Prenatal morphine alters the synaptic complex of postsynaptic density 95 with N-methyl-D-aspartate receptor subunit in hippocampal CA1 subregion of rat offspring leading to long-term cognitive deficits.

Infants who are passively exposed to morphine or heroin through their addicted mothers usually develop neurobiological changes. The postsynaptic density 95 (PSD-95) protein, a submembranous cytoskeletal specialization, is dynamically linked with N-methyl-d-aspartate receptors (NMDARs) to form a synaptic complex in postsynaptic neurons. This complex serves important neurobiological functions, including mammalian learning and memory. However, the effects of prenatal morphine exposure on this synaptic complex are not well understood. In this study, we determined whether prenatal morphine exposure altered the synaptic complex association between PSD-95 and three major NMDAR subunits (NR1, NR2A, and NR2B), at the mRNA and protein levels, within the hippocampal CA1 subregion (an important integration area for mammalian learning and memory) of rat offspring along with the performance of long-term cognitive functions. Sprague-Dawley rat offspring from morphine-addicted mothers were studied at a younger age (postnatal day 14; P14) and at an older age (P45). Subsequently, an eight-arm radial maze task was applied to analyze the working and cued reference memory in such offspring (P45). The real-time polymerase chain reaction results showed that prenatal morphine exposure caused significant decreases in mRNA levels of the PSD-95 and three NMDAR subunits (NR1, NR2A, and NR2B) in offspring (P14 and P45). Similarly, at the protein level, immunoblotting showed that decreased whole levels of PSD-95 and NMDAR subunits were seen in offspring subjected with prenatal morphine. Furthermore, the protein interaction of the synaptic complex between the PSD-95 and NMDAR subunit, as indicated by coimmunoprecipitation, was less in prenatal morphine samples than in vehicle controls (P14 and P45). The prenatal morphine group also showed poorer performance for an eight-arm radial maze task than the vehicle-control group. These results are particularly important for a better understanding of certain opioid-mediated neurobehavioral cognitive changes in offspring associated with altered protein interaction between PSD-95 and NMDAR subunits within the developing brain.

Nitroimidazole inhibition of lactate production in CHO cells.

Misonidazole (MISO) and desmethylmisonidazole (DMM) inhibit lactate production in CHO cells under aerobic conditions. This inhibition is time and dose dependent. At the 20 mM level DMM is stronger in producing this inhibition of lactate than is MISO. This inhibiton of lactate production suggests that these compounds are capable of inhibiting the glycolytic pathway. The resultant energy deficiency may be an explantation for the toxicity of these compounds under hypoxic as well as aerobic conditions. The increased dependency on glycolysis of hypoxic cells may correspond to the analogous increase in toxicity of hypoxic cells to nitroimidazoles.

The effect of nitroimidazoles on the oxygen consumption rate and respiratory control ratio of beef heart mitochondria.

The neurotoxic effect of the nitroimidazole radiosensitizers misonidazole (MISO) and desmethylmisonidazole (DMM) has seriously compromised their clinical effectiveness. We compare here the effect of MISO and DMM on oxygen consumption in purified beef heart mitochondria. MISO has been found to significantly increase the oxygen consumption rate and decrease the respiratory control ratio in isolated mitochondria when incubated in the presence of the NAD+ dependent substrate, beta-hydroxybutyrate. DMM has a similar but less pronounced effect than MISO on these respiratory parameters. When mitochondria were incubated in the presence of these radiosensitizers for 8, 15, 30, 45, and 60 minutes, the oxygen consumption rate was decreased when succinate, a FAD dependent substrate, was added following the incubation. This decrease, which is both time and dosage dependent, is equivalent for MISO and DMM.

The effect of nitroimidazoles on glucose utilization and lactate accumulation in mouse brain.

The radiation sensitizers misonidazole (MISO) and desmethylmisonidazole (DMM) can produce central and peripheral neuropathy in patients and laboratory animals. Behavioral and pathological investigations have indicated that in the central nervous system this primarily involves the cochlear and vestibular systems. Nitroimidazoles can also interfere with glycolysis in vitro under aerobic and anaerobic conditions. In the present work we have studied the effect of MISO or DMM on lactate production and glucose utilization in mouse brain. It is observed that these compounds result in a 25% inhibition of lactate production in brain slices relative to the control at a 10 mM level. Additionally, MISO (1.0 mg/g/day) or DMM (1.4 mg/g/day) were administered daily (oral) for 1, 4, 7, or 14 days to examine the effect of these two drugs on the regional glucose utilization in C3Hf mouse brain. Five microcuries of 2-deoxy[14C]glucose was given following the last drug dose and autoradiographs of serial brain sections were made and analyzed by a densitometer. Following a single dose of either MISO or DMM, no significant differences in glucose uptake were observed when compared with controls. However, following 4, 7, and 14 doses the rate of glucose utilization was significantly reduced in the intoxicated animals. Larger reductions were measured in specific regions including the posterior colliculus, cochlear nuclei, vestibular nuclei, and pons with increasing effects observed at later stages. These results share a degree of correspondence with the regional brain pathology produced by these nitroimidazoles.

Behavioural and ultrastructural studies of desmethylmisonidazole-induced neurotoxicity in mice.

The hypoxic cell radiosensitiser desmethylmisonidazole (DMM, Ro-05-9963) was administered orally to C3Hf female mice at three different dose levels (1.4, 1.6 and 1.8 mg g-1 day-1). Behavioural and morphological end-points were used to evaluate the neurotoxicity of this compound at these doses. The behavioural changes present at all three dose levels appeared when the total dose approached approximately 19 mg g-1. The behavioural changes initially appeared and developed to a severe state rapidly (24 to 48 h). A pattern of haemorrhagic necrosis was observed in the brain stem and cerebellum, but no pathology was observed in the cerebrum. Peripheral nerve degeneration was also evident and appeared concurrent with the central pathology. DMM appears to affect both the central and peripheral nervous systems at the same time at these dose levels. This is in some degree different from previous experience with misonidazole where peripheral damage distinctly precedes central effects. The development of the disorder appeared to be more rapid and severe when compared with earlier experience with misonidazole.

Thromboxane and prostacyclin in maternal and fetal circulation in pre-eclampsia.

OBJECTIVES: A major pathophysiologic change of pre-eclampsia has been attributed to the overproduction of thromboxane A2 (TXA2) mainly from activated platelets. On the other hand, increased biosynthesis of TXA2 has also been reported from preeclamptic placentas. The systemic role of these different sources of TXA2 has not been clarified. The purpose of this study is to define the changes of TXA2 and the antagonizing prostacyclin (PC) in maternal and fetal circulations. METHODS: The stable metabolites of TXA2 and PC [Thromboxine B2 (TXB2) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), respectively] in the cord and maternal blood of nine patients with pre-eclampsia and nine normal parturients were measured by radioimmunoassay. RESULT: In normal pregnancy, the cord blood contained much higher TXB2 (1697+/-898 vs. 267+/-128 ng/ml, P < 0.01) and 6-keto-PGF1alpha (266+/-263 vs. 12.5+/-3.9 ng/ml, P < 0.05) levels than the maternal blood. In the preeclamptic state, a marked increase of TXB2 was noted in both maternal and cord blood, reaching levels which were significantly higher than during normal pregnancy (2995+/-1103 vs. 267+/-128 ng/ml in maternal blood, P < 0.0001, and 3197+/-1288 vs. 1697+/-898 ng/ml in cord blood, P < 0.005). A less significant increase in 6-keto-PGF1alpha (134+/-10.8 vs. 12.5+/-3.9 ng/ml, P < 0.05) was also noted in the maternal blood. Moreover, the level of TXB2 correlated with the diastolic blood pressure of preeclamptic patients before and after delivery. CONCLUSION: The results suggest an abundant source of eicosanoids in the feto-placental circulation, which does not readily cross the placental barrier. In pregnancy complicated with pre-eclampsia, thromboxane level of both fetal and maternal circulations are markedly increased, which may be responsible for the pathophysiologic changes. The lack of adverse systemic effects on the fetus highlights a placental source of TXA2 of transient bioactivity which is rapidly hydrolyzed to non-active TXB2. Federation of Gynecology and Obstetrics

Haemolytic anaemia in rhesus monkeys induced by methylcellulose.

The rhesus monkey was evaluated in its haemopoietic and histological response to intraperitoneal injections of methylcellulose. The haematologic alterations included a mild haemolytic anaemia, lymphopaenia, monocytosis, a shortened survival of Cr51 -labelled autologous erythrocytes (17 . 1 vs 13 . 3 days, P less than 0.025) and normoblastic hyperplasia of the bone marrow. There was a diffuse sequestration of the polymer in the phagocytic cells of the spleen, liver, bone marrow, lymph nodes and adrenal glands. The renal glomerular endothelium also consistently stored this material. Overt splenomegaly was not induced. The monkey appears to present, along with other animals, a rather species-specific response to methylcellulose that is accompanied by fundamental responses observable in all subjects.

The induction and characterization of antiserum against human placenta 17-beta-hydroxysteroid dehydrogenase.

17-beta-Hydroxysteroid dehydrogenase was purified from human placenta. The purification process included the following steps: 50% saturated ammonium sulfate precipitation; heat treatment; affinity column chromatography; and preparative SDS gel electrophoretic elution. This enzyme was also characterized by HPLC gel filtration and two dimensional gel electrophoresis with isoelectrofocusing. The results indicate that the molecular weight of these enzymes in their native condition is around 68 kDa and 34 kDa in SDS PAGE. Therefore, the active form of the enzyme is an identical dimmer in nature. There are three charged isomers as demonstrated by isoelectrofocusing. The antiserum against the 17-beta-HSD was induced by injection of purified human placenta 17-beta-HSD in rabbits. The antiserum was collected and characterized by ELISA, immunohistochemistry, immunoblot and immunoprecipitation tests. The results showed that the antibody titer was over 1:12,800, and specificity against human placenta 17-beta-HSD was also observed.

Growth and metabolism of cultured bone cells using microcarrier and monolayer techniques.

The traditional in vitro cell culture methods provide bone cells on matrix-coated Petri dishes to grow the cells in a monolayer. This limits the usefulness in situations where there is a need to suspend anchorage dependent cells. To promote the massive production of the cultured cells and to enable the suspension of the cells in a medium, microcarrier cell culture was developed and evaluated. Isolated bone cells from rat calvaria were incubated in a microcarrier culture flask with Cytodex 1. The microcarrier cell morphology was examined by a phase contrast microscope, a scanning electron microscope, and a transmission electron microscope. Cell growth, enzyme markers, and biosynthetic characteristics were examined and compared for microcarrier and monolayer methods. The results indicate that all the characteristics of the bone cells, whether cultured in the Petri dish or microcarrier, were the same. Therefore, the studies of the function and behavior of bone cells still remain useful using the microcarrier system culture.

Comparison of endothelin binding sites in cultured aortic smooth muscle cells from spontaneously hypertensive and normotensive rats.

Endothelin-1 (ET-1), which is secreted from vascular endothelial cells, is not only a potent vasoconstrictor but also a vascular smooth muscle cell growth factor. The direct effect of ET-1 on vascular smooth muscle cells, mediated via its specific receptor may therefore play an important role in hypertension and atherosclerosis. Our previous studies indicated that ET-1 secretion from cultured aortic endothelial cells from spontaneously hypertensive rats (SHRs) at the prehypertensive stage (4 weeks old) was not significantly different from that of cells from age-matched Wistar-Kyoto (WKY) rats. In this study, the binding of ET-1 to cultured aortic smooth muscle cells from SHRs and WKY rats was studied. Using tissue explant techniques, rat aortic smooth muscle cells from SHRs and age-matched WKY rats of different ages (4 and 24 weeks old) were successfully cultured in vitro. The maximum binding capacity (Bmax) and binding affinity (Kd) of ET-1 to cultured aortic smooth muscle cells were evaluated by a receptor-binding assay. The data revealed that the affinity of ET-1 binding to smooth muscle cells was similar in all 4 groups of experimental rats. However, the Bmax of cultured smooth muscle cells from 24-week-old SHRs was 2.5 times higher than of smooth muscle cells from age-matched WKY rats (8.64 +/- 0.72 vs 3.69 +/- 0.10 fmol/10(6) cells) and 1.5 times higher than in aortic smooth muscle cells from 4-week-old SHRs (8.64 +/- 0.72 vs 5.36 +/- 0.36 fmol/10(6) cells). These results suggest that hypertension in SHRs may be related to a high density of ET-1 receptors on arterial smooth muscle cells.

Functional evaluation of cultured rabbit osteoblast-like cells.

For the improvement of the adult osteoblast culture, the osteoblasts of young adult rabbit endosteal from long bones were isolated by collagenase digesting separation. 0.1% of type-I collagen precoated culture flasks were used as substrate for isolated bone cell growth. Morphological examination of cultured cells under a phase-contrast microscope, SEM and TEM observations showed a structure similar to osteoblast in vivo. Histochemical examination of alkaline phosphatase demonstrated 97% purity of cultured osteoblasts. The presence of calcium deposit activity in cultured cells was demonstrated by Van Kossa stain. High activity of alkaline phosphatase and inorganic pyrophosphatase in cultured osteoblasts as determined by biochemical analysis. High calcium uptake in cultured osteoblasts was demonstrated by radioisotope labelled 45CaCl12. According to these methods, it was indicated that the cells isolated from young rabbit long bone endosteal were osteoblast-like and still maintained their biological function. Our system for culturing osteoblast-like cells is a successful attempt in growing bone tissue in vitro starting from isolated bone cells. Therefore, this modified method for bone cell culture on collagen precoated culture flasks could be used as the experimental model in studies concerning the osteoblasts in vitro.

Heat shock proteins and biological response to hyperthermia.

Thermotolerance is of significant interest because the resistance of pre-heated cells greatly surpasses the resistance of control cells. The induction of protein synthesis following a temperature shock (12 min, 45 degrees C) which produces thermotolerance is examined using SDS polyacrylamide gel electrophoresis. It was observed that the synthetic rates of certain proteins following heat shock significantly exceeded their synthetic rates in control (non-heat shocked) cells. The most strongly induced bands were observed at 68,000, 89,000 and 110,000 daltons. The induction of these proteins was blocked by inhibitors of transcription (actinomycin D) or translation (cycloheximide). This phenomenon strongly resembles the heat shock induction of gene transcription well studied in Drosophila melanogaster. The strong induction of gene transcription well studied in Drosophila melanogaster. The strong correlation between the kinetics of the induction of these heat shock proteins and the kinetics of thermotolerance induction is discussed.

The porcine sperm motility inhibitor is identical to beta-microseminoprotein and is a competitive inhibitor of Na+,K(+)-ATPase.

We have isolated a small porcine seminal protein called SMI-1. Our results from peptide sequence, amino acid composition and mass spectral analyses reveal that SMI-1 is identical to porcine beta-microseminoprotein, a protein with unknown function. We also report here that this protein inhibits competitively the activity of Na+,K(+)-ATPase purified from porcine cerebral cortex in a dose-dependent manner. The inhibitory effect could be reversed by the addition of ATP. The half-maximal inhibition was achieved at an inhibitor concentration of 90 microM.

Establishment and characterization of the transfectable golden hamster embryo fibroblast cell line.

A diploid, continuous cell line, Golden Hamster Embryo Fibroblast-III (GHEF-III), which had been passaged for one year, was established essentially by a 3T3 protocol from primary culture of 14-day-gestation Golden Hamster embryo fibroblast cells. The cultured fibroblast exhibited monolayer growth and had contact inhibition. In morphological identification by light microscope (LM), transmission electron microscope (TEM) and immunofluorescence examination (IF), these multipolar and spindle-shaped cells had a large ovoid nucleus, enriched rER and mitochondria in the cytoplasm. On the other hand, the vimentin presented in the cell with a random network and capped around the nucleus. The results indicated that the cultured GHEF-III cells were fibroblast in origin. The cells were free of bacterial and mycoplasma contamination. The doubling time in GHEF-III was about 15 hours. Chromosomal analysis of GHEF-III presented a diploid stem cell line with a modal number of 44. No evidence of transformation of GHEF-III was shown by properties of contact inhibition, no colony formation in soft agar, and no tumor growth in nude mice. The transformation of GHEF-III after transfection with pT24-C3, an oncogenic plasmid, was shown by the evidence of loss of contact inhibition, growth in low serum medium, colony formation in soft agar and tumor growth in nude mice. In vitro transformation testing of these cells may provide valuable data in studying the role of tumor transforming genes in carcinogenesis. Owing to the genetic stability and less spontaneous transformation, this GHEF-III cell line can be utilized as a source of recipient cells in transfection assay.

The correlation of blood pressure, age and endothelin-1 binding sites in aortic smooth muscle cells of rats.

Spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD) rats at the ages of 4, 8, 12, 16 and 24 weeks were used to examine the effects of age on the density of endothelin-1 (ET-1) binding sites in aortic smooth muscle cells and systolic blood pressure (SBP). The SBP of the 3 different rat strains was measured, and the maximum binding value (Bmax) and dissociation constant (Kd) of ET-1 binding sites in smooth muscle cells of the thoracic aorta were determined. The results showed that the SBP and Bmax values of SHR, WKY and SD rats increased with age; the SBP and Bmax value at each corresponding age were significantly higher in SHR than in WKY and SD rats, however, there was no significant difference between WKY and SD rats. The relationship of age vs SBP, age vs Bmax, and Bmax vs SBP showed significantly positive correlation in all 3 rat strains. The regression line in the Bmax of endothelin binding sites against SBP in the 3 different rat strains presented a similar slope. These results indicate that SBP, which increased with age, could be related to an increased density of ET-1 binding sites on vascular smooth muscle cells in these 3 different rat strains.

The secretion of two sperm maturation-related glycoproteins in BALB/c mouse epididymis.

A well-developed Golgi apparatus and rough and smooth endoplasmic reticulum in the principal cells of the mouse epididymis indicate active protein synthesis. Studies have shown that epididymal secretions are essential for sperm maturation. In a previous study, two wheat-germ agglutinin (WGA)-binding glycoproteins, GP-49 and GP-83, were identified on the surface of mature mouse sperm. In this study, synthesis and secretion of these two glycoproteins were investigated. Apparent WGA-binding was found on the stereocilia and in the apical region of principal cells in the corpus and cauda of epididymis. Post-fixation and pre-embedding cytochemical localization revealed that WGA-binding sites were situated in the Golgi apparatus, multivesicular bodies and stereocilia of principal cells. GP-49 and GP-83 were identified in the Nonidet P-40 homogenates of corpus and cauda epididymidis. In the epididymides of which ductuli efferentes had been ligated for more than 4 weeks, no sperm were found in the lumina of epididymal tubules. WGA-binding sites were present in the corpus and cauda; GP-49 and GP-83 were identified in tissue homogenates of the corpus and cauda as well. These findings suggest that GP-49 and GP-83 of mature sperm may be secreted by the principal cells of the corpus and cauda. These two molecules apparently conjugate to sperm whilst sperm transit through the epididymis.

Identification of two maturation-related, wheat-germ-lectin-binding proteins on the surface of mouse sperm.

Epididymal maturation is essential for mammalian sperm to develop fertility. Wheat germ agglutinin (WGA), a lectin which specifically recognizes N-acetyl-glucosamine and sialic acid, was used to investigate membrane characteristics of epididymal sperm in the mouse. Histochemical and cytochemical localization revealed that WGA-binding sites were increased as sperm became mature. The binding sites were mainly localized in the plasma membrane of the anterior acrosome and tails. On Western blots of NP-40 extracts, two WGA-binding glycoproteins, GP-49 and GP-83, were identified on the sperm recovered from both corpus and cauda epididymidis. GP-83 was removed from the sperm surface by centrifugation and resuspension in phosphate-buffered saline (PBS) three times. GP-49 was resistant to centrifuging at 400 g for 5 min up to seven times and the treatment with 1M NaCl in PBS. These observations suggest that GP-49 is very likely an intrinsic membrane protein of the sperm, whereas GP-83 is an extrinsic protein.

Paracrine function of cultured aortic endothelial cells in spontaneously hypertensive rats.

BACKGROUND: Vasoconstrictor and vasodilator release from vascular endothelial cells not only regulates vascular tone but also induces vascular smooth muscle cell proliferation. METHODS: In order to understand the role of vasoconstrictor and vasodilator release in the development of hypertension in spontaneously hypertensive rats (SHR), aortic endothelial cells were isolated and cultured from 4-week-old and 24-week-old SHR (SHR-4 and SHR-24) and age-matched Wistar-Kyoto rats (WKY-4 and WKY-24) used as control. Prostacyclin (PGI2), endothelin-1 (ET-1) and thromboxane A2 (TXA2) release from cultured endothelial cells in the culture medium, were measured after 30 min with or without treatment with acetylcholine, calcium ionophore A23187 or thrombin. RESULTS: The results showed that there was no significant difference in ET-1 secretion between SHR-4 and age-matched WKY rats, but ET-1 secretion was about twice as high in SHR-24 as in WKY-24. TXA2 secretion was significantly higher in SHR-4 than in WKY-4 and was also higher than in SHR-24, but there was no significant difference between SHR-24 and WKY-24. The secretion of PGI2 was higher in SHR-24 than in WKY-24 and also higher than in SHR-4 and WKY-4. The prostaglandin PGI2 and TXB2 secretions from all groups of cultured VECs treated with various reagents, acetylcholine, calcium ionophore A23187 or thrombin were increased in similar patterns. However, there was no significantly different response between SHR and WKY VECs. CONCLUSIONS: Similar levels of ET-1 secreted from endothelial cells between SHR-4 and WKY-4 indicated that ET-1 secretion seems not a crucial factor in early hypertension development in SHR. The high level of TXA2 secretion in SHR-4 may involve in early hypertension development in SHR.

On the biosynthesis of alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid catalyzed by the sialyltransferase of Escherichia coli Bos-12.

Escherichia coli Bos-12 synthesizes a heteropolymer of sialic acids with alternating alpha-2,9/alpha-2,8 glycosidic linkages (1). In this study, we have shown that the polysialyltransferase of the E. coli Bos-12 recognizes an alpha-2,8 glycosidic linkage of sialic acid at the nonreducing end of an exogenous acceptor of either the alpha-2,8 homopolymer of sialic acid or the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid and catalyzes the transfer of Neu5Ac from CMP-Neu5Ac to this residue. When the exogenous acceptor is an alpha-2,8-linked oligomer of sialic acid, the main product synthesized is derived from the addition of a single residue of [14C]Neu5Ac to form either an alpha-2,8 glycosidic linkage or an alpha-2,9 glycosidic linkage at the nonreducing end, at an alpha-2, 8/alpha-2,9 ratio of approximately 2:1. When the acceptor is the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid, chain elongation takes place four to five times more efficiently than the alpha-2,8-linked homopolymer of sialic acid as an acceptor. It was found that the alpha-2,9-linked homopolymer of sialic acid and the alpha-2,8/alpha-2,9-linked hetero-oligomer of sialic acid with alpha-2,9 at the nonreducing end not only failed to serve as an acceptor for the E. coli Bos-12 polysialyltransferase for the transfer of [14C]Neu5Ac, but they inhibited the de novo synthesis of polysialic acid catalyzed by this enzyme. The results obtained in this study favor the proposal that the biosynthesis of the alpha-2, 9/alpha-2,8 heteropolymer of sialic acid catalyzed by the E. coli Bos-12 polysialyltransferase involves a successive transfer of a preformed alpha-2,8-linked dimer of sialic acid at the nonreducing terminus of the acceptor to form an alpha-2,9 glycosidic linkage between the incoming dimer and the acceptor. The glycosidic linkage at the nonreducing end of the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid produced by E. coli Bos-12 should be an alpha-2,8 glycosidic bond and not an alpha-2,9 glycosidic linkage.

Effect of epidermal growth factor on the sheet formation of the human keratinocyte in cell culture.

Epidermal growth factor is an important element in maintaining keratinocyte proliferation and maturation. To evaluate its effect on keratinocyte growth in vitro, human foreskins were cultured. The epidermal keratinocyte growth in culture was separated into two stages by a conditional medium: the proliferation stage, in which the cells were maintained in a monolayer; and the differentiation stage, in which the cells grew to stratification and keratinization. The keratinocytes were cultured in various concentrations of epidermal growth factor, and their morphology and growth behavior were closely observed. Our results demonstrated that cultured keratinocytes grew in a confluent layer under the influence of epidermal growth factor. In contrast, in a culture without epidermal growth factor, the proliferation rate of cultured keratinocytes slowed down and eventually the cells stopped growing. During serum stimulation, with or without additional exogenous epidermal growth factor, the cultured keratinocytes grew continuously to the normal terminal differentiation. Under this two-stage culture model, the cultured keratinocytes could grow into an intact sheet of graftable epidermis.