacteria.

Basic features of the polyamide membranes.

Progress in membrane and membrane process development have contributed to the continuous improvement of the extracorporeal treatment of renal failure during the last 40 years. Today membranes can be adapted to the specific needs required by their clinical application. Synthetic structures like the polyamide membranes offer a wide range of possibilities in performance and hemocompatibility, due to: (1) the use of block polymers or polymer blends and mixtures, and (2) modification of membrane structure, pore size and porosity, due to changes of the preparation process. However, further improvements are still required to achieve the ultimate goal of matching the performance of biological membranes.

An archaebacterial cell-free transcription system. The expression of tRNA genes from Methanococcus vannielii is mediated by a transcription factor.

Our understanding of the mechanism of RNA biosynthesis in archaebacteria is limited, due in part to the inability of purified RNA polymerases to transcribe purified genes accurately in vitro. In the present study, we show that cell extracts of Methanococcus vannielii and Methanococcus thermolithotrophicus purified by gradient centrifugation synthesize a distinct transcript from templates harboring a cloned homologous tRNA(Val) and tRNA(Arg) gene. The in vitro transcripts initiate with GTP at the same sites as in Methanococcus cells. About 60% of the sequence of the in vitro RNA products was analyzed by dideoxyterminated primer extension and found to be identical with that of the precursors of tRNA(Val) and tRNA(Arg). This finding indicates that this RNA polymerase fraction both initiates and terminates transcription faithfully in vitro. After purification of a cell-free extract (S-100) of M. thermolithotrophicus by phosphocellulose chromatography, the endogenous RNA polymerase has lost its ability to transcribe the tRNA(Val) gene accurately. The activity directing specific expression of this template was reconstituted by the addition of a protein-fraction devoid of RNA polymerase activity. Thus, a transcription factor appears to be required for accurate cell-free expression of tRNA genes from M. vannielii.

PC-PE hollow-fiber membrane. Structure, performance characteristics and manufacturing.

Polyether-polycarbonate hollow fibers, spun by the phase inversion method, yield a dialysis membrane with a limited ultrafiltration coefficient but high diffusive permeability. Dialysers are made out of this membrane and are sterilized by gamma radiation. The dialysers show controlled ultrafiltration and clearance values for smaller molecular weight substances in the range of cellulosic membranes, but for larger molecular weight substances the clearances are higher. Correlation for ultrafiltration and clearance values, measured in saline and whole blood, indicate low interactions between blood and membrane.

Hyperthermic perfusion in malignant melanoma: 5-year results.

Between December 1975 and December 1980 a total of 154 patients with potentially curable malignant melanoma were treated by adjuvant hyperthermic perfusion. The basic therapy consisted in local excision of the primary tumor with elective dissection of the regional lymph nodes. The 4- and 5-year survival rates for our 103 patients with stage-I disease who were perfused are 90% +/- 9% and 80% +/- 17%. The 4- and 5-year survival rates for 51 patients with stage-II disease are 50% +/- 16% and 37% +/- 28%. Compared with historic control groups of patients who were treated at our hospital with the same surgical methods but without perfusion, essentially better results were achieved with adjuvant hyperthermic perfusion. It is concluded from our results that hyperthermic perfusion can further improve the prognosis for patients with malignant melanomas of the limbs.