Proteolysis in isolated autophagic vacuoles from rat liver : Effect of pH and of proteolytic inhibitors

Autophagic vacuoles (AV) were purified from livers of rats which were pretreated with vinblastine (VBL) to increase the occurrence of AV. To measure proteolysis in the isolated AV rats were labelled with [¹⁴C]leucine 2 or 16 h before sacrifice. The integrity of the AV was studied by measuring the leakage of hydrolytic enzymes during incubation at various pHs. VBL causes an increase in the degradation rate of liver homogenate and isolated AV. This increase was moderate if proteolysis was measured at neutral pH, whereas adjustment to acidic pH enhanced the rate of autodegradation in the AV several-fold. This indicates that the VBL-induced AV have acquired hydrolytic enzymes either by fusion with lysosomes or possibly by the sequestering endoplasmic reticulum (ER) membranes forming the limiting membranes of the AV. The internal pH is not optimal for degradation in vitro of sequestered proteins, indicating insufficient acidification of the isolated AV. Lysosomotropic inhibitors, like chloroquine and propylamine, but not asparagine, impede proteolysis in isolated AV, but not more than 40%.     

Visualization of reactive oxygen species formation by phagocytizing macrophages

Activated peritoneal macrophages exhibiting phagocytosing capacity produced an electron-dense precipitate of formazan in contact sites of macrophage plasmalemma and phagocytosed yeast cells. No production of formazan occurred, when non-opsonized latex particles were ingested by macrophages. Formazan precipitation could be prevented by anaerobiosis but not by addition of cyanide.     

Ploidy class-dependent variations during 24 h of glucose-6-phosphate and succinate dehydrogenase activity and single-stranded RNA content in isolated rat hepatocytes

The time-dependent variations over 24 h of glucose-6-phosphate dehydrogenase (G6PDH) activity, succinate dehydrogenase (SDH) activity and single-stranded RNA (ssRNA) content have been investigated by cytophotometric analysis of cytochemically stained isolated hepatocytes of different ploidy classes from adult male rats. A marked variation of 48 % over the day in G6PDH activity of the mononuclear diploid cells was revealed, but no significant variation in the binuclear tetraploid cells. The cells of the inbetween ploidy classes showed an amplitude of variation of 38 % (binuclear diploid cells) and 24% (mononuclear tetraploid cells), respectively. All cells showed a maximum activity of the enzyme at the middle of the day and a minimum during the night. The relative enzyme activity per mononuclear diploid cell was significantly higher than the relative activity in the other cells, especially at its maximum. The variation of the SDH activity in hepatocytes isolated from the same rats was similar in all cells, irrespective of their ploidy class. The activity was highest at the end of the activity phase of the animals. The SDH activity per cell was directly proportional to the quantity of genome copies. The ssRNA content of the hepatocytes showed a time-dependent variation with a maximum during the resting phase of the animals and a minimum during their activity phase. The variation was larger in the mononuclear diploid cells than in the cells of other ploidy classes and the ssRNA content was also significantly higher in these cells than in the hepatocytes of other ploidy classes when calculated on the basis of genome copies. It is concluded that the large amplitude of variation over the day and the high relative amount of G6PDH activity and ssRNA content in mononuclear diploid cells is related to the function of these cells as stem cells of the liver parenchyma.     

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

Studies on calmodulin isolated from Tetrahymena cilia and its localization within the cilium

Tetrahymena calmodulins from cilia, cell bodies and whole cells were isolated separately and compared. These calmodulins showed just the same properties: they co-migrated in SDS-polyacrylamide gel electrophoresis, had a Ca²⁺-dependent electrophoretic mobility change in alkali gel, held the same antigenic determinants in common, and activated brain cyclic nucleotide phosphodiesterase Ca²⁺-dependently with identical activation curves. Distributions of calmodulin and calmodulin-counterpart in Tetrahymena cilium were investigated by using alkali gel electrophoresis in the presence of Ca²⁺ or EGTA, and by immunoelectron microscopy. Calmodulin was detected in the membrane plus matrix fraction and outer-doublet microtubule fraction, and its Ca²⁺-dependent counterpart existed exclusively in the latter fraction. However, neither calmodulin nor its counterpart was detected in the crude dynein fraction. Immunoelectron microscopy revealed that calmodulin was localized along the longitudinal axis of outer-doublet microtubules at regular intervals of about 90 nm. The calmodulin-binding site in the ciliary axoneme was suggested to be interdoublet links.     

Fibronectin on the surface of rat mast cells

Rabbit antiserum against rat plasma fibronectin induced histamine release in isolated rat peritoneal mast cells. Immunofluorescence revealed fibronectin on the mast cells in rat mesentery and on the surface of the isolated mast cells. Mast cells adhered to collagen-coated dishes. This cellular adherence was inhibited by the addition of anti-fibronectin. Fibronectin on the surface of mast cells may play a role of attachment of the cells to collagenous connective tissues.     

Cartilage-derived factor (CDF) I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture

A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of ³⁵SO₄^?2, [³H]-glucosamine and [³H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

Considerations in the isolation of rat liver nuclear matrix,nuclear envelope,and pore complex lamina

A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.     

DNA synthesis catalysed by endogenous templates and DNA-dependent DNA polymerases in spermatogenic cells from rat

The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl₂ and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed:

     

12.

Fractionation and characterization of DNA at sites of replication from rat liver nuclei     

David G. Kaufman  Marila Cordeiro-Stone  Thomas H. Rude  Karen G. Nelson  William K. Kaufmann 《Experimental cell research》1983,146(2):339-347

Regenerating rat liver nuclei when sonicated and centrifuged in a Cs2SO4 gradient were fractionated into three distinct bands. These bands were designated as light band (LB), middle band (MB), and heavy band (HB) according to their density. LB and MB were shown to consist of large granular particles with varying electron densities, but LB also contained remnants of nuclear membrane. When analysed by gel electrophoresis, LB and MB displayed more than 35 bands of proteins. The third fraction, HB, consisted largely of small chromatin fibers and its proteins were predominantly the four histones of the nucleosomal core particle. Following short pulses with [3H]thymidine in vivo, the specific activity of DNA in LB and MB was significantly higher than that of bulk DNA contained in HB. DNA in all three fractions became equally labelled as the duration of the labelling interval increased beyond 30 min. Newly synthesized DNA was characterized by electrophoresis on analytical 1.7% acrylamide -0.5% agarose composite gels. After a 1-min labelling interval in vivo, 17% of the rapidly labelled DNA from LB and MB was stationary at the gel origin like replication forks from E. coli, but only 3% of HB DNA had zero mobility. Electron microscopy confirmed the presence of DNA replication forks in LB, MB, and HB. With increasing time of synthesis the proportion of labelled DNA exhibiting zero mobility decreased in all three fractions. Denaturation of DNA or digestion of single-stranded DNA with S1 nuclease released newly synthesized DNA from the gel origin. Ribonuclease was without effect. DNA recovered from LB and MB also had a higher molecular weight than the HB DNA. Together these results indicate (1) that LB and MB are enriched in newly replicated DNA; (2) that an increased proportion of newly replicated DNA in LB and MB is associated with DNA replication forks; and (3) that the replicating DNA recovered in LB and MB may be associated with other nuclear constituents in situ because this DNA appears to be protected from the more frequent chain breaks introduced into the bulk of chromatin (HB) by sonication.     

13.

Purine metabolism and urate biosynthesis in isolated chicken hepatocytes     

R A Burns  P J Buttery 《Archives of biochemistry and biophysics》1984,233(2):507-514

The metabolism of some purine compounds to urate and their effects on de novo urate synthesis in chicken hepatocytes were investigated. The purines, listed in descending order of rates of catabolism to urate, were hypoxanthine, xanthine, inosine, guanosine, guanine, IMP, GMP, adenosine, AMP, and adenine. During a 1-h incubation period, conversion to urate accounted for more than 80% of the total quantities of guanine, guanosine, and inosine metabolized, but only 42% of the adenosine and 23% of the adenine metabolism. Adenine, adenosine, and AMP inhibited de novo urate synthesis [( 14C]formate incorporation into urate), whereas the other purines, especially guanine, guanosine, and GMP, stimulated de novo urate synthesis. When hepatocytes were incubated with glutamine and adenosine, AMP, guanine, guanosine, or GMP, the rates of de novo urate synthesis were lower than the additive effects of glutamine and the purine in separate incubations. Increasing phosphate concentrations had no effect on urate synthesis in the absence of added purines but, in combination with adenosine, AMP, guanosine, or GMP, increased urate synthesis. These results indicate that the ratio of adenine to guanine nucleotides and the interaction between substrates and purine nucleotides are involved in the regulation of urate biosynthesis in chicken liver.     

14.

Subcellular fractionation of isolated rat hepatocytes a comparison with liver homogenate     

Geneviève Krack  Odette Gravier  Marcel Roberfroid  Michel Mercier 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,632(4):619-629

An improved method for the homogenization and the subsequent subcellular fractionation of hepatocytes isolated from adult rat liver is described.The homogenization procedure developed in the present study allows the preservation of the integrity of subcellular structures, as demonstrated by measurement of the activities of representative enzymes as well as by determination of their latency.The activities of representative marker enzymes, as calculated on subcellular fractions obtained by differential centrifugation of the homogenate, are identical whether the homogenate arises from isolated hepatocytes or from the whole liver.Moreover, there is a close similitude between the kinetic parameters (K_{m and} V) of two microsomal cytochrome P₄₅₀-dependent mixed-function oxidases, namely aniline hydroxylase and aminopyrine demethylase determined on microsomal preparations obtained either from isolated cells or from the whole liver.     

15.

Enzyme-induced aziridine formation by isolated hepatocytes     

Masamichi Watanabe  Kanya Tonda  Masaharu Hirata  Yoshiteru Hata 《Biochemical and biophysical research communications》1983,112(2):356-360

The metabolism of 2-bromoethylaminonaphthoquinone in hepatocytes isolated from rats was studied. This compound was chemically inert in the reaction system used. However, in buffer solution containing isolated hepatocytes, it was gradually converted into aziridinylnaphthoquinone. Under the same reaction conditions, 4-chlorobutylaminonaphthoquinone also gave the cyclization products, pyrrolidinylnaphthoquinone. Cellular GSH decreased in both reactions.     

16.

Isolation of cloned variants of a rat hepatoma cell line with altered attachment to collagen, but normal attachment to fibronectin     

Eve Barak Briles  Nancy B. Haskew 《Experimental cell research》1982,138(2):436-441

Cloned variants of a rat hepatoma cell line have been isolated which exhibit normal attachment and spreading behavior on fibronectin substrata, but which are defective in their ability to attach to native collagen films. These clones should be useful for identifying specific macromolecules involved in the cell-to-collagen interaction.     

17.

Albumin secretion and protein synthesis by cultured diploid and tetraploid rat hepatocytes separated by elutriation     

E Le Rumeur  C Guguen-Guillouzo  C Beaumont  A Saunier  A Guillouzo 《Experimental cell research》1983,147(1):247-254

Diploid and tetraploid rat hepatocyte subpopulations were isolated by elutriation and cultured for 24 h. Albumin secretion and protein synthesis rates were two-fold lower in 2n than in 4n hepatocytes. [35S]methionine-labelled proteins analysed by acrylamide gel electrophoresis showed a strikingly similar pattern in the two cell subpopulations. No differences in cellular proteins or in the intensity of labelling were observed. These results show (1) that viable diploid and tetraploid hepatocyte subpopulations can be separated by elutriation under sterile conditions and then cultured; and (2) strongly suggest that the same genes are transcribed and further translated at the same rate in both hepatocyte subpopulations.     

18.

Localization of genes on fractionated rat chromosomes by molecular hybridization     

J. G. Collard  J. Schijven  A. Tulp  M. Meulenbroek 《Experimental cell research》1982,137(2):463-469

Chromosomes derived from rat kidney cells were separated in specially designed sedimentation chambers by velocity sedimentation at 30 g. The DNA of the chromosomal fractions was used in molecular hybridization experiments to localize single-copy genes on the fractionated rat chromosomes. By cross-hybridization with a mouse immunoglobulin light chain kappa c-DNA probe, the rat immunoglobulin genes were detected only on the DNA of chromosomal fractions highly enriched for chromosomes 3, 4 and 5. The rat albumin gene was detected on fractions greatly enriched for chromosomes 11, 13 and 14. The described method allows the localization of structural genes or introduced DNA sequences on the chromosomal level especially in those cell systems in which no suitable somatic cell hybrids are available.     

19.

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells     

Ø.W. Rønning  E.O. Pettersen 《Experimental cell research》1984,155(1):267-272

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

20.

Immunocytochemical localization of a calcium-activated protease in skeletal muscle cells   总被引：1，自引：0，他引：1  

W. R. Dayton  J. V. Schollmeyer 《Experimental cell research》1981,136(2):423-433

The 80 000-D subunit of a calcium-activated protease from skeletal muscle was purified to homogeneity using preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was used to elicit antibody production in rabbits. Antiserum was purified using affinity chromatography to yield a monospecific antibody fraction (anti-80K) directed against the 80 000-D subunit. Localization studies showed that the 80 000-D subunit is present in or near the sarcolemma of cultured myoblasts and sectioned muscle tissue, in discrete areas of the cytoplasm of myoblasts, and in the Z disks of the myofibril. The location of the calcium-activated protease in the cell suggests that the enzyme may be involved in myofibril degradation and in membrane alterations in developing and mature muscle cells.     

	Pre-leptotene primary spermatocyte %	Pachytene primary spermatocyte %	Round spermatid %	Elongated spermatid %
DNA polymerase α	25	42	30	3
DNA polymerase β	29	34	36	1

Fractionation and characterization of DNA at sites of replication from rat liver nuclei

Regenerating rat liver nuclei when sonicated and centrifuged in a Cs2SO4 gradient were fractionated into three distinct bands. These bands were designated as light band (LB), middle band (MB), and heavy band (HB) according to their density. LB and MB were shown to consist of large granular particles with varying electron densities, but LB also contained remnants of nuclear membrane. When analysed by gel electrophoresis, LB and MB displayed more than 35 bands of proteins. The third fraction, HB, consisted largely of small chromatin fibers and its proteins were predominantly the four histones of the nucleosomal core particle. Following short pulses with [3H]thymidine in vivo, the specific activity of DNA in LB and MB was significantly higher than that of bulk DNA contained in HB. DNA in all three fractions became equally labelled as the duration of the labelling interval increased beyond 30 min. Newly synthesized DNA was characterized by electrophoresis on analytical 1.7% acrylamide -0.5% agarose composite gels. After a 1-min labelling interval in vivo, 17% of the rapidly labelled DNA from LB and MB was stationary at the gel origin like replication forks from E. coli, but only 3% of HB DNA had zero mobility. Electron microscopy confirmed the presence of DNA replication forks in LB, MB, and HB. With increasing time of synthesis the proportion of labelled DNA exhibiting zero mobility decreased in all three fractions. Denaturation of DNA or digestion of single-stranded DNA with S1 nuclease released newly synthesized DNA from the gel origin. Ribonuclease was without effect. DNA recovered from LB and MB also had a higher molecular weight than the HB DNA. Together these results indicate (1) that LB and MB are enriched in newly replicated DNA; (2) that an increased proportion of newly replicated DNA in LB and MB is associated with DNA replication forks; and (3) that the replicating DNA recovered in LB and MB may be associated with other nuclear constituents in situ because this DNA appears to be protected from the more frequent chain breaks introduced into the bulk of chromatin (HB) by sonication.     

Purine metabolism and urate biosynthesis in isolated chicken hepatocytes

The metabolism of some purine compounds to urate and their effects on de novo urate synthesis in chicken hepatocytes were investigated. The purines, listed in descending order of rates of catabolism to urate, were hypoxanthine, xanthine, inosine, guanosine, guanine, IMP, GMP, adenosine, AMP, and adenine. During a 1-h incubation period, conversion to urate accounted for more than 80% of the total quantities of guanine, guanosine, and inosine metabolized, but only 42% of the adenosine and 23% of the adenine metabolism. Adenine, adenosine, and AMP inhibited de novo urate synthesis [( 14C]formate incorporation into urate), whereas the other purines, especially guanine, guanosine, and GMP, stimulated de novo urate synthesis. When hepatocytes were incubated with glutamine and adenosine, AMP, guanine, guanosine, or GMP, the rates of de novo urate synthesis were lower than the additive effects of glutamine and the purine in separate incubations. Increasing phosphate concentrations had no effect on urate synthesis in the absence of added purines but, in combination with adenosine, AMP, guanosine, or GMP, increased urate synthesis. These results indicate that the ratio of adenine to guanine nucleotides and the interaction between substrates and purine nucleotides are involved in the regulation of urate biosynthesis in chicken liver.     

Subcellular fractionation of isolated rat hepatocytes a comparison with liver homogenate

An improved method for the homogenization and the subsequent subcellular fractionation of hepatocytes isolated from adult rat liver is described.The homogenization procedure developed in the present study allows the preservation of the integrity of subcellular structures, as demonstrated by measurement of the activities of representative enzymes as well as by determination of their latency.The activities of representative marker enzymes, as calculated on subcellular fractions obtained by differential centrifugation of the homogenate, are identical whether the homogenate arises from isolated hepatocytes or from the whole liver.Moreover, there is a close similitude between the kinetic parameters (K_{m and} V) of two microsomal cytochrome P₄₅₀-dependent mixed-function oxidases, namely aniline hydroxylase and aminopyrine demethylase determined on microsomal preparations obtained either from isolated cells or from the whole liver.     

Enzyme-induced aziridine formation by isolated hepatocytes

The metabolism of 2-bromoethylaminonaphthoquinone in hepatocytes isolated from rats was studied. This compound was chemically inert in the reaction system used. However, in buffer solution containing isolated hepatocytes, it was gradually converted into aziridinylnaphthoquinone. Under the same reaction conditions, 4-chlorobutylaminonaphthoquinone also gave the cyclization products, pyrrolidinylnaphthoquinone. Cellular GSH decreased in both reactions.     

Isolation of cloned variants of a rat hepatoma cell line with altered attachment to collagen, but normal attachment to fibronectin

Cloned variants of a rat hepatoma cell line have been isolated which exhibit normal attachment and spreading behavior on fibronectin substrata, but which are defective in their ability to attach to native collagen films. These clones should be useful for identifying specific macromolecules involved in the cell-to-collagen interaction.     

Albumin secretion and protein synthesis by cultured diploid and tetraploid rat hepatocytes separated by elutriation

Diploid and tetraploid rat hepatocyte subpopulations were isolated by elutriation and cultured for 24 h. Albumin secretion and protein synthesis rates were two-fold lower in 2n than in 4n hepatocytes. [35S]methionine-labelled proteins analysed by acrylamide gel electrophoresis showed a strikingly similar pattern in the two cell subpopulations. No differences in cellular proteins or in the intensity of labelling were observed. These results show (1) that viable diploid and tetraploid hepatocyte subpopulations can be separated by elutriation under sterile conditions and then cultured; and (2) strongly suggest that the same genes are transcribed and further translated at the same rate in both hepatocyte subpopulations.     

Localization of genes on fractionated rat chromosomes by molecular hybridization

Chromosomes derived from rat kidney cells were separated in specially designed sedimentation chambers by velocity sedimentation at 30 g. The DNA of the chromosomal fractions was used in molecular hybridization experiments to localize single-copy genes on the fractionated rat chromosomes. By cross-hybridization with a mouse immunoglobulin light chain kappa c-DNA probe, the rat immunoglobulin genes were detected only on the DNA of chromosomal fractions highly enriched for chromosomes 3, 4 and 5. The rat albumin gene was detected on fractions greatly enriched for chromosomes 11, 13 and 14. The described method allows the localization of structural genes or introduced DNA sequences on the chromosomal level especially in those cell systems in which no suitable somatic cell hybrids are available.     

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

Immunocytochemical localization of a calcium-activated protease in skeletal muscle cells

The 80 000-D subunit of a calcium-activated protease from skeletal muscle was purified to homogeneity using preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was used to elicit antibody production in rabbits. Antiserum was purified using affinity chromatography to yield a monospecific antibody fraction (anti-80K) directed against the 80 000-D subunit. Localization studies showed that the 80 000-D subunit is present in or near the sarcolemma of cultured myoblasts and sectioned muscle tissue, in discrete areas of the cytoplasm of myoblasts, and in the Z disks of the myofibril. The location of the calcium-activated protease in the cell suggests that the enzyme may be involved in myofibril degradation and in membrane alterations in developing and mature muscle cells.