Reaction of monosaccharides with proteins: possible evolutionary significance.

Measurements were made of the rate of condensation of various monosaccharides with amino groups of hemoglobin to form Schiff base linkages. The reactivity of each sugar was dependent on the extent to which it exists in the open (carbonyl) structure rather than in the ring (hemiacetal or hemiketal) structure. Among the 15 monosaccharides tested, aldoses showed higher reactivities than ketoses. Glucose was the least reactive of the aldohexoses. The emergence of glucose as the primary metabolic fuel may be due in part to the high stability of its ring structure which limits potentially deleterious nonenzymatic glycosylation of proteins.

Characterization of glycosylated hemoglobins. Relevance to monitoring of diabetic control and analysis of other proteins.

Boronate affinity chromatography and ion exchange chromatography were used to measure the levels of glycosylated hemoglobins in normal and diabetic hemolysates, as well as the distribution of glucose adducts on alpha-NH2-valine and epsilon-NH2-lysine residues. When analyzed by ion exchange chromatography on BioRex 70 resin, the Hb Alc peak comprised 4.4 +/- 0.6% of 15 normal hemolysates and 9.1 +/- 2.1% of 15 diabetic hemolysates. The "Hb Alc" was rechromatographed on GlycoGel B boronate affinity resin that binds vicinal hydroxyl groups of covalently linked sugars. Only 70 +/- 5% of the hemoglobin adhered to the resin. Analysis by the thiobarbituric acid colorimetric test confirmed that the affinity resin effectively separated glycosylated from nonglycosylated hemoglobin. When corrected for nonglycosylated contaminants, the mean level of Hb Alc in normal hemolysates was 2.9 +/- 0.4%, a value considerably lower than those previously reported. In addition to Hb Alc, 5.2 +/- 0.5% of the remaining hemoglobin (Hb Ao) was glycosylated. In diabetics, glycosylated Ao was increased in parallel with Hb Alc. After reduction with [3H]borohydride and acid hydrolysis, glycosylated amino acids were first purified on Affi-Gel boronate affinity resin and then analyzed by ion exchange chromatography. The glucose adducts on Hb Ao were distributed as follows: alpha-chain N-terminal valine, 14%; alpha-chain lysines, 40%; beta-chain lysines, 46%. This study has revealed several pitfalls in the analysis of nonenzymatically glycosylated proteins. Peaks isolated by ion exchange chromatography or electrophoresis are likely to be contaminated by nonglycosylated proteins. Furthermore, both the thiobarbituric acid test and [3H]borohydride reduction show variable reactivity depending upon the site of the ketoamine-linked glucose.

Human keratinocyte culture. Identification and staging of epidermal cell subpopulations.

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics. The first subpopulation was composed of small basal keratinocytes with low RNA content and a long generation time. The second subpopulation consisted of larger keratinocytes, having higher RNA content and a significantly shorter generation time. Finally, the third subpopulation contained the largest cells, which did not divide, and represent the more terminally differentiated keratinocytes. This in vitro approach provides discriminating cytochemical parameters by which the maturity of the epidermal cell sheets can be assessed prior to grafting onto human burn patients.

Butyrate-induced cytoarchitectural reorganization of Mallory body-containing rat hepatic tumor cells.

Diethylnitrosamine (CAS: 55-18-5)-transformed 72/22 rat hepatic tumor cells undergo marked cytoarchitectural changes during exposure to sodium butyrate in vitro. Butyrate treatment of this cell line resulted in an increased cell size, volume, and protein content and in structural reorganization within both the intermediate filament and microfilament networks resulting in the generation of a more normal appearing hepatocytic phenotype. Induced changes in the microfilament system involved the accumulation of F-actin at the cellular margins in the form of a peripheral band and in the development of an extensive, predominantly centralized network of thickened cytoplasmic filament bundles. Such butyrate-induced changes in hepatic tumor cellular morphology and microfilament organization were reflected in a 26-51% increase in the amount of cytoskeletal-associated actin in 72/22 cells, as determined by flow cytofluorimetry of permeabilized intact cells or by scanning densitometry of the electrophoretically separated, detergent-resistant cytoskeletal protein fraction, respectively. It is unlikely that this increase in cellular microfilament content was due to a direct effect of butyrate on actin polymerization per se since butyrate (in final concentrations equal to that used in culture) did not alter either actin monomer-polymer transitions or the nucleation reaction in a defined in vitro polymerization assay. The available data suggest that butyrate may regulate the synthesis or modulate the actin-binding capacity of microfilament-associating proteins in cultured cells. Butyrate-induced "normalization" of 72/22 cytoarchitecture was previously shown to be reflected in a reduction or loss in the expression of specific growth traits characteristic of the transformed phenotype. The experimental reversal of defined cytoarchitectural abnormalities and transformed growth characteristics of 72/22 cells by butyrate provided an in vitro model to elucidate both particular cytoskeletal events associated with epithelial cell transformation and the mechanism of action of apparent differentiation-inducing agents, such as sodium butyrate, on responsive tumor cells.

Intermediate-sized filaments in cultured rat liver tumor cells with Mallory body-like cytoplasm abnormalities.

The endoskeletal structure of tumor cells with a characteristic cytoplasmic abnormality initiated in rat liver by the in vivo adminisration of the carcinogen diethylnitrosamine was studied in clonal cell lines established and propagated in vitro. The bulk of the cytoplasmic and plasma membrane protein was removed by extraction with Triton X-100, and subsequently the juxtanuclear detergent-insoluble fraction containing filaments of 100-150 A was released into citrate buffer at pH 2.8. Analysis of this fraction by sodium dodecyl sulfate acrylamide gel electrophoresis revealed the pressence of five major proteins that banded with apparent molecular weights of about 66, 57, 52, 48 , and 43 x 10(3), the last of which comigrated with actin. The proteins thus resembled those from intermediate-sized filaments of both the vimentin (57 x 10(3)) and the prekeratin types obtained from various vertebrate cells. They also appeared to be related to the polypeptides of intermediate-sized filaments from Mallory bodies induced by griseofulvin in the livers of mice and to some of the polypeptides seen in isolates of Mallory bodies from human alcoholics. These results indicated that a major component of the carcinogen-induced lesion consisted of intermediate-sized filaments. The possible significance in cell transformation of this stably maintained aggregate of filaments that binds concanavalin A and displaces the nucleus is discussed. The close resemblance of this lesion to that seen in the cells of cirrhotic livers of alcoholics (Mallory's alcoholic hyalin) raises a question regarding the possible oncogenic status of such cells in humans.

Properties and malignant transformation of established rat liver parenchymal cells in culture.

Epithelioid cells from the livers of normal and genetically impaired (Gunn) rats were established in long-term cultures in vitro. These cells grew as flat, epithelioid cobble-stone-type monolayers and showed a diploid karyotype. They secreted rat serum albumin and proteins into their growth media and contained aryl hydrocarbon hydroxylase. Such cells were transformed by treatment with methylazoxymethanol acetate; they then exhibited an irregular, piling growth pattern, acquired the ability to grow in soft agar, and thereafter grew as tumors in hamsters given cortisone and in nude mice. These malignant spindle-cell tumors were reestablished in culture and still secreted serum albumin. The transformed cells became highly multinucleate when exposed to cytochalasin B and thus behaved like tumor cells. This behavior was not shown by the original cells. Cells transformed by benzo[a]pyrene failed to grow in soft agar culture or as tumor in animals. Cells were not affected by diethylnitrosamine.

Effects of retinoic acid versus dimethyl sulfoxide on Friend erythroleukemia cell growth. II. Induction of quiescent, nonproliferating cells.

Treatment of Friend erythroleukemia (FL) cells in vitro with 10(-7) to 10(-5) all-trans-retinoic acid (RA) leads to a concentration-dependent accumulation of a subpopulation of quiescent cells. This subpopulation, termed "Q-cells," contained markedly reduced RNA and protein levels and had a cell cycle distribution with a predominance of cells in G1 phase, which was nearly identical to that found in fully differentiated dimethyl sulfoxide (CAS:67-68-5; methyl sulfoxide)-induced FL cultures. The G1 cells in this RA-induced subpopulation (G1Q cells), though viable, did not enter S-phase, whereas the small percentage of Q-cells with S and G2 DNA content progressed very slowly through the cycle. While the Q-cell population did not contain the differentiation-associated chromatin protein H1 degrees, the cells did manifest a more condensed nuclear chromatin, altered sensitivity to acid denaturation, and reduced accessibility of the DNA in chromatin to acridine orange. The extent of chromatin condensation and the number of free ribosomes versus polysomes in RA-treated FL cells were intermediate between those in untreated and fully differentiated cells, whereas viral budding and the number of nucleoli remained unchanged from those seen in the untreated cell state. The non-Q-cell population in RA-treated cultures, termed "T" (transitional) cells, had an intermediate RNA and protein content and a cell cycle distribution similar to those of control cultures nearing the plateau phase of growth. In the absence of any late markers of differentiation, the Q-cell population was tentatively identified as a unique, quiescent cell population not previously described in the FL cell system.

Proliferative and antigenic modifications in human epithelial cells in chronic atrophic gastritis.

For the study of both proliferative and antigenic changes in epithelial cells in a disease predisposing to gastric cancer, endoscopic biopsy specimens were analyzed following removal from individuals with chronic atrophic gastritis (CAG); comparisons were made with specimens from normal gastric mucosa. All subjects were from Nariño, Colombia, the population of which has a high age-adjusted incidence of gastric cancer (150/100,000 population) occurring mainly in gastric antrum. After pulse incubation of biopsy specimens with tritiated thymidine ([3H]dThd), microautoradiographic distributions of [3H]dThd-labeled cells in the epithelial lining of gastric pits were correlated with expression of serologically defined gamma-fetal antigen (FA) as a second marker. Measurements were done both in gastric corpus and in antrum for entire gastric pits and over multiple gastric pit compartments. Total numbers of cells per gastric pit column did not differ between the normal and the CAG specimens either in corpus or in antrum; however, both in corpus and in antrum mean numbers of [3H]dThd-labeled cells per gastric pit column and labeling index were almost twice as large for the CAG population (P less than .006). Labeling index differences also were significant over most gastric pit compartments (P less than .02). In antrum gamma-FA-positive lesions had an expanded proliferative compartment with labeling indices significantly greater than those of antigen-negative lesions (P less than .02). This correlation did not extend to biopsy specimens obtained from corpus of stomach where the frequency of carcinoma is low. Findings indicate a hyperproliferative state in CAG compared to the proliferative state in normal gastric mucosa and, in gastric antrum, a further correlation with expression of gamma-FA in hyperproliferating cells. The two markers can be used to aid definition of the gastric mucosa in a disease associated with the development of gastric cancer and in prophylactic dietary intervention programs.

Expression of murine gamma fetal antigen in adult hematopoietic tissue and during induced differentiation of Friend erythroleukemia cells.

Quantitative analysis of extracts of various normal adult CD-1 mouse tissues indicated that the serologically defined murine gamma fetal antigen (gamma-FA) was expressed at high levels in hematopoietic tissue in general and in bone marrow (BM) in particular. Metabolic labeling of isolated BM cells indicated that the BM was a site of gamma-FA synthesis in the adult animal. The size(s) of the antigen immunoprecipitated from labeled BM cells (35 and 27 kilodaltons) with anti-gamma-FA serum correlated well with molecular weight estimates of fibrosarcoma-fetal mouse-associated gamma-FA, as determined by molecular sieve chromatography. For ascertainment of the relationship between hematopoietic cell differentiation and gamma-FA content, a multiparameter flow cytometric approach was used to evaluate gamma-FA levels in Friend erythroleukemia (FL) cells as a function of growth state (blast or dimethyl sulfoxide-differentiated) and cell-cycle compartment. Differentiated G1-arrested FL cells (G1D) possessed significantly lower gamma-FA-associated immunofluorescence as compared to control cells in the G0-G1 substate. Remaining S- and G2 + M-phase cells in differentiated populations demonstrated an even greater reduction in gamma-FA content relative to control cells in the corresponding cell-cycle phases. The available data support the tentative classification of gamma-FA as a murine differentiation antigen.

Pleotrophic action of interferon gamma in human orbital fibroblasts.

Analysis of the two-dimensional electrophoretic patterns of total radiolabeled cellular proteins derived from human orbital fibroblast cultures revealed that interferon gamma (100 U/ml) elicited significant quantitative changes in 42% of 86 randomly-selected proteins relative to untreated cultures. The most substantial up-regulation involved a protein with pI/mw map coordinates of 5.9/54,000 and a heterogenous 5 isoform protein cluster (pIs = 6.1-5.6) of approximately 47- to 50-kDa. These proteins were identified as the previously described 54-kDa protein inducible in interferon gamma-sensitive cell types and type-1 plasminogen activator inhibitor (PAI-1), respectively. Definition of PAI-1 as an interferon gamma-responsive protein in orbital fibroblasts was confirmed by immunoprecipitation using PAI-1-specific antibodies. Induction of PAI-1 and the 54-kDa protein in orbital fibroblasts, moreover, was relatively specific for interferon gamma since interferon alpha failed to initiate a similar inductive response. The synthesis of a 170 kDa protein, tentatively identified as a collagen, was decreased by approximately 80%. Analysis of the labeled proteins secreted into the culture medium revealed that interferon gamma increased the medium content of fibronectin and decreased the secretion of collagen. It would appear from these data that the inflammatory cytokine can exert regulatory effects on the synthesis of many specific proteins in orbital fibroblasts.

Bidimensional gel electrophoretic analysis of protein synthesis and response to interferon-gamma in cultured human dermal fibroblasts.

Densitometric analysis of [35S]methionine-labeled dermal fibroblast proteins by two-dimensional protein gel electrophoresis revealed that cultures derived from abdominal wall and the anterior aspect of lower leg skin exhibit different patterns of specific protein synthesis. Moreover, the response to interferon-gamma is dependent upon the anatomic site of culture derivation. Plasminogen activator inhibitor type-1 is expressed constitutively in fibroblasts from both sites. However, interferon-gamma (100 U/ml) treatment for 48 h resulted in an up-regulation of the polypeptide in leg cultures and a marked inhibition of expression in fibroblasts from the abdomen. A number of other protein spots became more or less abundant in cultures from the two sites following treatment with the cytokine. It would appear that dermal fibroblasts from the abdominal wall and leg differ intrinsically in regard to their protein synthetic repertoires and their responses to interferon-gamma. These findings may be relevant to the pathogenesis of Graves' dermopathy where the skin of the lower leg becomes infiltrated with glycosaminoglycans and other extracellular material.

Enhanced albumin production by malignantly transformed hepatocytes during in vitro exposure to dimethylsulfoxide.

The murine BW 1 and rat 32III 6/d tumor cell lines, derived from a spontaneous mouse hepatoma and a carcinogen-induced rat hepatocellular carcinoma, were used to investigate the effect of dimethylsulfoxide (DMSO) on liver cells in vitro. After a 4-day exposure to DMSO in final concentrations of 0.5 and 1.0%, BW 1 cell-associated albumin increased by 41.6 and 94.2%; extracellular albumin levels in these same cultures rose by 131.4 and 214.2%. Exposure of 32III 6/d cells to 2% DMSO produced increases in cell-associated and extra-cellular albumin concentrations of 67.8 and 188.7%, respectively. The lack of inducible gamma-glutamyl transpeptidase in BW 1 cells and its decrease in 32III 6/d cultures following DMSO treatment suggests that the DMSO-mediated enhancement of albumin production is not reflective of a random increase in the expression of cellular genes.

Reaction of selenium with immunoglobulin molecules.

Radioactive selenite reacts with purified human and goat immunoglobulins at acidic and neutral pH. The antigenic properties of the immunoglobulins are retained during the selenium labelling as shown by immunoelectrophoresis and autoradiography. Pepsin digests of 75Se-labelled IgG possess 75Se both in the (Fab')2 fraction and in the low molecular weight peptides derived from the Fc domains. Alpha-1-acid glycoprotein, ribonuclease, and lysozyme are also labelled by this procedure. Enhancement of 75Se incorporation by urea, guanidinium chloride, mercaptoethanol, sodium sulfite and carrier selenite is interpreted as an effect of destabilization of IgG disulfide bonds. Up to 1.4 g atoms Se per mol IgG have been incorporated. We assume that selenite is cleaving disulfides by a process akin to sulfitolysis. The lability of the isolated 75Se-labelled IgG to high concentrations of mercaptans and sulfite is consistent with this idea. These 75Se-labelled proteins may be useful in structure studies and radioimmunoassay.

Glycosylated hemoglobin in human and animal red cells. Role of glucose permeability.

We have compared red cells from man and selected animals in order to determine the effect of glucose permeability on nonenzymatic glycosylation of hemoglobin. Glucose permeability was highest in the primates (human, baboon, rhesus monkey), lower in dogs and rabbits, and nearly zero in pigs. Glycosylation of hemoglobin was measured by three independent methods: cation-exchange chromatography on Bio-Rex 70 (Bio-Rad, Inc., Richmond, California), agar gel electrophoresis, and affinity chromatography. The colorimetric thiobarbituric acid test did not provide valid data on animal hemolysates. However, this test was useful for identifying glycosylated hemoglobin (HbA1c) components isolated on Bio-Rex chromatography. In all animals tested, levels of HbA1c (from Bio-Rex chromatography) and total glycosylated hemoglobin (from affinity chromatography) correlated well with glucose exposure, the product of intracellular glucose concentration, and red cell life span. These results indicate that nonenzymatic glycosylation of hemoglobin in mammals is determined by three major variables: mean plasma glucose concentration, red cell life span, and red cell glucose permeability.

Mechanical debulking versus balloon angioplasty for the treatment of true bifurcation lesions.

OBJECTIVES: The purpose of this study was to compare the immediate angiographic and long-term results of debulking versus balloon angioplasty for treatment of true bifurcation lesions. BACKGROUND: Previous studies have shown true bifurcation lesions to be a high risk morphological subset for percutaneous transluminal coronary angioplasty (PTCA). Although atherectomy devices have been used to treat bifurcation lesions, no studies have compared the outcomes of these alternative treatment modalities. METHODS: Between January 1992 and May 1997, we treated 70 consecutive patients with true bifurcation lesions (defined as a greater than 50% stenosis in both the parent vessel and contiguous side branch) with conventional PTCA (n = 30) or debulking (with rotational or directional atherectomy) plus adjunctive PTCA (n = 40). Paired angiograms were analyzed by quantitative angiography, and clinical follow-up was obtained in all patients. RESULTS: Acute procedural success was 73% in the PTCA group and 97% in the debulking group (p = 0.01). Major in-hospital complications occurred in two patients in the PTCA group and one in the debulking group. Treatment with atherectomy plus PTCA resulted in lower postprocedure residual stenoses than PTCA alone (16+/-15% vs. 33+/-17% in the parent vessel, and 6+/-15% vs. 39+/-22% in the side branch; p < 0.001 for both comparisons). At 1 year follow-up, the incidence of target vessel revascularization (TVR) was 53% in the PTCA group as compared with 28% in the debulking group (p = 0.05). Independent predictors of the need for repeat TVR were side branch diameter >2.3 mm, longer lesion lengths, and treatment with PTCA alone. CONCLUSIONS: For the treatment of true bifurcation lesions, atherectomy with adjunctive PTCA is safe, improves acute angiographic results, and decreases target vessel revascularization compared to PTCA alone. The benefits of debulking for bifurcation lesions were especially seen in lesions involving large side branches.

Interferon gamma regulation of de novo protein synthesis in human dermal fibroblasts in culture is anatomic site dependent.

The propensity of the skin of the lower anterior leg to be involved in Graves' dermopathy prompted an examination of the specific protein synthesis and response to interferon gamma in cultured fibroblasts from this area. Confluent cultures from normal skin of the lower leg and from the abdomen of the same three donors were pulse labeled with [35S]methionine for 3 h and subjected to two-dimensional protein gel electrophoresis and fluorography. Protein spots were mapped using a computer-driven program and the relative densities of the resolvable spots analyzed. Fibroblasts from the two anatomic sites display distinct patterns of de novo protein synthesis. Of the 157 abundant spots arbitrarily chosen for analysis, 31% varied substantially in levels of expression between the sites. A number of proteins appear to be expressed only in cultures derived from one of the two anatomic sites. Interferon gamma (100 U/ml) present in the culture medium for 48 h influenced the abundance of a number of proteins in a site-specific manner. Among them, plasminogen activator inhibitor type-1 was induced three to five times in the leg cultures, whereas this same polypeptide was down-regulated in abdominal fibroblasts. A 54-kD protein was induced in interferon-treated cultures from both sites at least 50 times. It appears that fibroblasts from different regions of the integument are intrinsically distinct in terms of both their protein synthetic programs and their responses to cytokines.

A specific dietary zinc requirement for the growth of Walker 256/M1 tumor in the rat.

To determine the specific effect of zinc status on the growth of Walker 256/M1 carcinosarcomas young male rats were pair-fed either a control or zinc-deficient diet for 14 days, were implanted with tumors and killed 7 days later. Half of the deficient rats were repleted with zinc for the 7 days after tumor implantation. In deficient rats, tumor weights were decreased 70% (p less than 0.005), tumor necrosis was 3-fold greater (p less than 0.05) and tumor zinc concentrations were decreased 23 to 37% (p less than 0.005). A specific zinc effect was observed by a 2-fold increase in tumor weights in repleted rats (p less than 0.05) with marked decreases in tumor necrosis (p less than 0.05) and 29 to 84% increases in tumor zinc concentrations (p less than 0.005). Since there were no decreases in organ weights of zinc-deficient animals and no correlation between final tumor weights and postimplant changes in carcass weights, the results indicate a specific inhibitory effect of zinc deficiency independent of a nonspecific malnutrition.

Histogenesis of benign pleomorphic adenoma (mixed tumor) of the major salivary glands. An ultrastructural and immunohistochemical study.

Twenty-two benign pleomorphic adenomas of the major salivary glands were studied by transmission electron microscopy and immunohistochemical techniques (three cases) in order to characterize the cell types comprising the epithelial and so-called mesenchymal regions of the tumors. Light- and electron-microscopic studies showed the tumors to consist of variable mixtures of neoplastic ductular epithelial cells, rare acinar cells, and metaplastic myoepithelial cells. Many of the loosely organized "stromal cells" contained structures indicative of their myoepithelial origin, e.g., perinuclear tonofilaments, ectoplasmic actin microfilaments, and remnants of basement membrane. Polyclonal antikeratin antisera strongly stained ductular epithelial and myoepithelial cells, squamoid cell nests, and periductular myoepithelial cells, whereas myxoid and chondroid cells were less intensely stained. Monoclonal cytokeratin antibody AE1 stained only the ductular epithelial cells in both the normal glands and tumors. In contrast, S-100 protein, which is present only in scattered acinar cells and myoepithelial cells in the normal parotid gland, was found in the ductular and periductular myoepithelial cells, isolated myxoid cells, and chondroid and cartilagenous cells in the tumors. Actin was found in all the cell types of the tumor but staining was strongest in the ducts. Double immunofluorescence staining for cytokeratin and vimentin revealed coexpression of both types of intermediate filaments in occasional normal acinar and intercalated duct myoepithelial cells, and in some cells in the myxoid and chondroid regions of the tumors. In the tumors, vimentin was present in occasional periductular myoepithelial cells, stellate myxoid cells, and especially in chondroid cells and chondrocytes. Our findings indicate that benign pleomorphic adenomas of the major salivary glands are pure epithelial cell tumors. The histologic complexity of these neoplasms is due to the ability of the neoplastic ductular myoepithelial cell to modulate its morphologic appearance and intermediate filament composition, and to produce large amounts of matrix substances. We further postulate that these tumors arise from neoplastically transformed intercalated ducts.

A recombinant soluble chimeric complement inhibitor composed of human CD46 and CD55 reduces acute cardiac tissue injury in models of pig-to-human heart transplantation.

BACKGROUND: Inasmuch as complement plays a critical role in many pathological processes and in xenograft rejection, efficient complement inhibitors are of great interest. Because the membrane-associated complement inhibitors are very effective, recombinant soluble molecules have been generated. METHODS: We tested the efficacy of complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), in two models of pig-to-human xenotransplantation in which tissue injury is complement mediated. The in vitro model consisted of porcine aortic endothelial cells and human serum, and the ex vivo model consisted of a porcine heart perfused with human blood. RESULTS: In vitro, addition of CAB-2 to serum inhibited cytotoxicity and the deposition of C4b and iC3b on the endothelial cells. Ex vivo, addition of CAB-2 to human blood prolonged organ survival from 17.3 +/- 6.4 min in controls to 108 +/- 55.6 min with 910 nM (100 microg/ml) CAB-2 and 219.8 +/- 62.7 min with 1820 nM (200 microg/ml) CAB-2. CAB-2 also retarded the onset of increased coronary vascular resistance. The complement activity of the perfusate was reduced by CAB-2, as was the generation of C3a and SC5b-9. The myocardial tissues had similar deposition of IgG, IgM, and Clq; however, CAB-2 reduced the deposition of C3, C4, and C9. Hearts surviving >240 min demonstrated trace to no deposition of C9 and normal histologic architecture. CONCLUSION: These results indicate that CAB-2 can function as an inhibitor of complement activation and markedly reduce tissue injury in models of pig-to-human xenotransplantation and thus may represent a useful therapeutic agent for xenotransplantation and other complement-mediated conditions.

Control selection for RNA quantitation.

The study of mammalian gene expression is often carried out at the level of mRNA. In such analyses, one usually measures the amount of an mRNA of interest under different conditions such as stress, growth, development, cell and tissue localization or as part of an evaluation of the effects of gene transfection. A variety of techniques exist to measure gene expression and most commonly involve Northern hybridization analysis, ribonuclease protection or RT-PCR. Common to all of these assays is the inclusion of a so-called loading or internal control (i.e., analysis of an mRNA that does not change in relative abundance during the course of treatments). Here, we discuss the uses and pitfalls of the most popular of these controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, with special emphasis on precautions associated with the use of GAPDH.