CELL PROLIFERATION IN THE RAT KIDNEY INDUCED BY FOLIC ACID

The changes in proliferative activity of tubular epithelial cells of the rat kidney following a single injection of folic acid (250 mg/kg body weight) have been studied. Autoradiography with tritiated thymidine revealed a large increase in numbers of labelled cells, beginning at about 18 hr, in each of the three kidney zones examined. In the cortex the maximum increase in labelling index (16 times normal) was found at 36 hr whereas that of the outer medulla (34 times normal) occurred at 24 hr; there was no clearly defined peak in the inner medulla, values of up to 36 times normal being found between 24 and 96 hr. These changes were followed several hours later by similar changes in mitotic index in the corresponding zones. All the indices, except the mitotic index of the inner medulla, had returned to normal by 6 days. Comparison of the curves of labelling index and mitotic index in each zone indicated that the number of cells induced to synthesize DNA was approximately similar to the number of cells which subsequently underwent mitosis. A large increase was also found in the specific activity of DNA extracted from homogenates of whole kidneys from folic acid-injected rats, again using tritiated thymidine as label. The increase began at about 18 hr, reached a maximum of 16 times normal at 32 hr and returned to normal by 6 days. These changes were similar to those of labelling index in the cortical zone.     

DNA synthesis during larval development and compensatory growth in the mesonephros of Xenopus laevis.

Autoradiography following tritiated thymidine administration to Xenopus laevis tadpoles of stages 45–48 of larval development has revealed that, as the cells of the mesonephric kidney differentiate during organogenesis, there is a marked decrease in the percentage of cells synthesizing DNA (from 100% at stage 45 to less than 9% at stage 48). In the adult this figure is of the order of 0.1%. This reduced DNA synthetic activity was found to take place in the cells of both the proximal and distal tubules of the nephrons. Special mucous cells which serve as markers of distal tubules were not observed to synthesize DNA after the onset of their differentiation at stage 48 of larval development.Through the partial extirpation of tissue in one kidney of adult Xenopus laevis males, DNA synthesis was reactivated in differentiated cells. The increased DNA synthetic activity following partial unilateral nephrectomy was found to be maximal after 6 days of recovery when 19% of cells synthesize DNA. This increased DNA synthetic activity was found to occur only in the cells of the distal tubules, both mucous and nonmucous cells, while the cells of the proximal tubules did not respond to this reactivation.The apparent inability of proximal tubule cells to synthesize DNA following partial unilateral nephrectomy is discussed.     

The regulation of growth in the mesonephric kidney of adult Xenopus laevis by an endogenous inhibitor of proliferation.

The effect of dorsal lymph sac implanted tissue fragments, of a 100,000g kidney supernatant, and of various kidney-derived ultrafiltrate fractions on the percentage of DNA synthesizing cells in the mesonephric kidney of Xenopus laevis following partial unilateral nephrectomy was investigated autoradiographically. Using Amicon filters with cut-off values of MW 50,000 and 10,000, three ultrafiltrate fractions were obtained: a fraction containing molecules of MW 50,000 and less, a fraction containing molecules of MW 10,000 and less, and one containing molecules in the range of MW 10,000 to 50,000. The ultrafiltrates containing molecules of less than 10,000 MW were found to depress DNA synthetic activity on the sixth postoperative day by 30 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 showed no significant effect. It has been concluded that an endogenous inhibitor of proliferation, with the attributes of a chalone, is present in the fraction of less than 10,000 MW. The loss of inhibitor action following Pronase treatment of the ultrafiltrate suggests that the inhibitor substance may be a protein or polypeptide, or that such constituent may be the carrier for the active agent. Since a depression in DNA synthetic activity of 60% was obtained in normal adult mesonephric kidneys following the injection of the ultrafiltrate, it is concluded that both compensatory growth and reparative growth in the kidney of Xenopus laevis are regulated by a G₁ kidney chalone of less than 10,000 MW.     

PHOTORECEPTOR-PIGMENT EPITHELIAL CELL RELATIONSHIPS IN RATS WITH INHERITED RETINAL DEGENERATION : Radioautographic and Electron Microscope Evidence for a Dual Source of Extra Lamellar Material

Protein synthesis and displacement in photoreceptor and pigment epithelial cells of inbred normal (Fisher) and mutant (RCS) rats with inherited retinal degeneration has been studied by light and electron microscope radioautography. Groups of animals 14, 15, 17, 19, 27, 35, and 50 days of age were injected with amino acids-H³ and killed at subsequent time intervals. In normal rats, radioactive protein synthesized in the rod inner segments was incorporated into outer segment saccules and displaced outward; the total renewal time of outer segments at all ages was approximately 9 days. In RCS photoreceptors, outer segment displacement was slowed from the normal rate before day 17 and at all subsequent stages. Most of the newly synthesized protein appeared to migrate only into the basal third of the outer segments. Labeling of pigment epithelial cells in RCS rats was always heavier than in controls. Labeled protein was displaced as early as 1 hr postinjection from pigment epithelial cell somas into the apical processes, and by 2 hr postinjection was located in the adjacent lamellar whorls characteristic of the mutant rat retina. After 1 day, radioactivity was present in the 14, 15, 17, and 19 day series of RCS rats in the apical third of the outer segment layer (occupied mainly by extra lamellar material) while there were few silver grains in the middle third of the layer (occupied mainly by distal parts of outer segments). The RCS pigment epithelial cells thus have an unusual synthetic role and appear to be a source of the extra lamellar material. Electron microscope examination revealed that many intact pigment epithelial cell processes were incorporated into the large whorls of extra lamellae. In addition, many disorganized outer segment saccules were observed in continuity with longer membranous lamellae and large lamellar whorls. The extra lamellar material therefore appears to be derived from both rod outer segments and pigment epithelial cells.     

Whole-mount autoradiography study of DNA synthetic activity during postnatal development and androgen-induced regeneration in the mouse prostate

Ventral and dorsolateral prostatic lobes (VP and DLP), obtained from mice at different ages and at different intervals after castration or treatment of castrated males with testosterone propionate (TP), were microdissected into two-dimensional arrays and incubated in vitro with 14C-thymidine. Labeled whole-mount specimens were fixed and dried onto glass slides, dipped into photographic emulsion, and processed autoradiographically. The morphological pattern of DNA synthetic activity was similar in the VP and DLP. During early postnatal periods (10-15 days after birth), DNA synthetic activity was highest at the distal ductal tips (near the capsule) and considerably lower in proximal ducts (near the urethra). At 30 days of age, DNA synthesis was almost totally confined to the distal ducts, with exceedingly low labeling in the proximal ductal areas. In the prostate of the intact or castrated adult, DNA synthesis was nearly absent throughout the gland, but silver grains were still observed on the ductal tips. During androgen-induced prostatic regeneration, DNA synthesis was detectable only in distal ducts 24 h after TP was administered. Labeling intensity reached a maximum on the third day of TP treatment in both distal and proximal ductal areas, thereafter, it subsided to focal labeling confined mostly to distal ducts. These results demonstrate that levels of DNA synthetic activity vary considerably within the prostate on a regional basis. Explanation of this heterogeneity in DNA synthetic activity within the prostate gland is fundamental to understanding the mechanism of androgenic regulation of prostatic growth and development.     

EPIDERMAL REGENERATION AFTER CELLOPHANE TAPE STRIPPING OF HAIRLESS MOUSE SKIN

After repeated applications of cellophane tape to the dorsal skin of hairless mice, the proliferative response in the treated epidermis was estimated by three different methods. The mitotic rate was determined in the interfollicular epidermis using the Colcemid technique, and the DNA synthetic activity was estimated after ³H-thymidine injection by counting labelled interfollicular cells in autoradiographs and by determining the specific activity of epidermal DNA. An initial 40–50% inhibition of DNA synthesis and mitosis was followed by an increase in the labelling index and the mitotic rate 8–10 hr after tape stripping. By 24 hr, peak values 5–6 times the controls were attained for both parameters. The labelling index and the mitotic rate were nearly normal at 3–4 days, but a second small peak was seen on day 5. Normal values were found on days 6 and 8. A similar pattern of response was found biochemically, but the peak of DNA specific activity was much broader and the extent of the increase was only about half as great as the increase in the labelling index. Possible reasons for these differences are discussed.     

Reaction products of the carcinogen N-hydroxy-4-acetylamino-4'-fluorobiphenyl with DNA in liver and kidney of the rat

The binding of AABP4'F and ABP4'F residues to rat liver and kidney DNA in vivo was studied at different periods of time after administration of N-[G-3H]hydroxy-AABP4'F at dose levels of 5 and 25 mg/kg body weight. DNA preparations from both organs were hydrolyzed enzymatically at pH 8--9 with mixtures of DNAase, snake venom phosphodiesterase and alkaline phosphatase from Escherichia coli. The enzymatic digests were analysed by Sephadex LH-20 chromatography using synthetic N-([G-14C] deoxyguanosin-8-yl)-AABP4'F as marker. Elution with 30% ethanol gave three major peaks of tritium activity. The first peak consisted largely of N-(deoxyguanosin-8-yl)-ABP4'F decomposition products, which were not further characterized. The second product has similar chromatographical and chemical properties as 3-(deoxyguanosin-N2-yl)-AAF; and was also persistent in liver as well as in kidneys. The third peak of tritium activity co-chromatographed with the marker compound N-([G-14C] deoxyguanosin-8-yl)-AABP4'F. Kinetic studies revealed that the latter product was removed rapidly from liver and kidney DNA at equal rates (t1/2 = 2 days). Approximately 80% of the total radioactivity bound to DNA consisted of deacetylated material, which was removed at a much slower rate (t1/2 = 10 days) in both organs. An initial rapid removal of all products in kidney during the first 7 days (t1/2 = 3.3 days) at dose levels of 25 mg/kg is probably due to toxic effects on the kidneys, because this phenomenon was not observed at dose levels of 5 mg/kg. The synthetic ester N-OSO3K-AABP4'F was at least twice as reactive towards L-methionine and guanosine as compared to the corresponding AABP derivative, but had 40% of the reactivity of N-acetoxy-AAF under similar conditions. The new compounds 3-methylmercapto-4-acetylamino-4'-fluorobiphenyl and N-(deoxyguanosin-8-yl)-4-acetylamino-4'-fluorobiphenyl have been characterized by means of their NMR and mass spectra. Attempts to devise an unambiguous synthesis for 3-(deoxyguanosin-N2-yl)arylamides have been unsuccessful.     

Relationship between membrane-bound protein kinase C activity and calcium-dependent proliferation of BALB/c 3T3 cells

Extracellular calcium-deprivation inhibited the proliferation of BALB/c 3T3 cells and this inhibition correlated with a loss of protein kinase C activity from the particulate fraction. Addition of calcium induced proliferation of the cells with the DNA synthetic activity returning to the control rate at 18 hours following calcium addition. The level of protein kinase C activity in the particulate fraction was monitored at various times after calcium addition and increased in parallel with the DNA synthetic activity.     

Kinetics of Mx expression in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar L.) parr in response to VHS-DNA vaccination

The duration of the Mx mRNA response to an intramuscular injection of the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) gene DNA vaccine as well as to the control plasmid was determined in rainbow trout at 14 degrees C over a period of 11 weeks. The Mx response was detectable on day 7, peaked on day 14 and returned to pretreatment levels on day 21 and thereafter. No increase in Mx expression was detectable to the control plasmid. In further experiments, the kinetics of the Mx response were compared in rainbow trout and Atlantic salmon parr kept at 10 degrees C and injected with the DNA vaccine or the synthetic double-stranded RNA, poly I:C. In both species there was a rapid response to poly I:C detectable from day 1, reaching maximum from days 3 to 9 and decreasing to background level by day 12. The peak level and return to background was reached slightly later in salmon. In both species the response to the VHS/DNA vaccine was slower to begin, not being detectable on days 1 and 3, but elevated levels were found on day 6. However, in the salmon parr, the peak level was on day 6 and the signal disappeared by day 12, while in the rainbow trout, the response peaked at day 12 and lasted until day 21. The kinetics of the Mx response to the VHS/DNA vaccine in rainbow trout correlate with the early non-specific protection against VHS in this species following vaccination. It is speculated that the more transient Mx response in Atlantic salmon parr to the DNA vaccine may be related to the innate resistance of salmon to VHS.     

DNA synthetic activity of nuclei isolated from differentiating cardiac muscle and association of DNA polymerase alpha with the outer nuclear membrane

[³H]dTMP incorporation into DNA of nuclei isolated from differentiating cardiac muscle of the rat has been characterized. Nuclei prepared at different times during the terminal phase of differentiation by a procedure not involving a detergent (Triton X-100) wash show a progressively diminished capacity to support in vitro [³H]dTMP incorporation; this diminution parallels the loss of DNA polymerase α from cardiac muscle. The rate of incorporation of [³H]dTMP into DNA of nuclei washed twice with 0.5% Triton X-100 does not correlate with the in vivo DNA synthetic activity. As determined by electron microscopy the Triton X-100 wash removes the outer nuclear membrane; the pellet obtained by centrifuging the Triton X-100 extract of these nuclei consists of circular membrane vesicles. The predominant DNA polymerase activity in these preparations was characterized using pH optimum, N-ethylmaleimide sensitivity, and correlation to in vivo DNA synthetic activity as criteria. DNA polymerase α activity predominated in the non-Triton X-100-extracted nuclei and in the outer nuclear membrane fraction; DNA polymerase β activity was the predominant activity observed in Triton X-100-extracted nuclei. These data emphasize that the procedure which is used to isolate nuclei from proliferating cells can greatly influence the nature of the DNA synthetic activity that is observed in vitro, suggest that DNA polymerase α is associated with the outer nuclear membrane, and add support to the idea that this enzyme is involved in eukaryotic DNA replication.     

Identification and characterization of arginine vasopressin-like substances in the rat testis

We have previously demonstrated the suppression of Leydig cell steroidogenesis by arginine vasopressin (AVP) in vitro. Since the circulating level of AVP is too low to mediate a testicular action of this peptide, we have conducted studies to identify testicular AVP-like substances. The supernatant of homogenized, acid-extracted rat testes was found to contain AVP immunoreactivity which showed parallelism with synthetic AVP in a specific radioimmunoassay for AVP. Chromatography of this extract on a Sephadex G-25 column produced three peaks of AVP immunoreactivity. The largest peak eluted close to the column void volume, a second smaller peak eluted at the total column volume, while a third peak co-eluted with synthetic AVP. Following acetone precipitation, ether extraction, and octadecylsilica (C18) adsorption chromatography of the acid extract, the third peak of AVP immunoreactivity (about 600 pg/testis) was fully retained by C18 chromatography and showed parallelism with synthetic AVP in both radioimmuno- and radioreceptor assays. This substance also co-migrated with synthetic AVP on both Sephadex G-25 and reverse-phase thin layer chromatography (RPTLC). The second peak was only partially retained by C18 adsorption chromatography, dilution curves were not parallel with synthetic AVP in radioimmuno- or radioreceptor assay, and this material failed to co-migrate with synthetic AVP on Sephadex G-25 and RPTLC. The first peak of apparent AVP immunoreactivity was associated with an enzyme(s) that degraded labeled AVP. This enzymatic activity, as well as the immunoreactivity, could be eliminated by heating the extract to 90 degrees C. These results demonstrate, by a number of independent criteria, that rat testes contain a substance which behaves like authentic AVP. Due to its high concentration, the AVP-like peptide may be synthesized or concentrated by testis cells. In addition, testis tissue contains enzymatic activity which degrades AVP and could represent a site of regulation of AVP action. Coupled with the previously demonstrated testis action of AVP, these results suggest a paracrine or autocrine role of the neurohypophysial hormone at the testis level.     

Aldose reductase and sorbitol dehydrogenase distribution in rat kidney.

The sorbitol pathway catalyzes the conversion of glucose to fructose via the intermediate sorbitol. It consists of aldose reductase (AR) and sorbitol dehydrogenase (SDH). In adult (44 day) kidney zones, AR was highest in the outer medulla. In substructures AR was highest in distal convoluted tubule. The AR was greatest in newborn and 8-day zones of developing rat kidney. Acute alloxan diabetes was associated with decreased AR in small arteries, but not glomeruli. The SDH was lowest in outer medulla. It was most active in glomeruli and distal convoluted tubules. The diabetic state leads to no change of SDH in arteries but an increase in glomeruli. SDH increased with development. This study demonstrates AR and SDH in substructures of the kidney. The pathway is present in developing kidney. In diabetes the enzymatic changes would tend to decrease accumulation of sorbitol.     

Production of urea from arginine in pars recta and collecting duct of the rat kidney

Urea production from arginine was studied in vitro in the kidney of normal rats in tubule suspensions of the four different renal zones (cortex, outer and inner stripe of outer medulla, and inner medulla), and in individual microdissected nephron segments. Tissue was incubated with L-[guanido-14C]-arginine to measure cellular arginase activity. Addition of urease to the incubate freed 14CO2 from the 14C-urea formed by arginase and released from the cells. CO2 was trapped in KOH and counted. These experiments revealed that significant amounts of urea are produced in the outer stripe and in the inner medulla. This intrarenal urea generation takes place mainly in the proximal straight tubule and in the collecting duct, with increasing activity in these two structures from superficial to deep regions of the kidney. Urea is known to play a critical role in the urinary concentrating process. The fact that some urea can be produced in the mammalian kidney, and that the two structures showing this capacity are straight portions of the renal tubular system descending along the corticopapillary axis suggest that this urea production might play a role in the formation and/or maintenance of the medullary urea concentration gradient.     

Changes in urine 8-hydroxydeoxyguanosine levels of super-marathon runners during a four-day race period

We have determined the urinary 8-hydroxydeoxyguanosine (8-OHdG) levels of five well trained supra-marathon runners during a four-day race. The daily running distances of the four-day race were the following; 93 km, 120 km, 56 km and 59 km, respectively. Pre-race and post-race urine samples were collected on each day and analyzed by a monoclonal antibody technique. The urinary 8-OHdG content increased significantly on the first day and tended to decrease from the third day. By the fourth day 8-OHdG content was significantly less than measured on the first three days. The serum creatine kinase activity changed in a similar fashion, showing a large increase (P<0.001) up to the third day when it decreased significantly from the peak value (P<0.05). We conclude that extreme physical exercise causes oxidative DNA damage to well trained athletes. However, repeated extreme exercise-induced oxidative stress does not propagate on increase of urinary 8-OHdG, but rather causes an adaptation leading to normalization of oxidative DNA damage.     

Inter and intra diurnal variations of DNA, RNA and protein synthetic activity in newborn, infant and young adult mouse livers.

The present report is a continuation of our previous studies on the observations of macromolecule precursors incorporated into the liver of newborn (1-10 days), infant (11-21 days) and young adult (41-50 days) old mice C57BLfemale X C3Hmale Fl and 3-hour interval circadian rhythm in livers of 15 day-old mice. 3H thymidine, 3H uridine and 3H leucine were used for studying incorporation into the macromolecules. DNA, RNA and protein were prepared by a modified Kirby's procedure. The data obtained indicate fluctuation of DNA activity approximately at 5-day intervals with a decreasing tendency up to 50 days of age. In 15-day-old mice, the peak of synthetic DNA activity was observed at 9.00 a.m. and the lowest values were found the next day at 6.00 a.m. Our results should provide fundamental data from which it would be possible to speculate whether the hepatocarcinogenic effect of different compounds with or without a special affinity to induce hepatoma may be correlated to different macromolecular synthetic activity.     

Senescence in the nongreening region of the rice (Oryza sativa) coleoptile

Summary The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from the imbibed seed on day 2 after sowing and ceases its growth on day 3. In cross section, the cells near the outer epidermis turn into green between days 2 and 3, while those near the inner epidermis remain colorless. In this study, the complete process of the development in the nongreening cells in the coleoptile was examined by fluorescence and electron microscopy. Embryonic morphology on day 0 was rapidly converted into the differentiated greening or nongreening cells between days 1 and 2. Senescence in the inner, nongreening region first appeared on day 4 in the third or fourth cell layer from the inner epidermis and then spread towards both the inner and the outer epidermis, and the inner cells collapsed completely before the outer cells senesced. Cells adjacent to the inner epidermis, which senesced slowly, followed a sequence of events during development: (1) degradation of plastid DNA; (2) dispersal of nuclear chromatin, differentiation of plastids into amyloplasts, degradation of mitochondrial DNA; (3) degradation of the starch in amyloplasts; (4) disorganization of plastids; (5) condensation of the nucleus, shrinkage of mitochondria; (6) complete loss of cellular components, distortion of cell walls. In the interior cells, the early events including degeneration of plastid DNA and mitochondrial DNA occurred in parallel with those in the cells adjacent to the inner epidermis, yet rapid collapse of all the cellular components proceeded between days 3 and 5, and nuclear condensation could not be detected.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - DiOC₇ 3,3-dihexyloxacarbocyanine - IE inner epidermis - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - OE outer epidermis - ptDNA plastid DNA - pt-nucleoid plastid nucleoid     

Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication

Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.     

Incorporation of Iododeoxyuridine into Cellular Deoxyribonucleic Acid Synthesized After Infection with Simian Virus 40

Primary monkey kidney cells (Cerocpithecus aethiops) in the stationary phase of growth were labeled with (14)C-thymidine for 24 hr prior to infection with simian virus 40 (strain 777). (3)H-deoxyadenosine and 5-iodo-2'-deoxyuridine (IUdR) were added to some of the cultures 24, 48, or 72 hr after infection; 24 hr later the deoxyribonucleic acid (DNA) was extracted from these cultures and centrifuged in a CsCl density gradient. The portion of DNA which had become heavier because of incorporation of IUdR could be seen as a second peak in the sedimentation profile. This peak contained (14)C as well as (3)H activity. The possibility that the (14)C-labeled cellular DNA might be degraded and used for the synthesis of viral DNA could be excluded. On the basis of these results, it must be assumed that the infection of monkey kidney cells with simian virus 40 induces the synthesis of cellular DNA.     

Function of 2-mercaptoethanol as a macrophage substitute in the primary immune response in vitro.

The mechanism by which 2-ME acts as a macrophage-substitute for the induction of a primary PFC response to SRC in vitro was studied in macrophage-depleted mouse spleen cell cultures. 2-ME could replace macrophages only in FCS-supplemented cultures. Evidence is presented that the function of 2-ME is independent of residual macrophages. Neither normal nor macrophage-depleted spleen cell cultures from congenitally athymic nude mice supplemented with 2-ME, with or without FCS, could give rise to a primary in vitro anti-SRC immune response. 2-ME, at an optimal concentration of 10(-5) M, induced DNA synthesis in normal and macrophage-depleted spleen cells in both FCS-containing and serum-free cultures. The peak response occurred on day 3. The stimulation was accompanied by a polyclonal B cell activation to antibody secretion which was much more pronounced in FCS-containing than in serum-free cultures. Spleen cells from nude mice showed a weaker DNA stimulation than did cells from normal mice in FCS-containing cultures, and nearly no response under serum-free conditions. T cells obtained by a nylon column adherence method from normal mouse spleen cells showed good DNA synthetic responses in FCS-containing, but no response in serum-free cultures. These results show that 2-ME has weak mitogenic activity for B cells, and in combination with FCS, strong mitogenic activity for T cells. Since the macrophage provides stimulation to the T cell in the primary anti-SRC PFC response in vitro, these results suggest that the direct mitogenic activity of 2-ME with FCS on T cells provides the functional substitution for macrophages.     

Fractionation and purification of DNA methylases and specific endonucleases from cells of Escherichia coli SK]

Fractionation and purification of DNA methylases and specific endonucleases from E. coli SK responsible for DNA specificity to host prokaryotic cells were studied. The most efficient purification was achieved by precipitation of proteins by 0.6 saturated ammonium sulfate with subsequent chromatography on KM-cellulose and concentration of fractions by dialysis against glycerol. Under these conditions the methylase activity produced 4 discrete fractions. Due to purification the specific activity of methylases increased 11--20-fold in various fractions. Methylase from the first (A) and fourth (BII) peaks catalyzed the methylation of cytosine to produce 5-methylcytosine; methylase from the third peak (BI) methylated adenine to produce 6-methylaminopurine. The chemical specificity of the second peak (B) methylase could not be established due to very high lability of the enzyme in this fraction. Specific endonuclease was found in the gradient zones eluted by 0.1--0.2 M and 0.65--0.75 M NaCl. It is assumed that those enzymes providing for DNA hydrolysis up to the formation of high--molecular discrete fragments, are restricting endonucleases of the SK system. The results obtained strongly suggest the existence of several types of methylases and restricting endonucleases in E. coli SK cells.