EARLY AND LATE INCORPORATION OF TRITIATED THYMIDINE INTO SKIN CELLS AND THE PRESENCE OF A LONG-LIVED G0-SPECIFIC PRECURSOR POOL

40 min after a single injection of 50 µCi of tritiated thymidine a 3 mm punch of DBA-1 mouse skin contains about 1000 dpm. This value remains constant for at least 48 hr after injection. 50 hair follicles contain about 40 dpm, and from these values the activity calculated to reside in the basal layer of a 3 mm punch of skin is 760 dpm. These values also remain constant with time after injection. Fresh punches of skin contain much more activity. The fixative-soluble fraction (the difference between fresh and fixed values) decays slowly with time. The values for DBA-2 mice are similar. Plucking the hair from the follicles appears immediately to increase the size of the fixative-soluble fraction and decrease the fixed tissue values to about 500 dpm per punch for whole skin and about 1 dpm per 50 follicles for DBA-1. Thus almost all the activity is restricted to the epidermis. The fixative-soluble fraction returns approximately to the unplucked value between 24 and 48 hr after plucking. However, during this period the fixed tissue values are rising rapidly as stimulated cells enter S. It appears that in both strains labeled material remains available for incorporation into stimulated cells for at least 48 hr after a single injection. The amount persisting appears to decrease with time. The whole-fixed skin, the hair follicles, and the epidermis all contain cells that are capable of becoming labeled after stimulation 8–48 hr after an injection. The label in question does not become incorporated into normal cycling skin or hair follicle cells. It is concluded that the DNA precursor pool is possibly connected with G₀ cells and that both the hair follicle and the basal layer of the epidermis contain these resting cells.     

Circadian-Stage Dependence of Methotrexate In A Keratinized Epithelium. an in-vivo Study Using Flow Cytometry On the Hamster Cheek Pouch Epithelium

The partially synchronized cell system of the hamster cheek pouch epithelium shows a characteristic diurnal rhythm of cell proliferation. Bolus injections of methotrexate (Mtx) in both lethal (10 g/m²) and non-lethal (2 g/m²) doses were found to inhibit cell-cycle progression primarily by impairing the G₁/S transition. the results were obtained by flow cytometric DNA analysis. the inhibitory effect of Mtx manifested itself as a relative decrease of the S fraction (drug-effector phase), and was found to be dependent both on the dose and on the time of the day it was given. A bolus injection of Mtx was given either at 1200 hr (when a minimal number of cells are in S phase) or at 0200 hr (when a maximum number of cells are in S phase). the greatest cumulative decrease in S fraction was seen when the injection was given at 1200 hr. the time between injection and the effect (seen as a decrease in S fraction) was independent of the time of the Mtx injection, but seemed instead to be related to the natural diurnal period of increasing flux from G₁ to S phase (at the onset of the dark period). the main effect (the relative decrease in S fraction) was repeated during the following 24-hr period, pointing to a protracted effect of Mtx on G₁ cells. G₁ cells affected by the initial high Mtx plasma concentration seem to be responsible for the reduced influx into S phase in both the first and second 24-hr period. In earlier toxicological studies, the survival rate of hamsters was dependent on the time of injection and was highest after injection at 1200 hr. Thus maximum cytokinetic effect on epithelial cells was found at the time of the day when there was a minimum lethal effect on the animal.     

The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. II. The kinetics of residual protein synthesis and transport

The kinetics of acidic residual chromosomal protein synthesis and transport were studied throughout the cell cycle in HeLa S-3 cells synchronized by 2 mM thymidine block and selective detachment of mitotic cells. Pulse labeling the cells with leucine-³H for 2 min and then "chasing" the radioactive proteins for up to 3 hr showed that the amount of protein synthesized, transported, and retained in the acidic residual chromosomal protein fraction is greater immediately after mitosis and later in G₁ than in the S or G₂ phases of the cell cycle. During S, only 20–25% of the proteins synthesized and transported to the acidic residual chromosomal protein fraction are chased during the first 2 hr after pulse labeling, whereas up to 40% of the material entering the residual nuclear fraction in mitosis, G₁, and G₂ leaves during a 2 hr chase. Polyacrylamide gel electrophoretic profiles of these proteins, at various times after pulse labeling, reveal that the turnover of individual polypeptides within this fraction has kinetics of synthesis and turnover which are markedly different from one another and undergo stage-specific changes.     

Secretion of chondroitin SO4 by monolayer cultures of chick embryo chondrocytes

Monolayer cultures of chick embryo tibial chondrocytes incorporate 35SO42- into chondroitin SO4 which is rapidly secreted from the cells into two extracellular pools. Part of the extracellular chondroitin SO4 is recovered in a soluble form in the culture medium, and the remainder is associated with the cell matrix from which it is released by isotonic trypsinization. At 38 degrees C labeled chondroitin SO4 appears in the cell matrix fraction within 5 min after addition of 35SO42- and in the culture medium fraction 15 min after 35SO42- is added. The intracellular pool of labeled chondroitin SO4 reaches a steady state level of 150 to 200 pmol of bound SO4 per 10(6) cells in 60 min, while the cell matrix and medium fractions increase at rates of 3 and 1 nmol of bound SO4 per h per 10(6) cells, respectively. After 4 h of labeling, less than 20% of the newly synthesized cell-associated chondroitin SO4 is in the intracellular fraction. By labeling cells for 15 min at 25 degrees C 80% of the cell-associated chondroitin 35SO4 is obtained in the intracellular fraction. This material is chased without lag into both the cell matrix fraction and the medium fraction. A mixture of NaF and NaCN, both at 30 mM, lowers the cellular ATP level to 15% of normal and blocks secretion of the intracellular chondroitin SO4 into both extracellular fractions. Colchicine at 10(-6) M gives a partial inhibition of both synthesis and secretion of chondroitinSO4. Sucrose density gradient sedimentation analysis of the intracellular chondroitin SO4 and the two extracellular fractions shows that all three fractions contain both a heavy and light proteoglycan fraction. The intracellular light proteoglycan fraction is secreted preferentially into the culture medium where it represents 30% of the total culture medium pool. The ratio of 6-sulfated GalNAc to 4-sulfated GalNAc in the heavy proteochondroitin SO4 fraction is approximately twice that found for the light fraction.     

Polychlorinated biphenyl uptake and transport by lymph and plasma components

The uptake and vascular transport of ingested Aroclor 1242, an isomeric mixture of polychlorinated biphenyls (PCB), was investigated in experimental animals. High concentrations of ingested PCB were found in the chylomicron fraction of thoracic duct lymph. When the lymph flow was exteriorized PCB were not subsequently found in the vascular circulation. When lymph was not exteriorized plasma PCB concentrations reached maximal levels 6 hr after ingestion. Less than 1% of total plasma PCB was detected in cellular fractions of blood over a 10-hr period following ingestion. Chylomicrons contained 31% of total plasma PCB 30 min after ingestion, decreasing to less than 6% at 4 hr. A maximum of 10% of plasma PCB at 1 hr, and less than 5% at 6 hr, after ingestion was associated with very low density lipoproteins (VLDL) or low density lipoproteins (LDL). Although PCB enter the vascular circulation with the chylomicron fractions of lymph, delipoproteinated plasma contained 52% of the total PCB in blood collected 30 min after ingestion. This level increased to 78% after 2 hr, and remained constant at about 80% for an additional 8-hr period. High performance liquid chromatographic (HPLC) examinations of delipoproteinated plasma from blood taken 6 hr after PCB ingestion showed elution of greater than 95% of plasma PCB to coincide with the albumin peak. Electrophoretic examinations of delipoproteinated plasma showed the association of PCB with albumin to be noncovalent. The results suggest that apolar PCB are absorbed into intestinal epithelial cells from which they are secreted into the lymphatic drainage sequestered within the apolar core of chylomicrons, that these PCB transit the thoracic duct and enter the vascular circulation within chylomicrons and are metabolized or otherwise released from chylomicrons during hepatic chylomicron clearance, and that resulting PCB or PCB derivatives circulate in association with plasma albumins.     

SYNTHESIS OF THE CARBOHYDRATE OF MUCUS IN THE GOLGI COMPLEX AS SHOWN BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY OF GOBLET CELLS FROM RATS INJECTED WITH GLUCOSE-H3

It is known that colonic goblet cells utilize glucose to synthesize the carbohydrate portion of mucus glycoprotein. To determine the intracellular site of this synthesis, glucose-H³ was injected into 10-g rats. At 5, 20, 40 min, 1, 1½, and 4 hr after injection, segments of colon were fixed and prepared for electron microscope radioautography. By 5 min after injection, label had been incorporated into substances present in the flattened saccules of the Golgi complex. At 20 min, both Golgi saccules and nearby mucigen granules were labeled. By 40 min, mucigen granules carried almost all detectable radioactivity. Between 1 and 4 hr, these labeled granules migrated from the supranuclear region to the apical membrane; here, they were extruded singly, retaining their limiting membrane. The evidence indicates that the Golgi saccule is the site where complex carbohydrate is synthesized and is added to immigrant protein to form the complete glycoprotein of mucus. The Golgi saccule, distended by this material, becomes mucigen granules. It is roughly estimated that one saccule is released by each Golgi stack every 2 to 4 min: a conclusion implying continuous renewal of Golgi stacks. It appears that the Golgi synthesis, intracellular migration, and release of mucus glycoprotein occur continually throughout the life of the goblet cell.     

Receptor mediated endocytosis of hemoglobin-haptoglobin, galactosylated serum albumin and polymeric IgA by the liver

Labelled hemoglobin-haptoglobin (ham-hap), galactosylated serum albumin (gal-SA) and polymeric immunoglobulin A (p IgA) were injected intravenously to rats or mice. The labels disappeared from the plasma with a half-time of about 5 min and were almost entirely found associated with the liver where degradation products progressively appear. The uptake of hem-hap and gal-SA are partially saturable as a function of the plasmatic concentration and the uptake of gal-SA can be completely inhibited by the simultaneous injection of asialofetuin. About 45 min after injection to rats, labelled material appears in the bile in amounts corresponding to 3.9% of the injected dose (hem-hap), 2.8% (gal-SA) and 60.1% (p IgA). The molecular weight of the labelled material transferred into the bile has been characterized: it consists almost entirely of intact IgA and for about 60% of intact hem-hap and gal-SA. Cell fractionation experiments indicate that 4 min after injection, the label is associated with components which equilibrate around a density of 1.13 g/cm3 and which dissociate from marker enzymes of Golgi complex, plasma membrane and lysosomes. Longer times after injection (from 20 min for hem-hap and gal-SA to 1 h for p IgA) labelled material appears, within lysosomes. To explain all these data, we suggest that after binding to plasma membrane receptors, the ligands are rapidly interiorized into pinocytic vesicles which fuse with lysosomes. Most of the hem-hap and gal-SA molecules but only part of p IgA would be released and subsequently digested; these vesicles would dissociate from lysosomes and fuse with the biliary membrane where the molecules still bound to the membrane of the vesicles would be detached and excreted into the bile.     

G2 Sub-Population In Mouse Liver Induced Into Mitosis By Lead Acetate

A single intracardiac dose of lead acetate (40 μ lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. the prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G₂ phase. the injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. the incremental labeled cells may have originated from a small fraction of the G₁ population that was ready to enter the S phase without the usual pre-synthetic delay.     

Spindle formation and cleavage in Xenopus eggs injected with centriole-containing fractions from sperm

The eggs of Xenopus laevis are capable of initiating spindle formation and cleavage in response to microinjection of partially purified components of sea urchin sperm. High activity was assayed from a sperm head fraction obtained after removal of the plasma and nuclear membranes, acrosome, midpiece mitochondrion, and tail. Chromatin, the basal plate, and the distal centriole comprised the components of the head fraction. Disruption of the chromatin did not impair activity and purified chromatin lacked activity, suggesting the centriole and basal plate as the active materials. Low doses of active material induced apparently normal cleavage at 90 min after injection, with 16% of the eggs reaching the late blastula stage. High doses of active material induced precocious multiple cleavage, with some eggs cleaving into 3–10 segments within 20 min after injection. These eggs contained numerous spindles and asters in the animal hemisphere, as judged by light microscopy of stained sections. Microinjection of eggs is presented as a semi-quantitative bioassay for agents initiating spindle formation and cleavage.     

THE ELABORATION OF PROTEIN AND CARBOHYDRATE BY RAT PARATHYROID CELLS AS REVEALED BY ELECTRON MICROSCOPE RADIOAUTOGRAPHY

The parathyroid glands of young rats were radioautographed after a single injection of the protein precursor tyrosine-³H in the hope of identifying the sites of synthesis and migration of newly formed protein in the gland cells. The same procedure was used after injection of the glycoprotein precursor galactose-³H. As early as 2 min after intravenous injection of tyrosine-³H, the label was mainly found in the rough endoplasmic reticulum suggesting that cisternal ribosomes are sites of protein synthesis. By 5 and 10 min, much of the label had migrated from the rough endoplasmic reticulum into the Golgi apparatus. By 20 and 30 min, some label had migrated from there into secretory granules. By 45 min and 1 hr, the label content of the cell had decreased, indicating release of labeled material outside the cell. At 2 min after intravenous injection of galactose-³H, the label was mainly present in the Golgi apparatus, where presumably galactose is taken up into glycoprotein. By 10 min, some label appeared in secretion granules and by 30 min release of the material to the outside of the cell was under way. In conclusion, it is likely that the tyrosine-labeled protein material consists mainly of the parathyroid hormone. The galactose-labeled carbohydrate material would be either associated with the hormone in the cell or be part of a distinct glycoprotein which may be the one present on the outer surface of the plasma membrane (cell coat).     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the ciliary epithelium of the eye: effects of microtubule-disrupting drugs

3H-fucose was injected intravenously or intravitreously into albino rats. After time intervals of 10, 40, and 50 min, 1, 1.5, and 4 hr, 1, 3, and 7 days, and 1, 2, and 4 weeks after injection, the animals were sacrificed by intracardiac perfusion with glutaraldehyde. Samples of the ciliary body were prepared for light and electron microscope radioautography. Light microscope autoradiographs showed that the cells of both the inner and outer layers of ciliary epithelium actively incorporated 3H-fucose label in a reaction that peaked in intensity at 4 hr after injection, and then progressively declined. Electron microscope radioautographs revealed that, at early time intervals, most of the label was localized to the Golgi apparatus. With time, the plasma membrane of both cell types became increasingly labeled, and accounted for 60-70% of the total silver grains at 4 hr after injection. Adjacent to the basal cell surface of the inner layer cells, the fibers of the zonula became increasingly labeled from 1.5 hr onwards, providing strong evidence that these cells secrete glycoproteins to the zonula. When vinblastine was administered 30 min before 3H-fucose injection, followed by sacrifice 1.5 hr later, a much larger proportion of label remained localized to the Golgi apparatus than in controls, and the plasma membrane and zonula were much less labeled. These results suggest that, as documented in other cell types, microtubules may play a role in the intracellular transport of membrane and secretory glycoproteins in these cells.     

Prostaglandin F2α induced luteolysis,hypothermia, and abortions in beagle bitches

Luteal phase plasma progesterone was radioimmunoassayed in samples collected before, during, and after a 72 hr treatment period during which Beagle bitches received repeated i.m. injections of prostaglandin F₂α (n=17) or saline (n=3). PGF₂α (20 ug/kg every 8 hr or 30 ug/kg every 12 hr) was administered to 7 pregnant and 8 nonpregnant bitches during the mid or late luteal phase of the cycle (Day 25–58) and to 2 nonpregnant bitches during the early luteal phase (Days 5 and 20). Progesterone was depressed from pretreatment levels (3 – 40 ng/ml) in each of the 15 bitches given PGF₂α after Day 25 of the cycle. Mean progesterone (ng/ml plasma) at ?24, 0, 12, 24, 36, 48, 60, 72 and 96 hr from the initial PGF₂α injection were 16.6, 15.6, 9.3, 5.1, 2.1, 1.5, 1.4, 1.1 and 1.1 (±0.9, n=15). Thereafter, progesterone was nondetectable in the 8 nonpregnant bitches and in 4 pregnant bitches that aborted. Abortions occurred when progesterone was depressed to 0.6 – 1.4 ng/ml, 56–80 hr after starting PGF₂α treatment on Days 33–53 of the cycle. Three pregnant bitches did not abort when progesterone was depressed to a mean low value of 2.1 ng/ml during PGF₂α treatments begun on Day 31 – 40 of pregnancy. Progesterone in these bitches recovered to 5 – 10 ng/ml and was maintained until the normal prepartum decline. Since PGF₂α can induce complete luteolysis it may be of use as an abortifacient in the bitch.A transient fall in rectal temperature occurred in each of 12 luteal phase bitches injected with PGF₂α (20 ug/kg, i.m.). The hypothermia was detectable within 15 min, maximal at 45 – 60 min, and averaged 1.39° C. No temperature changes were noted in eight ovariectomized bitches similarly treated. In six luteal phase bitches, plasma progesterone fell 20–45% within the 15 min required to observe a consistent decline in rectal temperature following PGF₂α administration. The transient hypothermia following PGF₂α appears to be secondary to the luteolytic effect and dependent on a fall in progesterone.     

Regulation of Renal Na-HCO3 Cotransporter: VIII. Mechanism of Stimulatory Effect of Respiratory Acidosis

We examined the effect of respiratory acidosis on the Na-HCO₃ cotransporter activity in primary cultures of the proximal tubule of the rabbit exposed to 10% CO₂ for 5 min, 2, 4, 24 and 48 hr. Cells exposed to 10% CO₂ showed a significant increase in Na-HCO₃ cotransporter activity (expressed as % of control levels, 5 min: 142 ± 6, 2 hr: 144 ± 13, 4 hr: 145 ± 11, 24 hr: 150 ± 15, 48 hr: 162 ± 24). The increase in activity was reversible after 48 hr. The role of protein kinase C (PKC) on the stimulatory effect of respiratory acidosis on the cotransporter was examined in presence of PKC inhibitor calphostin C or in presence of PKC depletion. Both calphostin C and PKC depletion prevented the effect of 10% CO₂ for 5 min or 4 hr to increase the activity of the cotransporter. 10% CO₂ for 5 min or 4 hr increased total and particulate fraction PKC activity. To examine the role of phosphotyrosine kinase (PTK) on the increase in cotransporter activity we studied the effect of two different inhibitors, 2-hydroxy-5-(2,5-dihydroxylbenzyl) aminobenzoic acid (HAC) and methyl 2,5-dihydroxycinnamate (DHC) which inhibit phosphotyrosine kinase in basolateral membranes. Cells were pretreated either with vehicle or HAC or DHC and then exposed to 10% CO₂ for 5 min or 4 hr. In cells treated with vehicle, 10% CO₂ significantly increased cotransporter activity as compared to control cells exposed to 5% CO₂. This stimulation by 10% CO₂ was completely prevented by HAC or DHC at 5 min (5% CO₂: 1.8 ± 0.2, 10% CO₂: 2.6 ± 0.2, 10% CO₂+ HAC: 1.6 ± 0.2, 10% CO₂: +DHC: 2.0 ± 0.3 pH unit/min) and also at 4 hr. The protein synthesis inhibitors actinomycin D and cycloheximide appear to prevent the effect of 10% CO₂ for 4 hr on the cotransporter. Our results show that early respiratory acidosis stimulates the Na-HCO₃ cotransporter through PKC and PTK-dependent mechanisms and the late effect appears to be mediated through protein synthesis. Received: 28 March 1997/Revised: 22 December 1997     

STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.     

The role of the plasma membrane in fatty acid uptake by rat liver parenchymal cells

1. Suspensions of isolated rat liver parenchymal cells incorporate [(14)C]palmitic acid into glycerides at about 40% of the rate obtained with liver slices. 2. At short time-intervals most of the incorporation is into phosphatidylcholine and this is recovered mainly in the plasma-membrane fraction. 3. At later times (5min to 2h) the [(14)C]palmitic acid is mainly found in triglyceride, but this is not recovered in the plasma-membrane fraction. 4. Addition of lysophosphatidylcholine increases incorporation of palmitic acid into both phosphatidylcholine and triglyceride, with maximum effect at about 0.1mm. 5. In vivo, 1min after injection of [(14)C]palmitic acid, radioactive phosphatidylcholine is concentrated in the plasma-membrane fraction, but the proportion present in this fraction declines rapidly. 6. The phosphatidylcholine of the plasma-membrane fraction has, at 1min after injection, a specific radioactivity 30-fold greater than that of the whole tissue. 7. This phosphatidylcholine reaches its maximum specific radioactivity before the tissue phosphatidic acid or diglyceride. 8. The phosphatidylcholine of the plasma-membrane fraction has a very rapid turnover. 9. It is proposed that the rapid formation of phospholipids in the plasma membrane is by acylation of their lyso-derivatives and the role of this process in fatty acid uptake is discussed.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

Circadian-stage dependence of methotrexate in a keratinized epithelium. An in-vivo study using flow cytometry on the hamster cheek pouch epithelium

The partially synchronized cell system of the hamster cheek pouch epithelium shows a characteristic diurnal rhythm of cell proliferation. Bolus injections of methotrexate (Mtx) in both lethal (10 g/m2) and non-lethal (2 g/m2) doses were found to inhibit cell-cycle progression primarily by impairing the G1/S transition. The results were obtained by flow cytometric DNA analysis. The inhibitory effect of Mtx manifested itself as a relative decrease of the S fraction (drug-effector phase), and was found to be dependent both on the dose and on the time of the day it was given. A bolus injection of Mtx was given either at 1200 hr (when a minimal number of cells are in S phase) or at 0200 hr (when a maximum number of cells are in S phase). The greatest cumulative decrease in S fraction was seen when the injection was given at 1200 hr. The time between injection and the effect (seen as a decrease in S fraction) was independent of the time of the Mtx injection, but seemed instead to be related to the natural diurnal period of increasing flux from G1 to S phase (at the onset of the dark period). The main effect (the relative decrease in S fraction) was repeated during the following 24-hr period, pointing to a protracted effect of Mtx on G1 cells. G1 cells affected by the initial high Mtx plasma concentration seem to be responsible for the reduced influx into S phase in both the first and second 24-hr period. In earlier toxicological studies, the survival rate of hamsters was dependent on the time of injection and was highest after injection at 1200 hr. Thus maximum cytokinetic effect on epithelial cells was found at the time of the day when there was a minimum lethal effect on the animal.     

Reduced oxygen tension increases atrial natriuretic peptide release from atrial cardiocytes

To test the hypothesis that reduced oxygen tension stimulates cardiac atrial natriuretic peptide (ANP) secretion, we measured ANP release and expression in neonatal rat atrial and ventricular cardiac myocytes exposed to 45 min and 3, 6, and 24 hr of 3% or 21% oxygen. In atrial cardiocytes, the percentage of increase in culture media ANP concentration from baseline was greater in cells exposed to 3% than in cells exposed to 21% oxygen after 3 hr (814% +/- 52% vs. 567% +/- 33%, P < 0.05) and 6 hr of exposure (1639% +/- 91% vs. 1155% +/- 73%, P < 0.05). No differences in the percentage of increase in culture media ANP concentration was seen at 45 min (284% +/- 27% vs. 201% +/- 16%, P = NS) or 24 hr (2499% +/- 250% vs. 2426% +/- 195%). There was a significant increase in cellular ANP content between 3 and 24 hr in atrial cardiocytes exposed to 21% oxygen (105% +/- 40% vs. 296% +/- 60%, P < 0.05), but not in atrial cardiocytes exposed to 3% oxygen (118% +/- 20% vs. 180% +/- 26%, P = NS). Steady-state ANP mRNA levels in atrial cardiocytes were not affected by oxygen tension. In ventricular cardiocytes, oxygen tension did not affect ANP secretion, cellular ANP content, or steady-state ANP mRNA levels. We conclude that reduced oxygen tension increases release of ANP from atrial, but not ventricular cardiocytes and that this mechanism may contribute to the elevation in plasma ANP seen during acute hypoxia.