Essential role for Bim in mediating the apoptotic and antitumor activities of immunotoxins.

Protein synthesis is crucial for regulating cell homeostasis and, when unrestricted, it can lead to tumorigenesis. Immunotoxins derived from Pseudomonas exotoxin are antibody-toxin fusion proteins that inhibit protein synthesis of mammalian cells via ADP-ribosylation of the eukaryotic elongation factor-2. Here we investigate the role of the Bcl-2 family proteins in the response of cancer cells to immunotoxin challenge. Besides the well-known reduction of the prosurvival Bcl-2 family member, Mcl-1, following inhibition of protein synthesis, we show for the first time that immunotoxins also reduce the levels of selected proapoptotic BH-3-only proteins. Among these, only Bim protein levels correlated with the ability of immunotoxins to induce an apoptotic response. To support our findings, we verified that a Bim knockout completely abolished immunotoxin-mediated apoptosis. Further, mice bearing either wild-type or Bid knockout tumors responded to immunotoxin treatment with a decrease in growth kinetics, whereas mice engrafted with Bim knockout tumors showed no reduction in tumor size or prolongation of survival following immunotoxin treatment. From these results, we conclude that Bim expression is a major susceptibility factor for tumor cell death and, as such, constitutes a potential biomarker that could be evaluated before immunotoxin treatment. In support of this hypothesis, clinically, we analyzed patient cells before immunotoxin treatment and report that samples of hairy cell leukemia with high levels of Bim protein responded with a greater decrease in leukemic cell count compared with those samples expressing a low level of Bim.

p47phox-deficient immune microenvironment signals dysregulate naive T-cell apoptosis.

The phagocyte NADPH oxidase is a multicomponent enzyme complex mediating microbial killing. We find that NADPH oxidase p47(phox)-deficient (p47(phox-/-)) chronic granulomatous disease (CGD) mice develop lymph node hyperplasia even without obvious infection, where increased number of T and B lymphocytes is associated with increased percent of naïve cells and a lower T : B cell ratio than wild type. Paradoxically, despite lymphoid hyperplasia in vivo, when lymphocytes are placed in culture, p47(phox-/-) CD8(+) lymphocytes progress more rapidly to apoptosis than wild type. This is associated in cultured p47(phox-/-) CD8(+) lymphocytes with the induction of proapoptotic Bim and Puma expression, increased mitochondrial outer membrane permeabilization and depressed Bcl-2 expression. Addition of IL-7 to the culture partially corrects Bcl-2 levels in cultured p47(phox-/-) CD8(+) lymphocytes and improves the survival. Adding glucose oxidase to the culture to generate hydrogen peroxide along with IL-7 further improves p47(phox-/-) CD8(+) lymphocyte survival, but only to 30% of wild type. We conclude that p47(phox-/-) CD8(+) lymphocytes have an intrinsic survival defect likely in part related to the oxidase deficiency, but in vivo in lymph nodes of CGD mice, there are microenvironmental factors yet to be delineated that suppress the progression of apoptosis and allow the accumulation of lymphocytes leading to lymphoid hyperplasia.

Antitumor action of seminal ribonuclease, its dimeric structure, and its resistance to the cytosolic ribonuclease inhibitor.

Bovine seminal RNase (BS-RNase) is a homodimeric enzyme with a cytotoxic activity selective for tumor cells. In this study, the relationships of its cytotoxic activity to its dimeric structure and its resistance to the cytosolic RNase inhibitor (cRI) are investigated systematically by site-directed mutagenesis. The results show that (1) the dimericity of BS-RNase is essential for its full cytotoxic action; (2) the role of the dimeric structure in the antitumor activity is that of making the enzyme insensitive to the cytosolic RNase inhibitor; (3) a RNase may not be completely insensitive to cRI to exploit a full cytotoxic potential.

In vitro evolution of a dimeric variant of human pancreatic ribonuclease.

Site-directed mutagenesis of human pancreatic RNase (HP-RNase) was used as a model system for investigating the genetic events underlying the evolutionary origins of protein oligomers. HP-RNase is a monomeric enzyme with no natural tendency to oligomerize (K(d) for any dimers in solution of >280 mM). Nevertheless, deletion of five amino acid residues in the loop linking the N-terminal helix of HP-RNase to the rest of the protein was found to drive polypeptide chains to fold into dimers. These dimers could not be dissociated by heating at 70 degrees C, and small amounts of monomer were detected only in highly diluted samples. Measurement of dimer and monomer concentrations under equilibrium conditions yielded a K(d) of 1.5 microM. This implies that the deletion increases the protein propensity to dimerize at least 5.2 orders of magnitude. Moreover, the HP-RNase dimers were found to be over 4.6 orders of magnitude more stable than the dimers of bovine pancreatic RNase A obtained by lyophilization from acetic acid (K(d) > 73 mM). Cross-linking experiments with divinyl sulfone indicated that the HP-RNase dimers are stabilized by the exchange between subunits of their N-terminal helices. This generates composite active sites, i.e., each contributed by two subunit chains, that retain full enzymatic activity. Overall, these results show that a deletion of few residues in a key region of a monomeric protein can be the primary event irreversibly leading to oligomerization of the protein through the swap of a secondary structure element between protomers.

NADP+-dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1. Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases.

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+-dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five-residue insertion in a loop between two alpha-helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold-active enzymes.

Predictors of mortality in hemodialysis patients.

Serum biochemical measures suggestive of undernutrition have been reported to correlate with 1-yr mortality risk in prevalent groups of hemodialysis patients. The predictive power of these variables has not been reported in newly diagnosed patients or in patients whose dialysis prescription is guided by urea kinetics. The relationship of these predictors to mortality over periods of longer than 1 yr is also unreported. Therefore, the survival of 184 hemodialysis patients was examined for up to 44 months (1987 to 1991) with the Cox proportional hazards model. Baseline demographic, clinical, and biochemical parameters were used as independent variables. To adjust for bias in patient selection, the survival of patients with 12 months or less of prior dialysis at the time of enrollment ("new cases") was analyzed separately from that of patients with more than 1 yr of prior treatment ("long-standing cases"). Serum albumin was less than 3.5 g/dL in 31% of new cases and in 12% of long-standing cases. Adjusting for the other variables, low serum albumin was the strongest mortality risk predictor in both new and long-standing cases. Low serum cholesterol was an independent risk predictor in both groups. Diabetes and race were not significant predictors. Mean age at enrollment was nearly a decade higher for nonsurvivors than for survivors, in both new and long-standing groups. Yet, age was not an independent risk predictor in the Cox model for the new group because of an unexpectedly high death rate among young black men. Female gender, which was confounded by increased age, took the place of age in the model for the new group. For each model, there was good agreement between observed and predicted mortality for up to 24 months. To assess the influence of dialysis treatment time and dose (measured as pre-to-post treatment urea ratio) on risk, survival was examined in a subset of 139 patients monitored for up to 22 months, from 1989 to 1991, a period when the urea ratio was used routinely. Adjusting for the other variables, low serum albumin and cholesterol again independently increased risk. The urea ratio was also a significant independent predictor. The pattern of mortality by urea ratio was U shaped, with minimum risk for values between 2.5 and 3.4 Treatment time did not influence risk. It was concluded that baseline serum values of albumin and cholesterol strongly influence survival for up to 2 yr in new and long-standing hemodialysis patients.(ABSTRACT TRUNCATED AT 400 WORDS)

The uremic dyslipidemia: a cross-sectional and longitudinal study.

Patients on maintenance hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) exhibit numerous disturbances of serum lipids and apoproteins that may contribute to their high cardiovascular mortality. Cross-sectional studies have found that lipid levels are inversely related to time on dialysis. However, it is not known whether this association is the result of the attrition of hyperlipidemic patients or a decrease in lipid levels over time in all patients. Additionally, few studies have investigated the effect of dialysis modality on the lipoprotein disturbances of uremia adjusting for the confounding influences of demographics, or nutritional and endocrine status. To address these issues, we undertook a cross-sectional and longitudinal study of lipids, apoproteins, and atherogenic risk ratios in patients maintained on HD and CAPD. Patients were enrolled in annual cohorts from 1987 to 1990 and monitored until 1991. A total of 196 HD and 77 CAPD patients were studied. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), apoprotein (apo) A-I, and apo B were measured on enrollment and remeasured annually in survivors through 1990. Using multivariate methods, we examined the relationship of the lipids, apoproteins, their respective ratios, and their changes over time, to a broad range of clinical factors and to mortality. Compared with HD patients, CAPD patients had significantly higher TC, apo A-I, and apo B, and a significantly lower apo A-I/apo B ratio. Serum albumin correlated directly with TC and apo B and inversely with apo A-I/apo B. For patients with normal serum albumin (> or = 3.5 g/dL [35 g/L]), CAPD patients had a significantly higher TC/HDL-C than HD patients; otherwise the ratios were similar for CAPD and HD. Independent influences on lipoprotein levels in HD and CAPD patients were also demonstrated for race, gender, and diabetes, but not for parathyroid hormone (PTH) levels. For both dialysis modalities, patients who died had significantly lower TC and apo B, and significantly higher apo A-I/apo B throughout their entire courses compared with survivors. In the subset of patients followed longitudinally for 2 or more years, apo B tended to decrease with time, but TC, HDL-C, and apo A-I were stable. The longitudinal changes in lipoproteins did not correlate with outcome or other factors. In conclusion, CAPD patients have more atherogenic lipoprotein profiles than HD patients. Improved visceral protein nutritional status, as defined by serum albumin level, is associated with hyperlipidemia and, especially vor CAPD, worsened atherogenic risk ratios.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipid abnormalities in black renal patients.

Racial differences in lipoprotein (LP) and cardiovascular (CV) abnormalities have been noted in the general population and in the population of patients on dialysis. Few studies have investigated the interaction of race and LP and CV disturbances in other renal disease groups. We studied lipid profiles and risk ratios (total cholesterol (TC)/high density lipoprotein-cholesterol) (HDL-C) and apolipoprotein (apo) A-I/apo B (A-I/B)) and the influence of race across a spectrum of renal disease groups (normal renal function (NRD), nephrotic range proteinuria (NS), hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD), post-transplant (TR), renal insufficiency (RI)). We also performed a longitudinal study of lipid profiles in patients with end stage renal disease (ESRD) and the relationship of these profiles to race and other variables. There was a general tendency towards a better CV risk profile for blacks than whites in all the groups. Blacks tended to have lower TC, higher HDL-C, lower TC/HDL-C, higher apo A-I, lower apo B, and higher A-I/B. We analyzed four yearly cross-sections of the HD and CAPD populations using ANOVA with adjustment for appropriate covariates. Whites had lower HDL-C and a higher TC/HDL-C risk ratio than blacks. HD patients had lower TC, TC/HDL-C, apo A-I, and apo B than CAPD patients, and women had higher TC than men. When lipid profiles were studied longitudinally by yearly intervals, no consistent significant changes were seen, but over two years, levels of apo B fell and A-I/B rose. Race had no significant effect on any of the longitudinal data.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodialysis and the elderly patient: potential advantages as to quality of life, urea generation, serum creatinine, and less interdialytic weight gain.

A total of 204 patients treated by maintenance hemodialysis or continuous ambulatory peritoneal dialysis (CAPD) were studied to ascertain how advancing age influences adaptation to uremia therapy. No difference in Karnofsky score was noted among patients over 70 years of age and two groups of patients, 16 to 59, and 60 to 69 years of age, respectively. In a subset of 33 hemodialysis patients studied midweek, it was noted that increasing age is associated with a lower serum creatinine concentration, lower interdialytic weight gain, and a lower urea generation rate. These three findings contribute to a relative ease in treating older uremia patients with hemodialysis or CAPD, as they tend to be stable and compliant relative to younger patients.

Longitudinal survey of apolipoproteins and atherogenic risk in hemodialysis and continuous ambulatory peritoneal dialysis patients.

Total cholesterol (TC) and HDL-cholesterol (HDL-C) have been studied in dialysis patients, but a systematic study of apolipoprotein (apo) A-I, apo B, and the anti-atherogenic risk ratio, apo A-I/apo B, over time has not been done. We report lipid and apo values over 12-14 months in 55 hemodialysis (HD) and 40 continuous ambulatory peritoneal dialysis (CAPD) patients. For HD patients, mean TC fell, but not significantly, and HDL-C and TC/HDL-C, an atherogenic risk ratio, did not change over time. Apo A-I/apo B correlated with months on HD (r = 0.30, p less than 0.04) and rose significantly (p less than 0.005) during the study period. Paired t-test analysis by race, gender, and diabetes showed that in nondiabetics, apo A-I rose, apo B fell (p less than 0.05), and apo A-I/apo B improved (p less than 0.002). Similar trends were seen in all subgroups except for diabetics. For CAPD patients, total months of treatment correlated with TC/HDL-C (r = 0.46, p less than 0.05) and with HDL-C (r = -0.53, p less than 0.02), but paired t-test analysis of longitudinal data showed no significant changes in TC, HDL-C, apo A-I, apo B, TC/HDL-C, or apo A-I/apo B. The lipoprotein patterns of all patients who died were not significantly different from those of the surviving patients. Our longitudinal data reveal that lipids, apolipoproteins, and risk ratios remain stable over time on HD and CAPD. In fact, the anti-atherogenic index of apo A-I/apo B improved in HD patients, especially in nondiabetics.

Urinary red cell size: diagnostic value and determinants.

Urinary red blood cells (RBC) are usually small and morphologically abnormal in glomerular (GN) hematuria, and slightly enlarged and morphologically normal in nonglomerular (NG) hematuria. This study was performed to evaluate the diagnostic value of urinary red cell size and morphology and to investigate the mechanism of the alteration in cell size. In 34 consecutive patients with hematuria we examined the urinary RBC size distribution and mean corpuscular volume (MCV) by electronic sizing of suspensions of RBC in an isotonic medium and, in 28 cases, compared it with the presence of 50% or greater dysmorphia. In 15 consecutive cases, we correlated MCV values with urine chemistries. In two GN cases we recorded the urine MCV serially during a furosemide-induced diuresis. In vitro incubations of peripheral or urinary RBC in various electrolyte solutions prior to sizing were also performed. The MCVs were significantly lower in GN (p less than 10(-6)) and probable GN (p less than 10(-4)) than NG hematuria. A cutoff of 72 fl completely separated GN and probable GN from NG cases. Fifty percent or more 'dysmorphic' RBC were seen in 12 of 13 GN, 3 of 4 probable GN but in no NG sediments. In patients with NG hematuria, the ratio of urinary to peripheral MCV tended to be greater than unity and correlated strongly with pH (r = -0.97; p less than 0.002). The effect of pH was confirmed in vitro. Furosemide diuresis induced a partial correction of the microcytosis of GN RBC, which correlated with the changes in urine composition. Furosemide had no effect on GN cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

The impact of CAPD treatment on lipid metabolism and cardiovascular risk.

The metabolism of lipids in CAPD has not been fully elucidated. To further clarify the behavior of dyslipidemia in this setting we followed the values of total cholesterol (TC), HDL-cholesterol (HDL-C) and apolipoprotein (apo) parameters over time (12-24 months) in 40 patients and correlated these values and their ratios with clinical (age, gender, race, weight, diabetes, etc.) and biochemical (multiphastic screen) information. Mean HDL-C was lower in men (p less than 0.04), in whites, (p less than 0.03) and in diabetic patients (p less than 0.05), but there were no group differences for mean total cholesterol, mean apolipoprotein values, the atherogenic risk ratio TC/HDL-C, or the anti-atherogenic ratio apo A-I/apo B. Total months on CAPD was found to correlate positively with TC/HDL-C (p less than 0.05), an atherogenic risk factor, and to correlate negatively with HDL-C (p less than 0.02), an anti-atherogenic index. There was also a negative correlation with another anti-atherogenic index, apo A-I/apo B, which did not reach statistical significance (r = -0.41, p = NS). Counterbalancing this apparently increased atherogenic risk is the stability of individual parameters for each patient over time in this study. In fact, the good news appears to be that TC, HDL-C, apolipoproteins and the risk ratios TC/HDL-C and apo A-I/apo B all remained stable over 12-24 months (p = NS by paired t-test for all). Thus, we find no evidence for worsening of the uremic dyslipidemia over time with CAPD treatment.

Cholesterol and lipid disturbances in renal disease: the natural history of uremic dyslipidemia and the impact of hemodialysis and continuous ambulatory peritoneal dialysis.

Lipid abnormalities have been postulated to contribute to renal insufficiency by a mechanism that is analogous to atherogenesis. The majority of patients treated for chronic renal failure die of cardiovascular complications. Lipid abnormalities in this group are thought to contribute to this high mortality. Proving a causal association between dyslipidemia and accelerated atherosclerosis in the end-stage renal disease population has been confounded by the presence of other pro-atherogenic conditions in this population. The current study compiles the lipid data we have accumulated from our renal population for the years 1987 to 1989. The report is divided into three main parts: The first is a survey of lipid levels and atherogenicity indicators in groups with different types of renal disease or modalities of treatment. The second is a multivariate analysis of the relationship of clinical and biochemical variables (and their interactions) to the serum lipid and apolipoprotein levels and their ratios and their change over time in a large dialysis population. In the third study, we quantitate the peritoneal clearances of apolipoproteins A-I and B in patients undergoing continuous ambulatory peritoneal dialysis and assess the relationship of these clearances to serum lipid and lipoprotein levels and risk ratios.

Fall of cholesterol with time on dialysis: impact on atherogenicity.

The majority of patients treated for chronic renal failure die from cardiovascular complications. Lipid abnormalities in this group are thought to contribute to this high mortality. The authors have shown that hemodialysis patients with longer months on dialysis tend to have lower total cholesterol (TC) levels. This study extends data to examine lipid disturbances in 53 continuous ambulatory peritoneal dialysis (CAPD) and 126 hemodialysis patients using multivariate analysis. Longitudinal values after 9-10 months were measured in 16 CAPD patients and 52 hemodialysis patients. Analysis of covariance for all patients demonstrated an inverse correlation of TC (p = 0.003), high-density lipoprotein (HDL) cholesterol (p = 0.01), and apolipoprotein A-I levels (p less than 0.02) with months on dialysis. In addition, the TC level was higher in women (p less than 0.001) and CAPD patients (p less than 0.001), and correlated with age (p = 0.02) and albumin (p less than 0.001). HDL cholesterol was higher in blacks (p less than 0.001) but was not affected by gender, dialysis modality, age, or albumin. Diabetic status, serum parathyroid hormone, and blood pressure were not significant variables for TC or HDL-C. The atherogenic risk indicators, TC/HDL cholesterol (p less than 0.001) and apolipoprotein B (p less than 0.02) were higher in whites than in blacks. Longitudinal study revealed lower TC levels in individual hemodialysis patients (p less than 10(-6) by paired t-test), the fall being greater for patients with fewer total months on dialysis (p less than 0.04, by analysis of covariance). CAPD patients had no change in TC by paired t-test.(ABSTRACT TRUNCATED AT 250 WORDS)

Variability of peritoneal clearances for apolipoprotein and its relationship to susceptibility for atherosclerotic changes in CAPD.

Apolipoprotein Clearance and Atherogenicity in CAPD: Protein and lipoprotein loss is one of the disadvantages of CAPD. The impact of these losses on serum constituents is not fully understood. Lipoprotein disorders are observed in patients with chronic or acute renal failure or undergoing dialytic therapy with resultant increase in atherosclerotic clinical events yet these phenomenon are poorly understood, underinvestigated and underreported. Thus the impact of dietary and pharmacological steps to prevent these events are limited by lack of clinical facts. The recent emergence of effective lipid lowering agents makes a rapid analysis of parameters important. We studied the relationships between peritoneal clearance of apolipoproteins and serum atherogenicity indicators in a preliminary study of 10 CAPD patients with and without peritonitis. We measured total cholesterol (TC), HDL-Cholesterol (HDL-C), Apo A-I and Apo B and dialysate levels of Apo A-I and Apo B. Apo levels were determined immunotubidimetrically, and dialysate was concentrated by ultrafiltration. A subsequent prospective group of 10 additional patients was studied to test the relationship found in the preliminary study. In both preliminary and prospective nonperitonitis groups, the ratio of peritoneal clearance of Apo A-I to Apo B correlated strongly with the serum TC/HDL-C (r = 0.9 preliminary, r = 0.78 prospective group). There was an inverse correlation between the clearance ratio and both serum HDL-C (r = -0.71 preliminary, r = -0.77 prospective group) and serum Apo A-I/Apo B (r = -0.74 preliminary, r = -0.62 prospective group).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipids in diabetic and nondiabetic hemodialysis and CAPD patients.

To examine the question of "accelerated atherogenesis" in ESRD patients, the authors conducted a lipid survey among their hemodialysis and CAPD populations and evaluated data relevant to the lipid and diabetic status of these groups. Interestingly, longer duration of hemodialysis in nondiabetic patients was associated with lower cholesterol but not HDL, which may reflect diminished cardiac risk and mortality in the long-term survivors. Diabetic patients showed lower HDL but no cholesterol change with longer duration on hemodialysis. Type I diabetics showed the highest HDL and lowest risk ratio and a strong inverse correlation between PTH and HDL. This study highlights the role of lipids in the atherogenesis of uremia and serves as a baseline for a longitudinal study.

Specificity and biological activity of the protein deposited on the hydrogel surface. Relationship of polymer structure to biofilm formation.

The in-situ lens-bound protein layer (LBPL) was characterized on hydrogels of varying water content and ionic-binding capacity. The LBPL proved to be critically dependent on the ionic binding capacity of a given hydrogel. On nonionic polymers the LBPL invariably was thin and largely insoluble. Histochemical staining allowed the detection of all major types of tear proteins. Amino acid analysis revealed a variable composition. Extractable protein proved devoid of active lysozyme. Electrophoresis of pooled samples revealed a variable mixture of acidic, neutral, and basic bands. To what extent variability is dependent on tear film composition and lens structure awaits use of more sensitive analytic procedures. On anionic hydroxyethylmethacrylate copolymer lenses, the LBPL proved radically different. Here the LBPL invariably was much thicker and composed primarily of loosely bound protein. Electrophoresis and enzymatic analysis revealed a homogenous layer consisting primarily of lysozyme much of which retains enzymatic activity. The amino acid analysis of the insoluble protein suggests a similar composition. Specificity of deposition can be attributed to ionic affinity. Conformational integrity can be attributed partly to the unique stability of lysozyme. Electrophoresis of a pooled anionic lens extract revealed an unknown, highly mobile, basic protein. This presumably represents the selective accumulation of a highly basic trace or transient constituent of the tear film. The specificity and biological activity of the LBPL on the anionic lens may modify hydrogel biocompatibility affecting risk of spoilage, microbial colonization, and propensity to trigger an inflammatory and immune response.