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The initial step in the formation of thrombin via the intrinsic pathway is the activation of Hageman factor (factor XII). Some, but not all, studies have shown that this activation may be brought about by collagen. We examined the effect of three types of soluble human placental collagen on Hageman factor. Collagen types III, IV, and V did not appear to activate Hageman factor under the conditions tested. To the contrary, these collagen species inhibited activation of Hageman factor by glass or ellagic acid. These studies suggest that some types of collagen may play an inhibitory role in blood coagulation.

Modulation of vascular endothelial cell growth by basement membrane heparan sulfate was investigated using four lines of normal and transformed cells. The growth of transformed endothelial cells, but not normal cells, on reconstituted basement membrane was severely suppressed when heparan sulfate, one of the components of the membrane, was specifically degraded by an enzyme, heparitinase. Similarly, when cells were grown on surfaces coated with heparan sulfate, as little as 60 pg/cm2 of heparan sulfate caused growth enhancement of transformed cells, but suppression of normal cells. These results together with our previous observations (IMAMURA, T and MITSUI, Y. (1987) Exp. Cell Res., 172: 92-100) argue that transformed cells have reversed a mechanism by which basement membrane heparan sulfate functions as a physiological suppressor for the growth of normal endothelial cells.

Activation of the neutrophil respiratory burst by the supernatant fraction from an alkali-treated collagen preparations (SAC) was enhanced by longer durations of exposure to alkali (1 N NaOH for 0.5-24 hr). The concentrate obtained from ultrafiltration (greater than 30,000 molecular weight) of SAC (1 N NaOH for 24 hr) retained the stimulatory factor. Fractionation of this ultraconcentrate by high-performance liquid chromatography showed that the stimulatory activity resided in the void volume (highest molecular weight). The amino acid composition of this active fraction revealed that this proteinaceous stimulant was not derived from the collagen molecule. Treatment of the SAC with ultrapure bacterial collagenase increased its stimulatory capacity, confirming its noncollagenous nature. Alkali treatment of whole cornea also released a similar large molecular weight, noncollagenous protein that stimulated the respiratory burst of polymorphonuclear leukocytes. Enhanced stimulation after prolonged NaOH treatment of the collagen preparation or collagenase treatment of SAC suggests that the stimulant might reside between collagen fibrils and then be released as the matrix is degraded.

In the present study rat hepatocytes isolated either by trypsin or by collagenase were investigated concerning its cultivability and its morphological properties. The cultivation was carried out as monolayer for 24 h. The yields of cells prepared by trypsin or collagenase amounted to 8 x 10(7) cells per liver and 15-30 x 10(7) cells per liver, respectively, with viabilities measured by trypan blue exclusion test of 70-80 and 90-95%, respectively. Nonhepatocytes were not taken into consideration. Using the electron microscopy it could be established that both freshly isolated and 24 h cultured hepatocytes were intact. There were no morphological differences between cells isolated by trypsin and cells isolated by collagenase. After 24 h cultivation hepatocytes prepared by trypsin showed a little tendency in forming a slightly flattened appearance and in forming intercellular contacts.

Single segments of rat nephron contain two distinct ouabain-insensitive, K-independent, Na-dependent ATPase activities: a Na-stimulated ATPase and a Na-inhibited ATPase. Na-inhibited ATPase activity is found in the proximal tubule and the thick ascending limb of Henle's loop but is absent in the collecting tubule whereas Na-stimulated ATPase is exclusively located in the proximal convoluted tubule. Na-inhibited ATPase, but not Na-stimulated ATPase, is totally abolished in the presence of 100 microM Ca2+. Conversely, Na-stimulated ATPase, but not Na-inhibited ATPase, is curtailed when nephron segments are preincubated at pH 7.2 whereas it is activated at pH 7.8. Finally, Na-stimulated ATPase displays an apparent Km for Na+ of approximately 10 mM, and is dose-dependently inhibited by the diuretic triflocin (IC50 approximately 6 x 10(-6) M).

The microcirculation contains mononuclear phagocytes, with features characteristic of macrophages, adhered to luminal capillary surfaces by intercellular adhesion plaques. These pulmonary intravascular macrophages may play an important role in regulating lung vascular tone and capillary permeability, and may modulate capillary endothelial cell growth and replication by the secretion of soluble mediators (i.e., arachidonate metabolites, cytokines). This study describes a technique which utilizes in situ lung perfusion to remove intravascular macrophages in large numbers from the microcirculation of porcine lung (n = 26). This technique yielded 3.8 +/- 0.5 x 10(8) (mean +/- SEM) mononuclear cells which were highly phagocytic toward particulate carbon (phagocytic index, 80 +/- 6%). Harvested mononuclear phagocytes reestablished intercellular adhesion plaques when placed on small vessel porcine pulmonary artery endothelial cell monolayers and exhibited histochemical characteristics typical of monocyte/macrophage lineage cells. Mononuclear cells obtained from lung microcirculation displayed size heterogeneity varying from 10.4 to 16.5 microns in diameter. Both large and small cell populations phagocytosed particulate carbon. Morphometric studies performed on collagenase-treated lung demonstrated that in situ perfusion removed significant numbers of intravascular macrophages in lung capillaries. The technique described permits the rapid removal of anchored mononuclear phagocytes from lung capillaries with minimal postmortem delay.

One of the few disinfectants known to inactivate the causative agent of Creutzfeldt-Jakob Disease is NaOH. In this study, NaOH was evaluated as a possible routine treatment for human dura mater alloimplants. Use of high concentration NaOH (1 M) resulted in protein loss and macroscopic changes to the tissue. Lower concentrations (0.1 M), although exhibiting little direct detrimental effect, greatly increased the susceptibility of the tissue to collagenase digestion. The use of NaOH treated commercial or institutionally prepared human dura mater should be approached with caution.

Primary cultures of adult rat hepatocytes were established using two different isolation procedures: a two-step collagenase perfusion and a method using ethylenediaminetetraacetate (EDTA) as the dissociating agent. Both techniques provided good yields of hepatocytes with comparable viability. The evolution of hepato-specific protein levels and several drug-metabolizing enzyme activities were followed for 8 days in cultured hepatocytes obtained by both methods. EDTA-isolated hepatocytes maintained a low gamma glutamyltransferase (GGT) activity, whereas collagenase-treated cells acquired a high GGT level. Transferrin secretion and tyrosine aminotransferase (TAT), alanine aminotransferase (ALT), and microsomal epoxide hydrolase (mEH) activities were stable in both EDTA- and collagenase-isolated hepatocytes, whereas albumin secretion, aspartate amino transferase (AST) activity, total cytochromes P-450 content, IA1 and IIB1 P-450 isoenzymes, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) levels, and bilirubin glucuronidation decreased faster in collagenase-treated cells. The most important difference observed was the maintainance of the mixed-function oxidase system in EDTA-isolated hepatocytes. These results emphasize the critical role of isolation technique in stabilization of differentiated hepatocytes in primary culture.

The expression of type VIII collagen is restricted, in adult mammals, to specialized extracellular matrices and to a select subset of blood vessels. We have examined the distribution of type VIII collagen in sequential stages of mouse and chicken embryos and found a temporal and spatially restricted pattern of expression during cardiogenesis. Type VIII collagen was first detected by immunocytochemistry on Day 11 in the developing mouse embryo and at stage 19 in the chicken embryo. The distribution of this protein was rapidly modulated during cardiac morphogenesis. Initially (Day 11 in the mouse embryo), type VIII collagen was associated with cardiac myoblasts. From Days 15 to 18, the immunoreactive component was progressively diminished in the myocardium; however, this collagen was observed in the subendocardial layer of the atrioventricular canal and later in the cardiac jelly (or the myocardial basement membrane, an area associated with the formation of cardiac valves). On Day 17, type VIII collagen was also detected in the subendothelium (intima) and tunica media of large vessels. Neonatal and adult hearts contained low to undetectable levels of type VIII collagen. The presence of type VIII collagen was confirmed by immunoblot analysis of heart extracts at different stages of development. A major 185-kDa component, as well as polypeptides of 68 and 15 kDa, reacted with anti-type VIII collagen IgG. Exposure of heart extracts to hyaluronidase or reducing agent eliminated immunoreactivity of the 185-kDa component but not that of the 68- and 15-kDa polypeptides. Type VIII collagen therefore appears to be associated with a hyaluronidase-sensitive component of the extracellular matrix during a temporally restricted stage of embryonic cardiogenesis. The contribution of this collagen to cardiac morphogenesis might reside, in part, in its ability to influence the differentiation of the myocardium and formation of the cardiac valves.

We investigated the in vitro mechanism of platelet aggregation induced by HMV-I human melanoma cells. HMV-I cells, in the absence of exogenous plasma proteins, induced platelet aggregation, followed by the release reaction. Heparin at an anticoagulant concentration had no effect on the aggregation. Calcium ion was essential for this tumor cell-platelet interaction and could not be replaced by magnesium. Among the adhesive proteins containing RGD sequences that have been reported to enhance experimental metastasis, fibrinogen and thrombospondin significantly enhanced the aggregation induced by HMV-I cells, fibronectin and von Willebrand factor inhibited it, and vitronectin had no effect. To identify the platelet-aggregating factor(s) of the tumor cells, we have developed a monoclonal antibody against HMV-I cells that can inhibit HMV-I cell-induced platelet aggregation. Immunoprecipitation analysis revealed that this antibody recognized an Mr 71,000 membrane protein. These results suggest that the association between the tumor cells and platelets is mediated by the Mr 71,000 membrane protein recognized by this monoclonal antibody.

We established five clones (ML-01, ML-02, MH-01, MH-02, MH-03) from murine 3-methylcholanthrene-induced fibrosarcoma A (Meth A), and investigated their experimental metastatic potentials in relation to their platelet-aggregating activities. A clone with a high metastatic potential (MH-02) showed a characteristic biphasic pattern of platelet aggregation, of which the first peak was not present in the aggregation patterns of the clone with low metastatic potential (ML-01). The first peak was eliminated by treatment of the cells with apyrase, indicating that adenosine diphosphate (ADP) was the causative substance of this particular peak. The metastatic potential of clones correlated well with the ADP concentration of the culture media. These results suggest that the increased ADP production and consequential enhancement of platelet-aggregating activity are closely related to the increment of pulmonary metastatic potential of MH-02 clone.

The effects of pH, ionic strength and proteinases on the destruction of bovine incisor root collagen were studied. Experiments were done with powdered and intact root specimens. Completely demineralized root powder was subjected to solutions of varying pH and ionic strength: (a) 0.1 M acetic acid, pH 4.0, (b) 0.1 M acetic acid + 0.15 M KCl, pH 4.0, (c) 0.1 M Hepes, pH 7.0 or to (d) 0.1 M Hepes + 0.15 M KCl, pH 7.0 at 37 degrees C. The surfaces of intact root specimens were exposed to 0.1 M acetic acid, pH 4.0 (which resulted in erosive lesions) or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0 (which produced subsurface lesions) at 37 degrees C. After incubation, the extracts were analysed for soluble collagen and the insoluble matrices were treated with trypsin at 15 degrees C to determine the denatured collagen. To estimate sensitivity to non-specific proteinases, demineralized root powder was also treated with trypsin under physiological conditions of temperature, pH and ionic strength. The denaturation and subsequent solubilization of collagen material from the fibrils could be influenced by variations in pH and ionic strength but these effects were small when compared to proteolytic degradation under physiological conditions. This supports the hypothesis that, in root caries, destruction of exposed matrix collagen depends largely on the presence and activity of proteinases.

The effect of 2-O-stearoyl glycerol-1,3-bisphosphate (Glydip) on caries lesion formation in root surfaces of sound human third molars was investigated in vitro. For this purpose parts of the root surfaces were treated with Glydip. Adjacent parts of the surfaces were not treated and served as control. Lesions were obtained by demineralization with an acetate buffer of pH 5.0. It was found that Glydip had no inhibiting effect on the rate of lesion formation. Additionally, pretreatments were performed with lauryl sulphate, a chloroform-methanol mixture, an aqueous solution of sodium hypochlorite, and collagenase prior to the treatment with Glydip to enhance the accessibility of the tissue for Glydip. None of these pretreatments or combinations of them revealed an inhibiting effect of Glydip on the rate of caries lesion formation. This result is in contrast to the effect of Glydip on the demineralization of enamel.

The effects of the calcium channel blockers, diltiazem and verapamil, on osteoblastic functions were investigated in cultured osteoblastic cells MC3T3-E1. DNA synthesis was evaluated by the incorporation of [3H]thymidine, and collagen synthesis by measuring the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). Diltiazem inhibited the DNA synthesis of osteoblastic cells by up to 57.6 and 54.6% at concentrations of 25 and 50 microM. Verapamil also significantly inhibited DNA synthesis by up to 61.6 and 40.9% at concentrations of 25 and 50 microM. The percent control of CDP formation were decreased by up to 76.7% in 5 microM and 44.3% in 50 microM of diltiazem. Verapamil also decreased CDP synthesis to 49.7% at 10 microM and 32.6% at 50 microM. NCP synthesis was decreased by the calcium channel blocker but inhibition of the CDP formation was greater than that of NCP. The calculated percent collagen synthesis was decreased at a calcium channel blocker concentration of 10 microM. The inhibitory effects of diltiazem and verapamil on percent collagen synthesis were not reversed by increasing the calcium concentration of culture media by either 1 or 5 mM. From this study, we conclude that calcium channel blockers have a direct inhibitory effect on osteoblastic function. Long-term administration of diltiazem or verapamil produces adverse effects on normal bone metabolism.

The expression of two matrix glycoproteins, tenascin and cellular fibronectin (cFN), has been studied in fully developed human permanent teeth, periodontal ligament, and alveolar bone, in both frozen and paraffin-processed material. Polyclonal antibodies to tenascin and a monoclonal antibody recognizing the ED sequence specific to at least some forms of cFN were used. Staining for both tenascin and cFN was positive in the dental pulp, odontoblastic layer, cementoblast-pre-cementum zone, and on the periosteal as well as endosteal surfaces of the alveolar bone. In the periodontal ligament, cFN was evenly distributed, whereas tenascin was accumulated in the attachment zones. Pre-dentin stained for tenascin but not for cFN. Mineralized dentin and cementum were tenascin- and cFN-negative. The relative staining intensity for tenascin was greater than that for cFN in the cementoblast-pre-cementum layer and in the attachment zones of the periodontal ligament, whereas cFN stained more intensely in the pulp. In frozen material, antigenicities were well-preserved. Paraffin processing facilitated precise recognition of tissue morphology, but the antigenicity of cFN was lost. The co-expression of tenascin and cFN in the dental pulp, cementogenic zone, and on the surfaces of the alveolar bone may reflect the ability of the cells to deposit mineralized tissue matrices. The pronounced expression of tenascin in the interfaces between mineralized and non-mineralized tissues suggests that it is functionally associated with mechanical stress and may thus have at least two distinct functions. The relative amounts of the two matrix glycoproteins may contribute to regulation of tissue structure.

Norepinephrine (NE) uptake into a heart synaptosomal-mitochondrial fraction was assessed under conditions where neuronal uptake (type 1) was linear with respect to both time and protein concentration. The NE accumulation process was sensitive to incubation temperature, sodium ion concentration and medium osmolality. Furthermore, NE uptake was attenuated by the neuronal uptake inhibitor desmethylimipramine (DMI) in a concentration dependent manner; the IC50 value was approximately 10 nM and maximum inhibition was obtained at 100 nM. In contrast, the extraneuronal uptake inhibitor, metanephrine did not significantly attenuate NE uptake. Kinetic analysis demonstrated that the DMI sensitive NE accumulation is saturable with a KM of approximately 400 nM and that NE uptake occurs via a single uptake process. This demonstration of neuronal type NE uptake by a synaptosomal-mitochondrial fraction constitutes a successful demonstration of the preparation of a rat heart subcellular fraction containing functional synaptosomes.

Basement membranes (BM) are elements of the extracellular matrix that are essential for growth and differentiation of tissues. Several collagenolytic enzymes of tumor cells are involved in degradation of the extracellular matrix; growth and inhibitor factors [e.g. Epidermal Growth Factor (EGF), Transforming Growth Factors alpha and beta (TGF-alpha, beta)] seem to be involved in the extracellular matrix formation and degradation. To establish a possible association between the presence of collagenase (C), urokinase-type plasminogen activator (uPA) and the neoplastic growth of the endometrium, 44 endometrial specimens (14 proliferative, 11 secretive, 7 adenomatous hyperplasia, 12 adenocarcinoma) were studied using immunohistochemistry with antisera for C, uPA, EGF receptors and TGF-alpha. Immunostaining for collagenase revealed a positive reaction in moderately differentiated adeno-carcinoma without staining the normal and hyperplastic endometrium. A progressive increase in uPA immunostaining was observed in proliferative and neoplastic endometrium. TGF-alpha and its receptor (EGFr) were stained in proliferative and more clearly in hyperplastic and carcinomatous endometrium. In conclusion, BM play an important role in proliferation and differentiation of human endometrium; their degradation influences estrogen transportation from blood to the stroma. Endometrial BM degradation is associated with the presence of collagenolytic enzymes and growth factors.

Fibronectin fragments generated by Achromobacter iophagus collagenase exhibit a gelatinolytic activity. This activity is inhibited by phenyl-methyl-sulfonyl fluoride and pepstatin A. After separation of this collagenase digest of fibronectin on heparin Ultrogel, a laminase activity was also evidenced using laminin and the synthetic peptide Gly-Pro-Ala-Gly-Pro-Arg as substrates. Different results were obtained with a cathepsin D digest of fibronectin that exhibited gelatinolytic and laminolytic activities only after incubation with Ca++. This suggests that the proteinases produced by hydrolysis of fibronectin enhance the effect of collagenase on extracellular matrix proteins.

Chick basilar papillae were incubated in collagenase, using concentrations previously employed for the isolation of viable hair cells. When assessed by scanning electron microscopy, the hair bundles had a normal conformation, with no loss of tip links compared with control incubations. The results suggest that collagenase dose not destroy the integrity of structures on the apical surface of hair cells, and that tip links are not composed of collagen. The results are in agreement with the hypothesis that tip links are involved in mechanotransduction.

Oocytes of 40% of Xenopus laevis frogs respond to acetylcholine (ACh). Oocytes of the majority of responders exhibit the common two-component depolarizing muscarinic response (mean amplitude of the rapid component, 54 nA). Oocytes of approximately 10% of the responders ("variant" donors) exhibit a muscarinic response characterized by a very large transient, rapid current (mean amplitude 1242 nA, reversal potential -33 mV). Responses in oocytes of variant donors exhibit further qualitative differences: pronounced desensitization (absent in oocytes of common donors), characteristic prolonged latency (5.4 vs 0.9 s in oocytes of common donors) and marked inhibition of the response by activators of protein kinase C. Rapid responses in oocytes of variant donors are usually increased by treatment with collagenase, which, in common oocytes, often results in a complete loss of the response that correlates with the loss of muscarinic ligand binding. The number of muscarinic receptors was similar in oocytes of both types of donors (2.2 vs 3.0 fmol/oocyte). Also, the responses of oocytes of variant donors to microinjections of CaCl2 or inositol 1,4,5-trisphosphate were similar to those found in cells of common donors. These findings imply that altered receptor number, calcium stores and/or chloride channel density are not responsible for the variant responses. However, ACh caused an sixteen-fold greater efflux of 45Ca in oocytes of variant donors (35 vs 2.2% of total label in oocytes of common donors). Hence, the characteristics of the variant response may be related to a more efficient coupling between receptor stimulation and the mobilization of cellular calcium.

Epithelia and fibromuscular walls were dissociated from the vaginae of ovariectomized BALB/cCrgl mice (ca. 41 days old) exposed neonatally to diethylstilbestrol (DES) or sesame oil and purified by centrifugation through Percoll density gradients. Neonatal exposure to DES caused the vaginal epithelium to become permanently proliferated and partially keratinized; the control epithelium was low cuboidal. The major cellular proteins expressed in each tissue compartment were examined by two-dimensional gel electrophoresis of [35S]methionine-labeled tissues. The epithelia and fibromuscular walls displayed distinctive two-dimensional protein patterns. In the DES-exposed vaginal epithelium, the expression of two proteins (one had a molecular size of 65 kDa and a pl of 6.0, and the other had a molecular size of 38 kDa and a pl of 6.3) was increased, while the expression of three proteins with molecular sizes of 25, 30, and 140 kDa and pls of 5.6, 5.6 and 6.7, respectively, was reduced, relative to the control epithelium. In the DES-exposed vaginal fibromuscular walls, the expression of 9 proteins was increased whereas the levels of 21 specific proteins, distinct from those in the epithelium, were decreased. Thus, long-term tissue-specific alterations in the synthesis of a select number of cellular proteins occur in the DES-exposed vagina.

A novel polypeptide with a molecular weight of 17 kd (17k protein) was identified in bone marrow cell cultures. The synthesis of 17k protein is elevated in cell cultures maintained under Dexter conditions, which support myelopoiesis. The predominance of macrophages in the stromal layer of these cultures and the observation that a mouse myelomonocytic cell line P388D1 is capable of synthesizing large amounts of 17k protein led us to the study of its synthesis by bone marrow macrophages. Metabolic labeling with [14C]proline and partial amino acid analysis of 17k protein demonstrated that the polypeptide contains relatively high amounts of proline and is also sensitive to degradation with bacterial collagenase. However, no hydroxyproline is detectable in 17k protein, and it is extensively degraded with bacterial collagenase. However, no hydroxyproline is detectable in 17k protein, and it is extensively degraded by proteolysis with pepsin, using conditions under which collagen triple helices are resistant to degradation, suggesting that collagen-like structures are not contained in 17k protein. This polypeptide is found predominantly in the cellular layers of bone marrow macrophage cultures. Incorporation of [14C]proline into 17k protein is diminished by increasing concentrations of colony-stimulating factor 1 (CSF-1). The 17k protein may be involved in macrophage proliferation because its synthesis is inhibited by CSF-1, which is required for the maintenance of bone marrow macrophages in vitro.

Enzymes are often used for preparation of excitable tissues. The effects of papain, trypsin, pronase, collagenase and hyaluronidase on the photoreceptor function were studied by recording of scotopic PIII responses. Each enzyme treatment diminished the amplitudes of the PIII responses with a characteristic time course. The effects of papain, collagenase and hyaluronidase were at least partly reversible, while trypsin and pronase irreversibly reduced the amplitudes of the PIII responses.

The authors used the principal features of a collagenase perfusion technique and an automated dissociation technique to determine if islets could be isolated from the large mammal pancreas and to compare the effects of the two methods on isolated islets. The pancreases of 16 dogs were cannulated and perfused with collagenase at 4 degrees C, then warmed to 37 degrees C. Group 1 (eight) pancreases were perfused at 37 degrees C until digested, then dissociated manually by teasing and trituration. Group 2 (eight) pancreases were transferred to a closed chamber for continued collagenase digestion and dissociation at 37 degrees C. Islets were purified using identical Ficoll gradients. Aliquots were stained with dithizone and evaluated for number, size and purity. Total islet volume was calculated. Group 2 pancreases were thoroughly digested leaving only a few residual ducts, but undigested fragments persisted in group 1 pancreases. Islet size was similar in both groups. There was a greater islet volume before and after Ficoll purification in group 2, but the difference was not significant. Purity was greater than 90% in both groups. Perifusion with 28 mM glucose elicited a biphasic insulin release from islets in both groups. The data show that the combined protocol enables mass isolation of purified islets from the canine pancreas. Compared with the manual technique, the automated protocol for pancreas dissociation tends to improve the yield of islets without compromising islet size and viability. It provides the advantages of a closed system with increased control over the extent of collagenase digestion.

The purpose of this study was to assess the migration, attachment, and orientation of human gingival fibroblasts to human dental roots over a period of 21 days in vitro. The fibroblasts were incubated with a total of 120 periodontally diseased and non-diseased root slices (200 microns thickness) which had been treated in the following manner: 1) Root planed diseased root (DT); 2) Root planed and citric acid demineralized diseased root (DTD); 3) Non-treated diseased root (DNT); 4) Citric acid demineralized diseased root (DNTD); 5) Non-diseased control (ND); 6) Citric acid demineralized non-diseased root (CA); 7) Citric acid and collagenase digested non-diseased root (CAC); 8) EDTA demineralized non-diseased root (E); and 9) EDTA-demineralized and collagenase-digested non-diseased root (EC). The results showed that that most active phase of cell attachment and orientation occurred during the first 10 days of the experiment. Statistical differences were observed between the variables, and, in terms of cell attachment and orientation to the root slices, it was concluded that: 1) Root planing improves diseased roots; 2) Acid demineralization subsequent to root planing improved diseased roots to such an extent as to render them comparable to non-diseased roots; 3) Citric acid demineralization alone improved diseased roots to the same extent as root planed diseased roots; 4) The exposure of collagen fibrils resulting from acid demineralization of the tools is not the sole reason for the improvement of the root surface, but rather a combination of the exposed collagen fibrils with the creation of a more hospitable environment was found to be responsible.

The relationship between the sensitivity (the pD2 value) of carbachol and the density (the total concentration of receptors) of muscarinic receptors using single cells from the guinea pig taenia caecum prepared with a mixture of crude collagenase and trypsin inhibitor, purified collagenase alone, and a mixture of purified collagenase and papain was examined. The sensitivity of the single cells prepared with a mixture of purified collagenase and papain was about 10 times more effective than that of the single cells prepared under other conditions. The dissociation constant of [3H]quinuclidinyl benzilate (QNB) and Hill's coefficient did not change in the single cells prepared under the three conditions, though the maximum binding sites were significantly greater in the cells prepared with the mixture of purified collagenase and papain than in those prepared by other means. These results suggest that the increase in the sensitivity of carbachol obtained in the single cells prepared with this mixture is due to the increase in the density of muscarinic receptors and also suggest that the effects of this enzyme mixture may be due to an increase in the incorporation of newly synthesized receptors and (or) changes in receptor turnover.

Microelectrode studies have shown that the basolateral membrane of the principal cells (PC) of the rabbit cortical collecting duct (CCD) contains Cl(-)-conductive pathways. To determine the properties of single Cl- channels we prepared the basolateral membrane for patch clamping by incubating the CCD in collagenase and/or tearing the basement membrane with a fine needle. When high concentrations of collagenase were used, only a small nonselective channel was observed. In low concentrations or the absence of collagenase, however, we identified a Cl- channel (g46) in both cell-attached and excised patches. The Cl- channel gated rapidly between three equally spaced substates of 0 (S0), 23 (S1), and 46 pS (S2) and slowly between states C (inactive) and S0. The conductance of each substate was not voltage dependent between pipette potentials from -60 to +60 mV (cell attached). The probability that the channel gated from C to S0 increased with hyperpolarizing potentials, but the probability that g46 was in substate S0 increased with depolarizing patch potentials. This channel was similar to that described by Miller for the nonexcitable membrane of the electric organ of Torpedo californica. Because g46 was the most frequently observed basolateral membrane channel and was voltage dependent at physiological potentials, it is probably the channel responsible for the dominant Cl- conductance of PC.

To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5-30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20-30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.

There is evidence to suggest that the B cell population in the marginal zone (MZ) of the spleen is responsible for the antibody response to thymus-independent type 2 antigens (TI-2). The macrophage (M phi) population in the MZ has been shown to take up TI-2 antigens selectively, and this uptake is potentially important in understanding antigen handling in TI-2 responses. Uptake has not been studied in vitro because isolation of MZ M phi completely abrogates uptake. We have adapted a technique for cutting thin slice of viable spleen which retained this M phi function under in vitro conditions and allowed manipulation of the system. This technique may have widespread application in the study of tissue M phi which are difficult to isolate. Using this method, we show that MZ M phi in splenic slices in vitro selectively take up a TI-2 antigen, in a way apparently identical to that seen in vivo. This is not due to high pinocytic activity by these cells nor to their anatomical location. We provide evidence for a receptor-mediated uptake system, whose function is sensitive to collagenase. The ligand specificity of the uptake system showed unexpected cross-reactivity with the mannosyl-fucosyl receptor with high affinity for mannan, but was not identical to it, and may be indicative of the natural ligands for the TI-2 antigen uptake system.

Tumor-associated macrophages (TAM) have peculiar membrane phenotype and functional properties. In an effort to unravel possible molecular determinants associated with the reprogramming of mononuclear phagocytes that infiltrate tumors, we have investigated the expression of immediate-early genes in TAM from murine sarcomas. c-fos is a prototypic immediate oncogene, with transregulatory function on gene transcription, expressed by myelomonocytic cells and induced by certain activation signals. TAM from three murine sarcomas expressed basal levels of c-fos transcripts considerably higher than those of peritoneal exudate macrophages (PEM). Activation of myelomonocytic cells by bacterial LPS is associated with an early transient increase in c-fos transcription. Unlike PEM, TAM did not show any increase in c-fos expression after exposure to LPS. A similar unresponsiveness to LPS stimulation was observed in macrophages isolated from peritoneal ascitic tumors. c-fos expression in macrophages can be induced via protein kinase C activation or via an increase in cAMP levels. Unlike PEM, TAM did not respond to the protein kinase C activator PMA and to cholera toxin. After culture for 18 to 20 h, c-fos expression in TAM declined, and concomitantly, TAM completely recovered responsiveness to LPS in terms of augmented oncogene mRNA levels. These results demonstrate that TAM from murine sarcomas have an altered expression of the c-fos proto-oncogene, with high basal levels and unresponsiveness to augmenting signals, reversible upon in vitro culture. The altered c-fos expression in TAM may reflect exposure to cytokines present in the tumor microenvironment and may underlie at least some of the peculiar properties of TAM.

Effect of HCl on the endothelium-dependent increase in mucosal blood flow and effect of endothelium-derived relaxing factor (EDRF) inhibitors or nitrites on the HCl-induced gastric lesion were studied to clarify the effect of EDRF on the formation of gastric lesion in rats. Topical application of 0.6 N HCl on the gastric mucosa inhibited the endothelium-dependent increase in mucosal hemodynamics estimated using organ-reflectance spectrophotometry induced by vagal stimulation or acetylcholine, but not by papaverine. Collagenase, gossypol, hemoglobin and ascorbic acid have been reported to inhibit the endothelium-dependent vasodilation, inhibited increase in mucosal hemodynamics induced by vagal stimulation and acetylcholine. These inhibitors and methylene blue significantly enhanced the gastric lesion induced by 0.45 N HCl. Intra-arterial or topical application of nitrites (sodium nitrite and isoamyl nitrite) increased mucosal hemodynamics. Oral administration of nitrites prevented the formation of 0.6 N HCl-induced gastric lesion. These results suggest that EDRF plays an important role in the protection of gastric mucosa against HCl. Reduced endothelium-dependent increase in mucosal blood flow may be an etiology of gastric lesion in rats.

Papain, trypsin and collagenase were used in the experiment for the study of their effects on the demineralization of enamel surface by organic acids in vivo. No effect of any of the enzymes was observed. The use of papain and trypsin in treating tooth surfaces in vitro, no matter whether sodium bisulfite was used, showed no effect on the demineralization of the enamel by mixed organic acids. But, by the analysis of amino acids in the enzyme solutions before and after treating tooth surface, it was shown that some proteinous substances were destroyed. Accordingly, it can be suggested that the enzyme takes no part in the destruction of the inorganic tooth substance.

1. The influences of enzyme treatments (trypsin and collagenase) on responses to perfused acetylcholine were examined on physically isolated single Aplysia neurons, using the voltage-clamp, internal perfusion, and rapid external perfusion technique. 2. During treatment with trypsin (0.025 to 0.1%) for 10 to 30 min at room temperature (22 to 25 degrees C), the peak amplitude of the Na current induced by acetylcholine increased in a time- and dose-dependent manner, and the decay in the continued presence of acetylcholine was slowed. This effect of trypsin treatment was irreversible after washing for 60 min without enzyme. 3. Edrophonium, a cholinesterase inhibitor, has previously been shown to augment the Na acetylcholine response in this preparation by inhibition of acetylcholinesterase. After treatment of the neuron with trypsin, the augmentation after edrophonium was abolished. Furthermore, in the presence of edrophonium, trypsin also failed to increase the response. The dose-response curve for acetylcholine after treatment of trypsin was similar to that in the presence of edrophonium. These results suggest that the modification of the current response by trypsin is a result of removal of cholinesterase activity from the membrane. 4. In contrast to the effects of trypsin, collagenase (0.03 to 0.1%) for 10 to 60 min did not change the current amplitude of the acetylcholine response. However, collagenase treatment did alter the kinetics of the acetylcholine response in a dose-dependent manner, in that the rate of decay was accelerated. A similar acceleration was seen in the acetylcholine responses on other neurons which were due to Cl or K currents, suggesting that the effect was independent on the type of channel. This effect of collagenase was reversible after 30 to 60 min of washing of the neuron. 5. In the presence of edrophonium or after the treatment with trypsin, collagenase still accelerated the current kinetics of the acetylcholine response, indicating that cholinesterase activity is not related to this effect. Furthermore, heated collagenase (presumably inactivated) had a similar action, suggesting that the enzymatic activity of collagenase is not related to the modification of the response. 6. These results suggest that Aplysia acetylcholinesterase is sensitive to trypsin but not to collagenase. However, the preparation of a collagenase used in these studies contains some factor which alters the response to acetylcholine, but this effect is reversible and unrelated to enzymatic activity.

The goal of the present investigation was to determine whether leukotriene D4 (LTD4) was capable of releasing endothelium-derived relaxing factor (EDRF) from perfused renal arterial (RA) segments and to begin to characterize any released mediator. To detect EDRF, a bioassay was used in which a prostaglandin (PG) F2 alpha precontracted, endothelium-denuded coronary artery (CA) ring was superfused either directly or with effluent from a perfused RA segment. Addition of LTD4 or acetylcholine (ACh) to the perfusate proximal to the RA segment resulted in CA ring relaxation, the degree of which was inversely related to transit time. Calculated half-life (t1/2) for the CA relaxing substance released by LTD4 and ACh from the RA segment was 7.5 +/- 1.4 and 7.4 +/- 0.9 s, respectively. Pretreatment of the RA segment with collagenase prevented relaxation of the CA ring evoked by RA effluent in response to either ACh or LTD4 that was administered into the perfusate proximal to the RA segment. Addition of superoxide dismutase (SOD) to the effluent distal to the RA segment markedly enhanced both ACh and LTD4-evoked relaxation of the CA ring, whereas reduced hemoglobin (Hb) virtually abolished these responses. When either LTD4 or ACh was superfused directly over the CA, no change in tone of the bioassay ring was observed. These data indicate that, similar to ACh, LTD4 has the ability to release a labile substance(s), presumably EDRF, from the endothelium-intact RA segment that evokes relaxation of the endothelium-denuded CA ring. Although apparently similar, further studies are required to confirm whether or not the mediator(s) released by LTD4 is identical to that which is released by ACh.

We describe a method for maintaining neonatal pig pancreatic isletlike cell clusters (as pseudo-islets) embedded in a collagen gel matrix for long periods. The pseudo-islets were formed from single cells of pig pancreas maintained in a suspension culture and then embedded in pepsin-solubilized type I collagen. When the pseudo-islets were cultured in the collagen matrix, the amount of collagen in the culture decreased gradually during the culture period as soluble hydroxyproline-containing material accumulated in the medium. A low concentration of collagen (0.16%) degraded the collagen gels more rapidly than did high concentrations of collagen (0.64%). The degradation of collagen depended both on the number of pseudo-islets embedded in the gel matrix and on the culture conditions used to maintain them. With added nicotinamide, the accumulation of hydroxyproline decreased in the medium and the structure of the gel matrix was well maintained. Hydrocortisone or a specific inhibitor of collagenase did not decrease the solubilization of embedded pseudo-islet cultures and did not help to maintain their structure. These observations indicate the possible utility of long-term maintenance of pseudo-islets in collagen gel matrix in the presence of nicotinamide.

The true surface of rabbit spinal ganglion neurons has been made directly accessible to scanning electron-microscope observation after removal of both the connective tissue and satellite cells that normally cover it. The neuronal surface is characterized by a profusion of slender projections whose shapes have been determined and whose length and width have been quantified. Controls carried out with transmission electron microscopy demonstrate that the procedure employed in this study satisfactorily preserves neuronal structure.

Previous studies have shown that prostaglandin E2 (PGE2) has both inhibitory and stimulatory effects on the incorporation of proline into collagenase-digestible protein (CDP) in cultured fetal rat calvaria. The present studies were undertaken to analyze further these biphasic effects of PGE2. PGE2 increased [3H]thymidine incorporation at 24 h, and this effect was enhanced in the presence of cortisol (10(-8) and 10(-7) M). An inhibitory effect on CDP labeling was observed at 96 h with PGE2 (10(-6) M) in the absence or presence of indomethacin (10(-6) M), but not in the presence of cortisol (10(-8) or 10(-7) M). When the central osteoblast-rich bone and periosteum were analyzed separately, the inhibitory effect of PGE2, with or without indomethacin, was confined to the central bone. Addition of aphidicolin (30 microM), an inhibitor of cell replication, did not prevent the inhibitory effect of PGE2 on CDP labeling. Analysis of labeled collagen by polyacrylamide gel electrophoresis showed a decrease in labeling of type I collagen in central bone. Moreover, mRNA for alpha 1(I)procollagen was decreased, as measured by dot blot hybridization and Northern blot analysis. Cortisol (10(-8)-10(-6) M) decreased the labeling of CDP as well as noncollagen protein (NCP) at 96 h. In the presence of cortisol, PGE2 (10(-8)-10(-5) M) consistently stimulated labeling of CDP and NCP, with a greater increase in CDP, resulting in an increase in the percentage of collagen synthesized. In the presence of low concentrations of cortisol (10(-8) or 3 x 10(-8) M), PGE2 (10(-7) M) increased CDP labeling by 260-480%, and the absolute value was 145-160% of that in control cultures without any hormone addition. The stimulatory effect was seen in both central bone and periosteum, although absolute values for CDP and percentage of collagen synthesized were higher in central bone. PGE2 (10(-7) M) had similar effects on CDP at 24 and 96 h in the presence of cortisol, and the stimulation at 10(-7) M was the same in the presence and absence of aphidicolin, suggesting that it was not dependent on cell replication. Cortisol decreased labeling of type I collagen, determined by polyacrylamide gel electrophoresis, and alpha 1(I)procollagen mRNA levels, determined by both Northern and dot blot analysis. PGE2 reversed these effects, increasing both radiolabeled collagen type I chains and alpha 1(I)procollagen mRNA levels. These results indicate that PGE2 can regulate bone collagen synthesis at a pretranslational site.(ABSTRACT TRUNCATED AT 400 WORDS)

Deposits of IgG localized to collagen bundles/extracellular matrix components occurred in skin biopsies from patients with primary fibromyalgia (PF). None of these patients demonstrated a positive lupus band test. Control skin biopsies from healthy controls were negative but showed intense reactivity for IgG after collagenase treatment. PF-skin attached both homologous and heterologous serum IgG in indirect immunofluorescence, which may point to a qualitative alteration of dermal matrix components in PF. Skin from patients with systemic lupus erythematosus and rheumatoid arthritis showed a lower dermal fluorescence intensity than in PF patients. The cause of the presence of IgG in dermal tissue from PF patients is unclear. It may be caused by a non-specific attachment of IgG to the extracellular matrix related, for example, to tissue hypoxia and/or increased capillary leakage due to an increased number of mast cells in the PF-skin.

Cultures of rat myotubes from 18-day-old embryos produce both globular (G) and asymmetric (A) forms of acetylcholinesterase (AChE; EC 3.1.1.7), mostly G1, G4, and A12 and a small proportion of A8. We show that all forms are partly intracellular and partly exposed to the extracellular medium; the A forms and their intra- and extracellular distribution are not modified when myotubes are grown in the presence of spinal cord neurons. In these cocultures, however, AChE patches may be detected immunohistochemically at sites of neuromuscular contacts. These patches represent a very minor proportion of AChE activity. We found that collagenase removes AChE patches but not the acetylcholine receptor clusters with which they coincide. This digestion specifically decreases the level of the A12 form. cis-Hydroxyproline, an inhibitor of collagen synthesis, reduces the level of G1 and blocks the synthesis of A forms.

The effectiveness of the enzymatic and mechanical methods for isolation of hemopoietic islets (HI) from rat bone marrow was comparatively studied. It has been shown that the use of collagenase brings higher yields of HI than the mechanical treatment of bone marrow, mainly at the expense of associations containing no central phagocytizing macrophages. When the mechanical method of HI isolation was used, erythroid islets associated with mononuclear phagocytes prevailed, while with the enzymatic method mainly erythro-granulocytic islets associated with macrophages or reticular cells were isolated. It has been suggested that granulocytic and erythro-granulocytic associations are less resistant to the mechanical treatment.

Experiments were performed to examine longitudinal retractive force in pressurized arteries in vitro. This force opposes vessel elongation and prevents the development of tortuosity. Common carotid arteries were excised from six adult dogs and external iliac arteries were excised from six elderly male humans at autopsy. Each vessel was mounted in a tissue bath at in situ length and was pressurized. Longitudinal retractive force was measured under control conditions and after treatment with elastase or collagenase. Results showed that, in the dog vessels, elastin provides all of the longitudinal retractive force. In the aged human vessels, both elastin and collagen provide longitudinal retractive force, with elastin contributing the much greater part.

We have developed a retinal microvessel culture system which permits study of the initial events of endothelial cell activation and migration during the angiogenic response. Enzyme digest experiments indicate that Type IV collagen is the major basement membrane component regulating migration and growth of endothelial cells. Following removal of basement membrane collagen, further cell migration and proliferation require a suitable substrate. Laminin, fibronectin and fibrin(ogen) provide excellent substrates for endothelial cell outgrowths while Type I collagen, even if prepared as a three-dimensional gel, or Type IV collagen fails to promote typical cell growth. In contrast to fibrin and fibronectin, plasmin was a poor substrate for cell outgrowth and it is suggested that cell-associated protease activity may exert a regulatory role over endothelial cell-matrix interactions during cell migration.

The electric organs of two species of skate have been examined morphologically, physiologically and biochemically. They can be easily dissociated into innervated or denervated component electrocytes by a Torpedo Ringer's solution containing 1% collagenase. Collagenase treatment did not, however, separate the Schwann cell cover capping the synaptosomes. Isolated electrocytes generate normal MEPP frequencies and show evoked responses for two days in Torpedo Ringer's. The nerve terminals retain excitability and transmitter release properties up to the time of separation. Since isolated terminals and denervated electrocytes show normal ultrastructural characteristics for up to 12 h, the skate electric organ provides several preparations which are not attainable with Torpedo tissue. Acetylcholine (ACh) content of supernatant fractions containing the synaptosomes was comparable to that found in Torpedo (sps.). Collagenase specifically eliminates the basal lamina associated with the synaptic junctional region. Neuronal cell death and synaptic terminal degeneration were also noted in the adult organs of both species. The skate electric organ is ideally suited for the study of cholinergic development and transmission.

Elastic fibers comprise elastin and other proteins termed as elastin-associated proteins. The nature of association between the elastin and elastin-associated-protein is not known. We have isolated elastic fibers from 5-month-old porcine aorta and lung parenchyma using urea, dithiothreitol and 1% sodium dodecyl sulfate at 55 degrees C. Lysyl-derived crosslinks were stabilized by sodium borohydride reduction. The methionine residues and associated peptides were decreased by CNBr treatment. Limited proteolysis of elastic fibers obtained by this procedure by trypsin (TPCK) and chymotrypsin (TLCK), removed 3% and 11% of the elastic fibers from aorta and lung, respectively. Limited elastase digestion solubilized a further 12 to 14% of the elastic fiber from aorta and lung samples, respectively. The insoluble residue remaining after elastase digestion had amino acid composition similar to alkali extracted elastin and to tropoelastin. The material solubilized by chymotrypsin and trypsin contained lysinonorleucine, whereas desmosine crosslinks were present only in the elastase digests. Aorta and lung elastic fibers differ in their structures as indicated by quantitative differences in limited proteolysis. These results strongly indicate that elastin and elastin-associated proteins interact strongly through lysyl-derived crosslinks.

Intralesional injection of BCG into an established line-10 hepatocellular carcinoma in the strain-2 guinea pig causes regression of the tumor and induction of line-10 immunity. We found that the animals were already protected for a second challenge with line-10 tumor cells 7 days after BCG treatment. We studied whether this early induction of immunity was correlated with the expression of MHC class II antigens on line-10 tumor cells and was correlated with an increased expression of MHC class II antigens on leukocytes in the primary tumor and in the regional lymph node (Ln. axillaris accessorius). The MHC class II antigens and the leukocyte subpopulations were measured with monoclonal antibodies and flow cytofluorometry. In the draining lymph node the number of nucleated cells increased about 10-fold during the first 5 days after intralesional injection of BCG. At this time the MHC class II antigen expression of these cells was increased from 21%-32% in the naive controls to 39%-53% in animals with BCG-treated tumors. This implies that the number of MHC-class-II-positive cells increased about 20-fold in the draining lymph node. Surprisingly, the increase in percentage of MHC-class-II-antigen-positive cells was mainly due to an increase of IgM-positive B cells from 8%-11% to 22%-41% and an increase of IgG-positive B cells from 7%-27% to 25%-44%. In the tumor, BCG treatment induced a small increase of MHC-class-II-antigen-positive cells from 11%-12% to 15%-20%. Probably this increase came not from tumor cells but mainly from a BCG-induced infiltration of mononuclear cells, as an increase of T cells from 14% to 20%, an increase of macrophages from 8% to 18%, and an increase of B cells from 0 to 6% was observed. We conclude that the potentiation of anti-(line-10 tumor cell) immunity correlated with a 20-fold increase of MHC-class-II-antigen-positive cells in the lymph nodes and a small increase in the number of MHC-class-II-antigen-positive tumor-infiltrating cells.

Extracellular glycosylation of proteins in diabetics may contribute to observed impairments in wound healing. We investigated the interactions of blood glucose concentration and wound collagen glycosylation, collagen content, and proteolytic activity during wound healing in diabetic animals. Rats were made differentially hyperglycemic with intraperitoneal injection of streptozotocin (0-65 mg/kg body weight). Animals with blood glucose concentrations greater than 240 mg glucose/dL were classified as diabetic, those with blood glucose concentrations less than 160 were grouped as nondiabetic. Polytetrafluoroethelene (PTFE) wound cylinders were surgically implanted in 32 rats and removed on postoperative day 5. Harvested cylinders were analyzed for hydroxyproline content, collagen glycosylation, and collagenase and protease activity. Collagenase activity was 14% higher in diabetics than nondiabetics (P less than .001). Glycosylation of wound collagen averaged 48.0% higher in diabetics (P less than .001). Wound hydroxyproline content was 39% lower in diabetics (P less than .05). Studies show a high degree of correlation (Pearson) of wound collagen nonenzymatic extracellular glycosylation (NEG) with mean blood glucose concentration (r = .98, P less than .001). Wound collagen glycosylation correlates strongly with both protease activity (r = .86, P less than .001) and collagenase activity (r = .83, P less than .001). This study demonstrates a significant blood glucose concentration dependent increase of glycosylation in newly synthesized collagen in hyperglycemic animals that is associated with increased collagenase activity and decreased wound collagen content.

High yields of human hepatocytes (up to 23 X 10(6) viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham's F12 containing 0.2% bovine serum albumin, 10(-8) M insulin, and 10(-8) M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250 +/- 177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50 +/- 0.17 nmol glucose.mg-1.min-1) similar to that reported for human liver. Insulin at 10(-8) M activated glycolysis (X1.40) and glycogenesis (X1.34), and glucagon at 10(-9) M stimulated gluconeogenesis (X1.35) and glycogenolysis (X2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, haptoglobin, alpha 2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10(-9) M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol.mg-1 cell protein.min-1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol.mg-1). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.

Two different types of elastic behaviors could be distinguished in the twitch contraction curve of the isolated frog geniohyoid muscle under changing of chemical conditions; one was negative oscillation that occurred after completion of the relaxation phase which was sequential to a typical cyclic pattern demonstrated in the contraction and relaxation period, and the other was oscillation which occurred in the phase of plateau formation during relaxation. The former will relate to the parallel elastic component (the effect of caffeine combined with a low Ca++ condition) and the latter will to the series one (collagenase and dantrolene). The causal or genetic relationship is not yet clarified.

We evaluated colony formation in soft agar by cells obtained after mechanical and/or enzymatic disaggregation of 455 malignant human tumours. Counting and assessment of cell colonies in the agar plates were done by inverted microscopy, computerized image analysis, and inspection of serial photomicrographs of the agar plates. Our results indicate that standard methods of tumour disaggregation did not usually produce single-cell suspensions and that aggregates of tumour cells varying greatly in size were placed in the agar. Most groupings of cells identified as colonies 1-3 weeks after plating arose from enlargement of preexisting aggregates of cells.