Short-term Organ Culture Research Articles

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan.

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.

The apolipoprotein A-I gene is actively expressed in the rapidly myelinating avian peripheral nerve.

The expression of the apolipoprotein A-I (apo A-I) gene was investigated in the myelinating sciatic nerve. Hybridization analysis with an apo A-I cDNA probe obtained from a cDNA library of mRNA isolated from rapidly myelinating chick sciatic nerve indicated that apo A-I coding transcripts increase during development in the chick sciatic nerve in parallel with the increase of myelin lamellae. Substantial apo A-I-like immunoreactivity in chick sciatic nerve homogenates was detected by Western blotting. The amount of antigen increased from the 15-d embryonic stage to 1 d posthatch and then decreased. Two subcellular fractions corresponding to the cytoplasmic compartments were particularly enriched in apo A-I. apo A-I immunoreactivity was also found in highly purified myelin preparations. Immunohistochemical staining provided further evidence for the presence of apo A-I in the endoneurial compartment of the sciatic nerve. Electron microscopic examination of these fractions after negative staining showed the presence of spherical and disc-shaped particles resembling high density lipoproteins. The presence of apo A-I, cholesterol esters, phospholipids, and triacylglycerols in ultracentrifugal fractions corresponding to serum lipoproteins and the behavior of apo A-I on nondenaturing gradient gels implied that apo A-I was associated with lipid. Studies with short-term organ cultures of sciatic nerves from 1-d chicks strengthened the evidence for local synthesis and secretion of apo A-I and apo A-I-containing lipoproteins by this tissue. These results establish that the apo A-I gene is actively expressed in developing sciatic nerve during the period of rapid myelination. These findings support the hypothesis that apo A-I synthesized within the nerve participates in the local transport of lipids used in myelin biosynthesis.

T lymphocytes and monocytes bind to keratinocytes in frozen sections of biopsy specimens of normal skin treated with gamma interferon

When 2 mm punch biopsy specimens of normal skin are incubated with gamma interferon (IFN-γ), the keratinocytes are induced to express intercellular adhesion molecule-1 (ICAM-1). Lymphocytes and monocytes that express lymphocyte function-associated an-tigen-1 (LFA-1) bind to cultured keratinocytes expressing ICAM-1. We have developed an in vitro adherence assay using frozen sections of normal skin that have been incubated with IFN-γ, and then overlaid by peripheral blood mononuclear leukocytes. Although peripheral blood mononuclear leukocytes do not bind to the epidermal keratinocytes of untreated skin sections, after exposure of the skin to IFN-γ the peripheral blood mononuclear leukocytes prominently bind to the epidermal keratinocytes that express ICAM-1. The binding by peripheral blood mononuclear leukocytes is increased approximately twofold by activation with phorbol ester treatment. The temperature dependence and kinetics of this adherence reaction reveal no binding at 8° C, good binding at 24° C, and optimal binding at 37° C, reaching a maximal extent by 60 minutes. The adherence reaction is blocked either by pretreating the peripheral blood mononuclear leukocytes with LFA-1 antibody or the IFN-γ—exposed skin specimen with ICAM-1 antibody. The immunophenotypic analysis of the adherent peripheral blood mononuclear leukocytes to epidermal keratinocytes revealed that most cells activated by 12- O-tetradecanoyl-phorbol-13-acetate are T lymphocytes, with CD8 + T cells binding slightly better than CD4 + T cells, with a smaller population of monocytes. These results provide additional support for a role of LFA-1, ICAM-1, and IFN-γ in modulating ke-ratinocyte-lymphocyte interactions. This short-term organ culture—frozen section adherence assay system provides a new research tool to explore further possible molecular adhesion mechanisms and cellular interactions involved in trafficking of peripheral blood mononuclear leukocytes in the skin.

Characterization of intercellular adhesion molecule-1 and HLA-DR expression in normal inflamed skin: Modulation by recombinant gamma interferon and tumor necrosis factor

Lymphocytes bind to cultured keratinocytes that are treated with interferon gamma (IFN-γ) and tumor necrosis factor (TNF). When the lymphocytes are preincubated with antibody to lymphocyte function associated antigen—1 (LFA-1), this adherence is inhibited. Because intercellular adhesion molecule—1 (ICAM-1) is a ligand for LFA-1, we studied the cellular expression of ICAM-1, as well as two other IFN-γ-inducible antigens, (HLA) human lymphocyte antigens DR and DQ, in both normal and diseased skin. The modulation of these cell surface antigens by IFN-γ and TNF with the use of short-term organ cultures of skin was compared with isolated keratinocytes grown in a conventional tissue culture system. While in normal skin, keratinocytes did not express HLA-DR, DQ, or ICAM-1, when organ cultures were supplemented with IFN-γ, rapid induction of keratinocyte ICAM-1 expression occurred after 24 hours; HLA-DR but not DQ expression occurred after 48 hours. TNF also induced keratinocyte ICAM-1 expression (although to a lesser degree than IFN-γ) but did not induce either keratinocyte HLA-DR or DQ expression. There was good correlation of keratinocyte expression of ICAM-1 and HLA-DR by IFN-γ and TNF when the epidermis of the organ culture system was compared with the isolated keratinocytes grown in tissue culture. The presence of intraepidermal lymphocytes correlated extremely well with keratinocyte ICAM-1 expression but not with keratinocyte HLA-DR expression in psoriasis, atopic dermatitis, lichen planus, and mycosis fungoides. The intensity of endothelial cell expression of ICAM-1 correlated with the degree of dermal inflammation. We conclude that IFN-γ, once produced by activated T lymphocytes in the dermis, may be of importance in lymphocyte trafficking in the epidermis by the induction of keratinocyte ICAM-1 expression. The use of the short-term organ culture system, in which there is inducible ICAM-1 expression, provides an experimental bridge between purely in vitro and in vivo investigations to further our understanding of the molecular basis for lymphocyte apposition to keratinocytes in the skin.

Transport across rat trachea in vitro after exposure to cytoskeleton-active drugs in vitro or to ozone in vivo.

Full-length tracheas from Sprague-Dawley rats were exposed to cytoskeleton-active drugs in short-term organ culture, and the permeability of the tracheal epithelium was measured by instilling radiotracers into the lumen and assay of the radioactivity appearing in the external bathing medium. In vitro treatment with cytochalasin D (cyto D, 2-10 x 10(-6) M) increased the rate of movement of [14C]mannitol across the epithelium. Exposure to vinblastine (VB, 10(-4) M) alone had no significant effect. However, VB in combination with cyto D increased the permeability in a dose-dependent manner. In vivo exposure to ozone (O3, 0.8 or 2.0 ppm, 2 h) had only a slight effect on the rate of movement of the tracer as measured in vitro immediately after exposure. At 24 h postexposure there was no significant difference in permeability between ozone- and air-exposed tracheas. Prior in vivo O3 exposure sensitized the tracheas to the in vitro effects of cyto D; treatment of O3-exposed tracheas with cyto D immediately after O3 exposure produced a greater than additive effect on permeability measured in vitro. VB at concentrations up to 10(-4) M had no enhancing effect on permeability in O3-exposed tracheas. Sham exposure to clean air did not affect permeability compared to untreated (shelf) controls. Electron microscopic studies demonstrated penetration of horseradish peroxidase into intercellular spaces in the tracheas treated in vitro with cyto D or cyto D plus VB. Cyto D is known to affect intracellular microfilaments that have attachments at or near the cell surface, while VB affects microtubules associated with internal cellular structures. Therefore, the synergistic effect on tracheal permeability observed with O3 and cyto D, but not with O3 and VB, suggests that O3 may change cell surface structures associated with the microfilamentous cytoskeleton.

Secretion of plasminogen activators by human colorectal and gastric tumor explants.

Conditioned media from explants of human colorectal and gastric tumors in short-term organ culture were analysed for plasminogen activator activity, activity toward the synthetic urokinase substrate, Spectrozyme-UK, and for the presence of urokinase antigen using monospecific goat antibody, by enzyme-linked immunosorbent assay. Comparisons were made between primary tumors, adjacent normal mucosa and metastatic lesions. These analyses were carried out on unfractionated culture fluids and on fractions obtained by fast protein liquid chromatography separation using Superose 6 gels. Plasminogen activator activity, tested by azocaseinolysis in the presence of added plasminogen, was restricted to peaks of 55 kD and 155 kD. These were of the urokinase type as shown by specific immunoinhibition and by absorption by an antiurokinase antibody-Affigel 10 column. Spectrozyme-UK, in addition to these peaks, detected a series of higher molecular weight activities, the largest of which appeared in the void volume, and were therefore of greater than 10(6) molecular weight. These activities were greatly increased by inclusion of trace plasmin indicating that these components were mostly in their proenzyme forms. The characteristics of these very large enzymes were similar to those isolated earlier from a human lung cancer cell line. Comparison of the primary and metastatic tumors confirmed earlier observations showing that urokinase secretion by the metastatic tumors was greatly reduced in comparison with the primary tumors: in the colon carcinomas it was 10 per cent of the value for the primary, in the gastric tumors 3 per cent, whether means or medians were compared (P less than 0.0001). This large difference was characteristic only of plasminogen activator secretion assayable by azocaseinolysis; activities toward Spectrozyme-UK, and antigen reacting with anti-urokinase antibody, were considerably less different in the two groups. In individual tissues, no correlation was found between the amount of extractable plasminogen activator and amounts secreted, or between the latter and the amount of lactic acid released. It is postulated that the greatly reduced plasminogen activator secretion by explants of metastatic tumors may be a phenotypic characteristic of distinct advantage for cancer cells destined to initiate metastatic foci, and may contribute to the ability of circulating cancer cells to lodge in the blood vessels of the target organ.

Formation of DNA adducts in human skin maintained in short-term organ culture and treated with coal-tar, creosote or bitumen.

Adult and foetal human skin samples maintained in short-term organ culture were treated topically with solutions of coal-tar, creosote or bitumen, then DNA was isolated and analysed by 32P-post-labelling for the presence of aromatic DNA adducts. Autoradiographs of the 32P-labelled adducts resolved on polyethyleneimine-cellulose tlc sheets revealed a band of radioactivity indicative of the formation of adducts by a large number of components in these complex carcinogenic mixtures. Single doses of the materials, similar to those used to initiate tumours in experimental animals, resulted in the formation of approximately 0.3 fmol total adducts/micrograms DNA. The levels of adducts formed in human skin are thus similar to those formed in mouse skin after administration of doses known to be carcinogenic to the latter. The results provide direct evidence of DNA damage in human skin by materials strongly suspected of being carcinogenic to humans, and point to a method for evaluating other complex mixtures of aromatic chemicals for their potential carcinogenic hazard.

Hemorrhagic endovasculitis-like lesion induced in placental organ culture

In organ culture, human chorionic villi develop vascular changes that resemble so-called hemorrhagic endovasculitis. The latter is a morphologic finding more prevalent in placentas of stillborn infants but seen also in those of liveborn infants, in whom the lesion is localized rather than generalized. We compared the histologic vascular changes in short-term organ cultures of 15 placentas (10 term, 5 preterm) with the naturally occurring vascular lesion in 6 placentas (2 liveborn, 4 stillborn). All organ cultures of placentas from liveborn infants developed hemorrhagic endovasculitis-like lesions in the fetal stem arteries; these lesions were present as early as 1 day and persisted for 7 days in culture. A mechanism common to both the in vivo and in vitro systems depending on hypoxia and vascular smooth muscle contraction may explain both the naturally occurring and tissue culture-induced lesions.

Stereoselective aspects of the metabolic activation of benzo[ a]pyrene by human skin in vitro

Benzo[ a]pyrene (BP) is activated within tissues in both a regio- and a stereoselective manner and, since human skin is sensitive to tumour induction by polycyclic aromatic hydrocarbons (PAH), the stereoselective metabolism of BP in this tissue has been investigated. Samples of skin from eleven individuals were treated with [ 3H]BP in short-term organ culture. Two samples were also treated with mixtures of [ 14C](+)- and (−)- trans-7,8-dihydro-7,8-dihydroxybenzo[ a]pyrene (BP-7,8-dihydrodiol) in varying proportions. Following application of [ 3H] BP, more 7,8-dihydrodiol was recovered from the skin itself than from the culture fluid in ten cases; no 7.8-dihydrodiol was detected in extracts from the eleventh. The 7,8-dihydrodiol metabolite was extracted predominantly (range 74–99%) as the (−)-enantiomer in nine of these ten patients, although proportionately more (+)-enantiomer was recovered from the culture fluid than from the skin in each case. The relative proportions of [ 3H]BP tetrols derived from syn- and anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydroxybenzo[ a]pyrene (BPDE) detected in these extracts was more variable. When skin samples were treated with [ 14C]BP-7,8-dihydrodiol, more anti- than syn-BPDE-derived tetrols were extracted, irrespective of the optical purity of the dihydrodiol applied. These findings provide evidence for interindividual variations in the stereoselective metabolism of BP, which may be of some importance in determining individual susceptibility to PAH-induced skin carcinogenesis.

Induction of an activation antigen on postcapillary venular endothelium in human skin organ culture.

We studied the effects of the immune mediators interleukin 1, interleukin 2, tumor necrosis factor, immune interferon, and lipopolysaccharide on the expression of the endothelial activation antigen recognized by the murine monoclonal antibody H4/18 in short term organ cultures of newborn foreskins. No endothelial staining was detectable before culture. Interleukin 1, tumor necrosis factor, and lipopolysaccharide each induced 2+ to 3+ H4/18 staining of microvascular endothelium at 6 hr. Combining mediators produced additive (3+ to 4+) effects, and reactivity was lost or markedly diminished by 24 hr. Incubation with culture medium alone resulted in 1+ to 2+ H4/18 staining at 6 hr, and medium conditioned by cultured foreskins, but not mock-conditioned medium, could induce H4/18 binding in cultured human umbilical vein endothelial cells. The spontaneous expression of microvascular staining in the foreskins was markedly inhibited by cyclosporin A, but not polymyxin B sulfate or dexamethasone; cyclosporin A did not inhibit induction of staining by exogenous mediators. Both light level and immunoultrastructural studies demonstrated H4/18 expression to be associated predominantly with postcapillary venular endothelial cells of the superficial vascular plexus. We conclude that microvascular endothelium of skin can undergo activation in response to exogenous and endogenous cytokines, with the greatest changes occurring in those portions of the vessels most involved in leukocyte and lymphocyte trafficking.

Differential effects of Clostridium difficile toxins A and B on rabbit ileum

The pathogenesis of Clostridium difficile enterocolitis appears to involve colonization of the bowel followed by release of toxin A, an enterotoxin, and toxin B, a cytotoxin. The purpose of this study was to determine the effect of purified toxins A and B on intestinal secretion, epithelial permeability, and morphology in perfused rabbit ileal loops. Intestinal permeability after toxin exposure was assessed by blood-to-lumen clearance of [3H]mannitol. Toxin A at doses of 5–100 μg/10 cm ileal loop caused a threefold to fivefold increase in [3H]mannitol permeability (p < 0.001) vs. equal concentrations of toxin B or buffer control. In addition, perfusate from toxin A-exposed loops contained significantly more neutrophils (p < 0.001) than toxin B or control loops. Toxin A caused severe epithelial cell necrosis with destruction of villi and polymorphonuclear infiltration. Electron microscopy of mucosa subjected to a low dose of toxin revealed widespread nonspecific dilatation of endoplasmic reticulum and mitochondrial swelling. In contrast to these effects of toxin A in ileal loops, in vitro experiments with ileal explants in short-term organ culture revealed that toxin A had no effect on epithelial cell permeability, protein synthesis, release of alkaline phosphatase, or morphology. Our results show that purified toxin A but not toxin B causes severe inflammatory enteritis in rabbit ileal loops, but has no discernable effect on rabbit ileum in vitro. We speculate that toxin A may contribute significantly to intestinal damage in C. difficile-associated colitis and diarrhea.

Plasminogen activator content of gynecological tumors and their metastases

The plasminogen activator content of surgically excised gynecological tumors was measured with an azocaseinolytic assay. Ovarian (10 primary, 18 metastases) and uterine (5 primary and 6 metastases) tumors showed similar mean activator activities (21 CTA units/g tissue) mainly of the urokinase type with similar wide variations in each group. About 44% (14 of 32) of the tissues placed in short term organ culture were shown to produce and secrete urokinase activity. Results of the plasminogen activator activity found in the patient tumors or produced by the tumors during culture in the absence or presence of some drugs indicate a wide range of individual variations in sensitivity to these agents.

Transport and metabolism of l-glutamic acid by abdominal ganglia of the hawk moth, Manduca sexta

1. The transport of exogenous L-glutamate was characterized in an insect ganglion with an in vitro preparation consisting of whole ganglia in short-term organ culture. The characteristics studied include the ionic dependence and specificity of transport, as well as its rate of operation. 2. Rates for the Na +-dependent and Na +-independent components were measured, revealing lowaffinity carriers; the apparent K Ms are 0.48 and 1.27 mM, respectively. 3. Accumulation of exogenous glutamate is saturable, sensitive to metabolic inhibitors, and specific for certain dicarboxylic acids, thus exhibiting features of a carrier-mediated process. Transport is partially dependent on Na + in the medium, and it is reduced in the presence of the competitor amino acids, L-and D-aspartate. 4. Exogenous glutamate is metabolized by abdominal ganglia; the major amino acid metabolites were measured in extracts of the ganglia and in the culture medium. Major amino acid metabolites derived from glutamate and recovered in the ganglia are glutamine, proline, GABA, and small amounts of aspartate and alanine. 5. Proline and glutamine are spontaneously released by the ganglia. A portion of this material is newly synthesized from exogenous glutamate, and its release follows a different time course for proline and glutamine. 6. Metabolism of exogenous glutamate may be a biochemical aspect of the insect blood-brain barrier.

Low-density lipoprotein apheresis and regression of atherosclerotic plaque in vitro.

Short-term organ culture and primary cell culture of human aorta were used to study the effect of selective removal of low-density lipoproteins (LDLs) from the surrounding medium (LDL apheresis) on the lipid content of cultured tissue and cells, respectively. LDL apheresis was performed by passing the culture medium through immunosorbent containing agarose-bound goat antibodies against human LDL. LDL apheresis promoted the decrease of lipids of all classes in the cultured atherosclerotic plaque. However, only the cholesteryl ester level, but not free cholesterol, triglyceride, or phospholipid levels, was lowered in the cells cultured from the plaque. One may thus assume that LDL apheresis facilitates regression of lipoidosis by reducing the content of lipids in atherosclerotic lesions.

Metabolism of a cartilage matrix glycoprotein in normal and osteoarthritic canine articular cartilage.

We have recently described a 550,000-dalton noncollagenous cartilage matrix glycoprotein (CMGP), with subunits of 130,000, which is present in hyaline cartilage and fibrocartilage. Biosynthetic studies indicated that CMGP was synthesized by short-term organ cultures of normal canine articular cartilage, representing approximately 9% of the total 3H-leucine incorporated into protein in 24-hour cultures. There was no incorporation of 35S-sulfate or 3H-mannose into CMGP under these conditions, but the protein did incorporate 32P-phosphate. The majority of the 3H-leucine-labeled CMGP was removed after 24 hours of chase with unlabeled leucine, and only a small amount remained at 72 hours, which suggests that there was rapid metabolism of the protein. CMGP was not detected in cartilage after addition of cycloheximide to the culture medium; this confirms its short half-life. Cultures of osteoarthritic cartilage obtained from dogs 8-10 weeks after anterior cruciate ligament transection revealed no difference in the metabolism of CMGP in this tissue compared with that found in cultures of normal articular cartilage.

Short-term organ culture of the retinal pigment epithelium in microtitration plates: ultrastructural studies.

Microtitration plates were used to culture simultaneously multiple, small (6 mm diameter) explants of bovine retinal pigment epithelium (RPE). Evaluation of tissue by light microscopy and by scanning and transmission electron microscopy after various incubation periods up to 6 h showed that RPE maintained in this system retains near normal morphology. Initially, the explanted RPE lacks apical microvilli, but during the first 2-3 h in culture recovery of apical microvilli occurs. The results suggest that the system is suitable for short-term maintenance of RPE for experimental purposes. Moreover, the ability to culture up to 16 explants from one bovine eye aids statistical evaluation of RPE behaviour under varying experimental conditions.

Induction of rat E and chicken A-I apolipoproteins and mRNAs during optic nerve degeneration.

Apolipoprotein synthesis was measured in control optic nerves and optic nerves undergoing Wallerian degeneration. After short term organ culture with radiolabeled amino acid, optic nerve extracts were reacted with antiserum to rat or chicken apolipoproteins. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the degenerating rat optic nerve, apo-E synthesis increased from 0.30 to 0.90% of newly synthesized protein and from 0.45 to 1.4% of secreted protein. A DNA-excess solution hybridization assay was constructed to measure the absolute amount of apo-E mRNA in control and degenerating optic nerves. Paralleling the increase in apo-E protein synthesis, the absolute amount of apo-E mRNA was elevated 3- to 4-fold after enucleation. Similar to rat apo-E, apo-A-I synthesis was increased in degenerating chicken optic nerve. Chicken apo-A-I represented 0.65 and 3.5% of newly synthesized protein from control and enucleated optic nerves, respectively. Apo-A-I increased from 0.85 to 5.5% of secreted protein following enucleation. Using in vitro translation to quantitate relative amounts of chicken apo-A-I mRNA, enucleated optic nerve apo-A-I mRNA content was increased 5-fold. These results suggest that local apolipoprotein synthesis may be involved in the mobilization of myelin cholesterol which occurs during Wallerian degeneration. The similar response of the rat and chicken to increase optic nerve apolipoprotein synthesis during degeneration supports the idea that avian peripheral apo-A-I and mammalian peripheral apo-E may be performing functions common to both classes of animals.

Metabolism of arachidonic acid by hamster trachea lack of stimulation by A23187

The metabolism of arachidonic acid has been studied using hamster trachea in short-term organ culture. To study endogenous substrate utilization, tissue lipids were labeled with [ 3H]-arachidonic acid, whereas exogenous substrate turnover was assessed by the addition of 100 μM [ 14C]-arachidonic acid to the medium. Both exogenous and endogenous arachidonate were converted primarily to 6-keto-PGF 1α and PGE 2, with varying amounts of an unidentified non-polar product noted. Production of the prostanoids increased steadily with time up to 24 hours. No significant generation of lipoxygenase products was found. Release of incorporated labeled arachidonic acid was nearly linear with time, resulting in the transfer of about 10% of the total label into the medium after 24 hours. About 3% of the total label was converted to prostaglandins. In the presence of 10 μM A23187, release of label was increased by only 25 to 60% relative to the control. Analysis of labeled compounds in the medium showed that this increase resulted from increased release of unchanged arachidonic acid, and that the yield of oxygenated products was the same as from the control incubations.

Immunochemical measurement of apolipoprotein synthesis in cell and organ culture

Immunoprecipitation of radiolabeled proteins can be used to detect and quantitate the synthesis of specific proteins in tissues and cells. The basic strategy is to carry out metabolic labeling with radiolabeled amino acid or carbohydrate precursors in cell culture or short-term organ culture. A tissue extract is prepared, and the protein of interest is precipitated by reaction with specific antibody. The immunoprecipitate can be analyzed in a qualitative or quantitative fashion for the incorporation of radioactivity into the specific protein. Both direct and indirect immunoprecipitation have been used to study apolipoprotein synthesis. With direct immunoprecipitation the high-speed supernatant (HSS) is reacted with excess antibody to the protein of interest and the resultant immunoprecipitate is isolated by centrifugation. The advantages of direct immunoprecipitation are that it is simple, requires only one antibody, and yields very low background values because the mass of the immunoprecipitate is small. The disadvantages are that it usually requires carrier antigen for efficient precipitation and cannot be used with a nonprecipitating polyclonal or a monoclonal antibody. The following procedure has been used to immunoprecipitate newly synthesized apolipoprotein (apoB) from avian and mammalian liver.

Two Sensitive in Vitro Monitors of Chemical Toxicity to Human and Animal Skin (in Short-Term Organ Culture): I. Paranuclear Vacuolization in glycol Methacrylate Tissue Sections II. Interference with [14C]Leucine Incorporation

AbstractTwo sensitive in vitro tests have been developed to determine the toxicity of chemicals applied to full-thickness skin explants from fresh human surgical specimens and sacrificed guinea pigs and rabbits. Before incubation, dinitrochlorobenzene (DNCB), sulfur mustard, or resorcinol was topically applied to the exposed top surface of each explant. The explants were then organ-cultured for 24 hr in shallow Petri dishes.Toxicity to skin was assessed by two methods. The first was histologic and morphometric. Paranuclear vacuoles in the basal epidermal cells were counted microscopically in tissue sections prepared from the cultured ex-plants. The number of these vacuoles was found to increase with the toxicant concentration, in the range from low to full toxicity. The DNCB (0.5%) caused fewer vacuoles in guinea pigs than in the other two species. Resorcinol (50%) caused more vacuoles in rabbits than in the other two species. Control skin explants (normal skin and skin exposed only to diluents) contained...