Simultaneous ultrafiltration and affinity sorptive separation of proteins in a hollow fiber membrane module.

A protein separation scheme combining affinity or ion exchange sorption with hollow fiber cross-flow filtration is described. Sorptive gel particles were loaded into the shell side of a hollow fiber membrane module. In the adsorption step, crude protein mixtures were passed through the lumen and permeating proteins passed through the membrane to bind on the gel particles in the shell. During elution, a buffer of adequate ionic strength to desorb the bound proteins was passed through the lumen and permeated through the shell. The eluant was then collected at the outlet to the shell of the hollow fiber module. The concept is illustrated by two examples: the purification of butyrylcholinesterase (EC 3.1.1.7) from raw horse serum using the affinity gel procainamide-Sepharose as the packing and the separation of carboxylesterase (EC 3.1.1.1) from beef liver homogenate using DEAE-Sephadex as the packing. The technique has the advantage of high volumetric throughputs typical of hollow fiber membrane modules as well as the high capacity characteristic of chromatographic packings. In addition, cross-flow filtration of particulates, agglomerates, and debris in passing protein from lumen to shell side can help eliminate the need for extensive pretreatment.

Effects of a state law on rates of restraint on a child and adolescent unit.

Rates of restraint and seclusion on a child and adolescent unit were evaluated before and after the implementation of a restrictive state law which was designed to reduce the monthly rates of restraint. Overall, the total number of hours in restraint, corrected for mean daily census, decreased significantly. The average number of patients in chemical restraint stayed about the same. There was a significant increase in number of patients, number of episodes, and hours of mechanical restraint as expected. Rates of seclusion dropped to zero as mandated. A new category of physical restraint was defined by law and was used to limited extent.

Chlorophyll antenna proteins of photosystem I: topology, synthesis, and regulation of the 20-kDa subunit of Chlamydomonas light-harvesting complex of photosystem I.

The light-harvesting complex of photosystem I (LHCI) was isolated from wild-type cells of Chlamydomonas reinhardtii; the Chl a/b-protein complex contains four major polypeptides of approximately 27, 26, 24, and 20 kDa (polypeptides 14, 15, 17.2, and 22, respectively, in the nomenclature for Chlamydomonas thylakoid proteins). Antiserum against the 20-kDa subunit of LHCI was prepared and used to determine the membrane topology, subcellular site of synthesis, and cell-cycle regulation of this polypeptide. The results indicate that the 20-kDa subunit as well as the other major LHCI polypeptides are integral membrane proteins. Moreover, protease digestion experiments reveal that the 20-kDa polypeptide is completely protected by the membrane bilayer but the 27- and 26-kDa LHCI polypeptides are exposed at the membrane surface. In vivo synthesis of the 20-kDa polypeptide is sensitive to cycloheximide but not to chloramphenicol; the form of the polypeptide recovered from in vitro translations of polyadenylated RNA is approximately 24 kDa, 4 kDa larger than the mature polypeptide. It is concluded that this LHCI polypeptide is nuclear encoded and synthesized in the cytoplasm as a higher molecular weight precursor. Synthesis of the 20-kDa polypeptide is restricted to the light period in light-dark synchronized cells. Translatable mRNA for this polypeptide accumulates during the light but levels are dramatically reduced during the dark period. Thus, synthesis of the 20-kDa subunit of LHCI appears to be transcriptionally regulated during the cell cycle.

Regulation of genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase and the photosystem II polypeptides D-1 and D-2 during the cell cycle of Chlamydomonas reinhardtii.

Synthesis of the major chloroplast proteins is temporally regulated in light-dark-synchronized Chlamydomonas cells. We have used cloned chloroplast DNA probes, and in vitro and in vivo protein synthesis to examine the cell cycle regulation of photosystem II polypeptides D-1 and D-2, and the large subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase LS). Synthesis and accumulation of D-1 and D-2 mRNAs occurs during the first half of the light period (G1), correlating with increasing synthesis of the polypeptides. Rifampicin, added immediately before the light period, inhibited the normal increase in D-1, D-2 polypeptide synthesis. During the dark period D-1, D-2 mRNAs persist at high levels despite reduced rates of mRNA synthesis and translation during this period. Cell-free translation analyses indicate that the D-1 mRNA present during the dark period is efficient at directing synthesis of the D-1 precursor in vitro. We conclude that expression of the psbA (D-1) and psbD (D-2) genes are regulated primarily at the transcriptional level during the light-induction period but at the translational level for the remainder of the cell cycle. Transcripts of the RuBPCase LS gene (rbcL) are also found at high levels during the light and dark periods but, unlike D-1 and D-2, LS mRNA levels do not increase until the last half of the light period and measurable synthesis and accumulation of this mRNA occurs during the dark. Furthermore, induction of LS polypeptide synthesis during the light period is insensitive to rifampicin. We conclude that LS production is regulated primarily at the translational level during the cell cycle.

Hollow-fiber membrane bioreactors using immobilized E. coli for protein synthesis.

Actively growing Escherichia coli C600(pBR322), immobilized within the macroporous matrix of asymmetric-wall hollow-fiber membranes, has been propagated to extremely high densities, typically more than 10(12) cells/mL of accessible void volume, in some regions cells accounting for nearly 100% of the available macrovoid volume forming a tissue-like mass. Production rates of beta-lactamase, an enzyme used as an indicator of the culture's biosynthetic potential, remained at high and relatively stable levels for more than three weeks of continuous operation, and effluent supernatant enzyme activities attained 25% of the accumulated level measured in a 24-h shaker-flask culture. Based on the accessible void volume within the fiber wall, the beta-lactamase productivity was independent of the specific asymmetric membrane used. On a per cell basis, however, cells cultured using hollow-fiber membranes were only 10% as productive as those in the shaker-flask culture, possibly due to the high packing density or culture aging. By contrast, the hollow-fiber bioreactor was 100 times more productive than the shaker-flask culture on a reactor-volume basis, primarily as a consequence of the high cell densities. Reactor productivity was dependent on the number of cells in the reactor, suggesting that reactor performance was kinetically controlled and not mass transport limited.

Ethanol Production by Saccharomyces cerevisiae Immobilized in Hollow-Fiber Membrane Bioreactors.

Saccharomyces cerevisiae ATCC 4126 was grown within the macroporous matrix of asymmetric-walled polysulfone hollow-fiber membranes and on the exterior surfaces of isotropic-walled polypropylene hollow-fiber membranes. Nutrients were supplied and products were removed by single-pass perfusion of the fiber lumens. Growth of yeast cells within the macrovoids of the asymmetric-walled membranes attained densities of greater than 10 cells per ml and in some regions accounted for nearly 100% of the available macrovoid volume, forming a tissue-like mass. A radial distribution of cell packing existed across the fiber wall, indicating an inadequate glucose supply to cells located beyond 100 mum from the lumen surface. By comparison, yeast cell growth on the exterior surfaces of the isotropic-walled membranes resulted in an average density of 3.5 x 10 viable cells per ml. Ethanol production by reactors containing isotropic polypropylene fibers reached a maximum value of 26 g/liter-h based on the total reactor volume. Reactor performance depended on the fiber packing density and on the glucose medium flow rate and was limited by low nutrient and product transport rates. The inhibition of ethanol production and the reduction in fermentation efficiency arose primarily from the accumulation of CO(2) gas within the sealed reactor shell space.

Membrane lipid metabolism in Chlamydomonas reinhardtii 137+ and y-1: II. Cytochemical localization of acyltransferase activities.

We have cytochemically localized the acyltransferase activities in the alga Chlamydomonas reinhardtii. Glycerol 3-phosphate (G3P) acyltransferase and lysophosphatidate (LPA) acyltransferase activities were cytochemically assayed utilizing biochemically optimized reaction conditions (Jelsema et al., 1982). The cytochemical assays clearly demonstrate that in the wild-type cells (137+) and the y-1 mutant, both enzymes were present in the photosynthetic membranes, envelope, and pyrenoid tubules of the chloroplast. Activity was also localized to the Golgi apparatus and its associated vesicles. Additionally, LPA acyltransferase activity was found associated with the outer mitochondrial membranes. The cytochemical data from this study confirm the biochemical data obtained using purified chloroplast membranes (Jelsema et al., 1982) and establishes the presence of these glycerolipid-synthesizing enzymes in the photosynthetic membranes of the chloroplast. These findings support the earlier reports of the presence and activity of lipid-synthesizing enzymes in the chloroplast, and also is in agreement with the postulated role of the pyrenoid tubules in the synthesis of the thylakoid membranes. This report differs in that these lipid-synthesizing enzymes have been localized to the chloroplast photosynthetic membranes themselves as well as exhibiting significant levels of activity for both enzymes in the Golgi apparatus. During light-induced chloroplast biogenesis in the yellow mutant of C. reinhardtii (y-1), the photosynthetic membranes appear to be the primary site of acyltransferase activity, suggesting that in situ synthesis of the membrane lipids during this period of rapid membrane formation is the primary mode for the synthesis of the thylakoid lipids. That these intrinsic thylakoid enzyme activities are not exclusively a function of the growth phase of the cell, but are found in mature chloroplast of the 137+ cells as well, further supports the conclusion that the photosynthetic membranes of the chloroplast have the capacity for synthesis of their own membrane lipids.

Membrane lipid metabolism in Chlamydomonas reinhardtii 137+ and Y-1: I. Biochemical localization and characterization of acyltransferase activities.

The acyltransferases involved in the synthesis of the chloroplast membrane glycerolipids were analysed biochemically in dark-grown and greening Chlamydomonas reinhardtii y-1 as well as in the synchronous wild-type algae (strain 137+) and wild-type membranes. Using oleoyl-CoA as a substrate, three acyltransferase enzyme activities were detected. Glycerol-3-phosphate (glycerol-3-P) acyltransferase exhibited a pH optimum of 8.0 and was inhibited by addition of N-ethylmaleimide (MalNEt). Lysophosphatidate (PtdLys) acyltransferase exhibited a pH optimum of 7.0 and was not affected by the addition of MalNEt. From preliminary analyses, the activity at pH 5.5 appeared to be associated with dihydroxyacetone phosphate acyltransferase activity. Both glycerol-3-P and PtdLys acyltransferases were analysed further and found to be present in dark-grown and light-induced y-1 cells as well as in synchronous 137+ cells and their photosynthetic membranes. Both enzyme activities were enriched at least 10-fold in the photosynthetic membranes of 137+ chloroplasts relative to the activities present in the whole cells. This enrichment is indicative of their intrinsic localization in the thylakoids, suggesting that the photosynthetic membranes exhibit a greater degree of autonomy with respect to the synthesis of their membrane lipids than previously reported. A role for glycerol-3-P and PtdLys acyltransferases in the synthesis of the chloroplast membrane lipids is suggested further by the increases in both enzyme activities coincident with and preceding thylakoid biogenesis following light induction of dark-grown y-1 cells. Increased acyltransferase activity preceded the increase in the chlorophyll content of greening y-1 cells, which is a generally accepted marker for thylakoid synthesis. The increase in the PtdLys acyltransferase activity upon light-induction of the y-1 cells was both more immediate and more dramatic than the increase in glycerol-3-P acyltransferase activity. PtdLys acyltransferase activity was negligible in dark-grown cells and the dramatic increase upon light induction may be important in the subsequent initiation of chloroplast membrane lipid synthesis. On the basis of the localization of acyltransferase enzyme activities to the photosynthetic membranes of 137+ cells and the increase in acyltransferase activity both preceding and occurring in concert with thylakoid synthesis, we propose a direct role for the photosynthetic membranes in the synthesis of their membrane lipid components.

Mechanisms of proteinuria in diabetic nephropathy: a study of glomerular barrier function.

The fractional clearance of neutral dextrans (theta D) with Einstein-Stokes radii between 30 and 64 A was determined in normal subjects (controls, N = 15) and in diabetic patients with heavy proteinuria (advanced nephropathy, N = 16) or trace proteinuria (early nephropathy, N = 8). When plotted on log normal probability coordinates, the correlation between theta D and radius in controls and in early diabetic nephropathy was linear, suggesting that glomerular pores form one population with a normal distribution. In advanced diabetic nephropathy, however, theta D for large molecules (radius greater than 46 A) was elevated and departed from linearity suggesting a bimodal pore size distribution within the glomerular membrane. A mathematical model was devised, which revealed the mean fraction of glomerular filtrate permeating the upper pore mode to be 0.009 +/- 0.002, and the pores to be totally nondiscriminatory toward molecules with radii up to 64 A. We conclude that the development of large pores (or defects) within the glomerular membrane in advanced diabetic nephropathy permits the unrestricted passage of large plasma proteins into the urine.

Pathophysiology of hemodynamically mediated acute renal failure in man.

A tubular injury characterized by intraluminal obstruction and transtubular backleak of glomerular filtrate occurs in experimental acute renal failure (ARF) in animals. To determine whether these alterations also occur in human ARF, we studied 44 patients developing nonoliguric ARF following cardiac surgery. The delay in appearance of i.v. administered inulin in urine (Tu) was used as a measure of tubular fluid flow rate. Tu was not longer in 13 ARF patients than it was in control subjects (7.2 vs 9.0 min), suggesting that at least a subpopulation of tubules was widely patent. The fractional urinary dextran clearance profile (thetaD; radii, 20 to 40 A) was then determined in 20 patients with sustained ARF in whom inulin clearance averaged 11 +/- 1 ml/min/1.73 m2. A mass conservation model, which assumes that thetaD in Bowman's space in ARF is the same as that measured in controls, when applied to the experimental observations revealed that, on the average, 42% of filtered inulin was lost by transtubular backleak. A similar fractional inulin backleak (38%) persisted in 11 additional patients in whom ARF had begun to recover and in whom inulin clearance averaged 26 +/- 3 ml/min/1.73 m2. These findings suggest that in hemodynamically-mediated and nonoliguric ARF, (1) tubular obstruction is not homogeneous, and (2) backleak of glomerular filtrate contributes to but does not fully account for depression of inulin clearance.

Transtubular leakage of glomerular filtrate in human acute renal failure.

Ten postcardiac surgical patients with acute renal failure (ARF) were infused with inulin and dextran 40. Plasma and urine were then submitted to gel-permeation chromatography to ascertain the apparent fractional clearance profile for the dextrans. Compared to normal volunteer controls, the fractional clearance profile was substantially elevated for dextran molecules in the Einstein-Stokes radius (r) range 20-40 A. For the smaller molecules (r = 20-28 A), fractional dextran clearance in ARF was frequently in excess of unity. A simple mass conservation model which assumes that the "true" fractional dextran clearance profile for the glomerulus (in Bowman's space) in ARF is the same as that for normal controls, when applied to the experimental observations, revealed that in ARF, on the average, 50% of filtered inulin is lost by tubular backleakage. Furthermore, the model permitted an estimate of the permeability properties of the damaged tubular wall. This indicated tubular permeability not unlike that of the normal glomerulus to dextran molecules with r less than 30 A, but relative impermeability to larger dextran molecules.