Mutator Phenotype Induced by Aberrant Replication

We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures. Allele-specific mutants of DNA polymerase δ (polδ) and mutants of Polα, two Polδ subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats. Deletion of rad2⁺, which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats. The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2Δ background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2Δ was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization. Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent. The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2Δ. These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures. The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.     

Interplay between Pleiotropy and Secondary Selection Determines Rise and Fall of Mutators in Stress Response

Mutators are clones whose mutation rate is about two to three orders of magnitude higher than the rate of wild-type clones and their roles in adaptive evolution of asexual populations have been controversial. Here we address this problem by using an ab initio microscopic model of living cells, which combines population genetics with a physically realistic presentation of protein stability and protein-protein interactions. The genome of model organisms encodes replication controlling genes (RCGs) and genes modeling the mismatch repair (MMR) complexes. The genotype-phenotype relationship posits that the replication rate of an organism is proportional to protein copy numbers of RCGs in their functional form and there is a production cost penalty for protein overexpression. The mutation rate depends linearly on the concentration of homodimers of MMR proteins. By simulating multiple runs of evolution of populations under various environmental stresses—stationary phase, starvation or temperature-jump—we find that adaptation most often occurs through transient fixation of a mutator phenotype, regardless of the nature of stress. By contrast, the fixation mechanism does depend on the nature of stress. In temperature jump stress, mutators take over the population due to loss of stability of MMR complexes. In contrast, in starvation and stationary phase stresses, a small number of mutators are supplied to the population via epigenetic stochastic noise in production of MMR proteins (a pleiotropic effect), and their net supply is higher due to reduced genetic drift in slowly growing populations under stressful environments. Subsequently, mutators in stationary phase or starvation hitchhike to fixation with a beneficial mutation in the RCGs, (second order selection) and finally a mutation stabilizing the MMR complex arrives, returning the population to a non-mutator phenotype. Our results provide microscopic insights into the rise and fall of mutators in adapting finite asexual populations.     

A recA-dependent mutator of Escherichia coli K12: Method of isolation and initial characterization

A number of mutator strains of E. coli were isolated using histochemical techniques which allow the identification of a single mutator colony on agar plates with as many as 2000 colonies. Several mutators isolated in this way were found by P1-mediated transduction to map to the proA-proB region of the E. coli chromosome. The map position of these mutators is very close to that of the conditional mutator, mutD. However, in contrast to mutD, one of these newly isolated mutators was suppressed in thermosensitive recA strain at 43°C, but not at 30°C. This mutator mutation has been named mut-8. Besides being dependent upon recA, mut-8 is also dependent upon growth in enriched medium for the expression of its mutator activity. The mutator activity of mut-8 was found to be recessive to the wild-type allele.     

Panicum maximum (Jacq.) density effect upon macrofauna structure in sediments of pilot-scale vertical flow constructed wetlands treating domestic wastewater

Plant density may have an impact on macrofauna structure in constructed wetlands (CW). In this study, Panicum maximum density effect on macrofauna structure in pilot-scale vertical flow CW treating domestic wastewater was estimated. Two beds were planted with P. maximum at 10 roots/m² (low density) and two others at 20 roots/m² (high density). Two unplanted beds were used as controls. After six months of wastewater treatment, macrofauna was collected by taking five cores of sediment samples at the corners and the centre of each bed following three layers in the vertical profile. Plant density seems not to impact significantly the CW performance. However, the plant density of 10 roots/m² (low density) provided higher wastewater treatment efficiency. Concerning macrofauna, 11 taxa belonging to 6 classes and 11 orders were collected. Macrofauna was significantly more diversified in the planted beds than in the control. But the two plant densities investigated do not show significant difference between macrofauna diversity of the planted beds. Macrofauna abundance was two- and three-fold higher, respectively, in the planted beds at high and low densities than in the controls. From the upper surface to the bottom of the beds, macrofauna diversity and abundance decreased, and were heavily dominated by Annelida.     

Acidic Conditions Are Not Obligatory for Onset of Butanol Formation by Clostridium beijerinckii (Synonym, C. butylicum)

Factors that may initiate the metabolic transition for butanol production were investigated in batch cultures of Clostridium beijerinckii (synonym, Clostridium butylicum) VPI 13436. Cultures maintained at pH 6.8 produced nearly as much butanol as those incubated without pH control, indicating that neither a change in the culture pH nor acid conditions per se are always required to initiate solvent formation. Acetate and butyrate levels at the onset of butanol production were dependent on the pH at which the cultures were maintained. Cultures maintained at pH 6.8 could be accelerated into solvent production by artificially lowering the pH to 5.0 or by the addition of acetate plus butyrate without a pH change (but neither acid alone was effective). Solvent production was associated with slower rates of growth and general metabolism, and it did not show a requirement for mature spore formation. We speculate that a slowdown in metabolism, which may be brought about by several conditions, is mechanistically related to the onset of butanol production. Extracts of solvent-producing cells contained acetoacetate decarboxylase activity as well as higher NADP⁺-linked butanol dehydrogenase and lower hydrogenase activities than extracts of acid-producing cells. Solvent production did not appear to involve an enhanced ability to catalyze H₂ oxidation.     

The post-replication repair RAD18 and RAD6 genes are involved in the prevention of spontaneous mutations caused by 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae

7,8-Dihydro-8-oxoguanine (8-oxoG) is an abundant and mutagenic lesion produced in DNA exposed to free radicals and reactive oxygen species. In Saccharomyces cerevisiae, the OGG1 gene encodes the 8-oxoG DNA N-glycosylase/AP lyase (Ogg1), which is the functional homologue of the bacterial Fpg. Ogg1-deficient strains are spontaneous mutators that accumulate GC to TA transversions due to unrepaired 8-oxoG in DNA. In yeast, DNA mismatch repair (MMR) and translesion synthesis (TLS) by DNA polymerase η also play a role in the prevention of the mutagenic effect of 8-oxoG. In the present study, we show the RAD18 and RAD6 genes that are required to initiate post-replication repair (PRR) are also involved in the prevention of mutations by 8-oxoG. Consistently, a synergistic increase in spontaneous Can^R and Lys⁺ mutation rates is observed in the absence of Rad6 or Rad18 proteins in ogg1 mutant strains. Spectra of Can^R mutations in ogg1 rad18 and ogg1 rad6 double mutants show a strong bias in the favor of GC to TA transversions, which are 137- and 189-fold higher than in the wild-type, respectively. The results also show that Polη (RAD30 gene product) plays a critical role on the prevention of mutations at 8-oxoG, whereas Polζ (REV3 gene product) does not. Our current model suggests that the Rad6–Rad18 complex targets Polη at DNA gaps that result from the MMR-mediated excision of adenine mispaired with 8-oxoG, allowing error-free dCMP incorporation opposite to this lesion.     

The Efflux of Substances from Frog Ventricles to Sucrose and to Ringer's Solutions

The frog ventricle in sucrose solution contracts for several hours at 25°C, and for as long as 24 hours at 5°G. The possibility that a fraction of the extracellular fluid remains outside of the excitable membrane was examined by measuring the efflux of tracers. The half-time for the efflux to sucrose solution at 25°C of C¹⁴ sucrose is about 1 minute, for Na²⁴ is 6.5 minutes, and for Cl⁸⁶ is 4 minutes. There is no evidence for the retention of an extracellular Na fraction. The Q₁₀ for Na and Cl efflux is about 1.3. The half-time for K⁴² efflux is about 180 minutes; the Q₁₀ is 1.7. The efflux rates of Na²⁴, Cl³⁶ and K⁴² to sucrose and to Ringer's solutions are quite similar. Ca⁴⁵ efflux is only one-fifth as fast to sucrose solution as to Ringer's; the retention of Ca⁺⁺ may be important for maintaining excitability in sucrose solution. P³² efflux is five times faster to sucrose solution than to Ringer's solution, and there is a similar increase in the rate of inosine loss to sucrose solution. The Q₁₀ for efflux to sucrose solution is 2.2 for P³²O₄ and 2.4 for inosine. We suggest that energy metabolism is abnormal in ventricles in sucrose solution and that low temperature prolongs excitability by slowing the metabolic change.     

Incorporation of [S]Sulfate and [C]Bicarbonate into Karyotype-specific Polysaccharides of Chondrus crispus

Gametophytic and sporophytic tissues of Chondrus crispus (Stackhouse) cultured in vitro were labeled with ³⁵S and ¹⁴C. The major sulfated polysccharides isolated from the two karyotypes were characterized by KCI fractionation, immunoprecipitation, and infrared spectroscopy. Reproducibility of data has been demonstrated by an experiment using gametophytic T₄ strain with five replicates per time point. The rate of sulfate uptake was similar in haploid and diploid plants from a given area cultured for a similar time. Cultures from different sources cultured for different times showed different uptake and incorporation levels. Although sulfate uptake did not appear to be karyotype-related, the pattern of incorporation of ³⁵S and ¹⁴C into polysaccharides was ploidy-specific.     

Relationship of Temperature to Stomatal Aperture and Potassium Accumulation in Guard Cells of Vicia faba

Epidermal strips of Vicia faba were floated on 10 millimolar KCl at various temperatures and for several time periods. The diameter of the stomatal aperture was determined microscopically and K⁺ content was estimated and expressed as the per cent of the guard cell stained. Stomatal opening was associated with increased K⁺ in guard cells, but the quantitative association was modified both by time and temperature. At low temperatures (0-20 C) there was a prolonged Spannungsphase while at higher temperatures (30-45 C) motorphase was exhibited. During the motorphase there was a rapid opening of the stomates which was highly correlated with K⁺ influx. At treatment periods of 360 minutes and temperatures higher than 25 C there appeared to be a maintenance phase during which K⁺ concentration of the guard cells decreased without an equivalent decrease in aperture.     

Chronic effects of low insecticide concentrations on freshwater caddisfly larvae

The insecticide load in surface waters does not ordinarily reach concentrations acutely toxic to aquatic fauna. The effects of the low insecticide concentrations typical of natural habitats are still not clear, for they often appear only after relatively long exposure times. To test such a situation, the insecticides lindane and parathion were introduced into a static-with-renewal outdoor aquaria system at concentrations about four and five orders of magnitude lower than their respective 96-h LC₅₀s, and their chronic (about 90 days) effects on the survival rate of freshwater caddisfly larvae were observed. The emergence and hence survival rate of Limnephilus lunatus Curtis was significantly reduced by lindane at 0.1 ng l^–1, a value nearly five orders of magnitude lower than the 96-h LC₅₀. Parathion, with acute and subacute toxicity similar to that of lindane, did not significantly alter the emergence rate of this species. In contrast, this substance did produce a significant reduction in emergence rate of the closely related species Limnephilus bipunctatus Curtis at 1 ng l^–1, even though this species was significantly less susceptible than L. lunatus to parathion at high concentrations. We conclude that chronic insecticide exposure can be hazardous to freshwater macroinvertebrates even at unexpectedly low concentrations. The low-concentration effects may depend on both species and substance and therefore cannot be predicted from toxicity data at higher concentrations.     

N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA

In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N⁶-methyl-adenosine (m⁶A) (Sommer et al., 1976; Moss &; Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of ³H from methyl-labeled methionine and ¹⁴C from uridine into Ad-2²-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins &; Darnell, 1978a), the rate of accumulation of [¹⁴C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [³H]methyl label in m⁶A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m⁶A.In accord with the accumulation kinetics, the ratio of ³H to ¹⁴C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA.A mathematical model was designed to evaluate the accumulation of methyl label in m⁶A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time ($\text{t}\text{1}\text{2}$) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from ¹⁴C label to be about 30 and 70 minutes, respectively.The model gave an excellent fit to the data when the $\text{t}\text{1}\text{2}$ for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, $\text{40.5–52.6}$, which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from $\text{38, 40, 43, 45}$, and $\text{48}$ was analyzed, it appeared that m⁶A was conserved.Finally, m⁶A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m⁶A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA.     

Compartmentation in Vicia faba Leaves: II. Kinetics of C-Sucrose Redistribution among Individual Tissues following Pulse Labeling

Leaflets of Vicia faba L. were pulse labeled with ¹⁴CO₂ and the kinetics of ¹⁴C-sucrose redistribution among individual tissues was followed. Sucrose specific activity in the whole leaf peaked about 15 minutes after labeling and declined with a half-time of about 80 minutes. In one experiment, leaflet discs taken at various times during the ¹²CO₂ chase were quick frozen, freeze-substituted, and embedded in plastic. The tissue was sectioned paradermally and sections of palisade parenchyma, of spongy parenchyma, and of spongy parenchyma that contained veins were collected. Water extracts from these sections were assayed for sucrose specific activity. Sucrose specific activity in the palisade parenchyma was higher than that of the spongy parenchyma and reached a maximum in both tissues 9 to 15 minutes after labeling. Sucrose specific activity initially declined rapidly in the palisade parenchyma followed by a period during which little or no loss occurred. Sucrose specific activity in sections containing veins peaked at 15 minutes with a maximum value substantially higher than either mesophyll tissue, indicating that recently synthesized sucrose was preferentially exported from the mesophyll. Decline of activity in these sections containing veins continued for the remainder of the experiment. Sucrose specific activity in lower epidermal peels peaked several minutes after that of the whole leaflet and remained lower. Sucrose specific activity in upper epidermal peels was variable (probably due to contamination), but the limited data suggest that the sucrose specific activity there reached somewhat higher values than those of the lower epidermis. The experiments indicate that each leaf tissue contains a kinetically identifiable sucrose pool (which we refer to as “histological compartmentation”), and that further compartmentation may occur at the intracellular level. A simulation of leaf sucrose compartmentation is presented.     

Root morphology and architecture respond to N addition in Pinus tabuliformis, west China

Belowground dynamics of terrestrial ecosystems are responding to global increases in anthropogenic N deposition with important consequences for productivity and ecosystem health. We compared root characteristics across five root orders in Pinus tabuliformis plantations treated for 3 years to a gradient of N addition (0–15 g m^?2 year^?1). In reference plots, the roots of P. tabuliformis were finer and with higher specific root length than reported for other pine species, suggesting severe N limitation. Addition of N resulted in slightly reduced fine root biomass and significant changes in root morphology, responses that were associated primarily with first and second order roots. In particular, root number, cumulative root length, individual root length, and specific root length all declined with increasing N addition for first and second order roots, with most of the responses elicited at <9 g m^?2 year^?1 N addition. These responses (1) support the concept of ephemeral root modules consisting of first and second orders and (2) are consistent with a change in functional demand from uptake to transport with increasing soil resource availability. Traditionally, fine roots have been identified by a somewhat arbitrary diameter cut-off (e.g., 1 or 2 mm); as an index of fine root function, diameter would fail to reveal most of the functional response.     

Biosynthesis of poly-β-hydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN)

The aim of this study was to characterize the influence of the aeration conditions on the production of PHB and its molecular mass in a mutant strain of Azotobacter vinelandii (OPN), which carries a mutation on ptsN, the gene encoding enzyme IIA^Ntr, previously shown to increase the accumulation of PHB. Cultures of A. vinelandii wild-type strain OP and its mutant derivative strain OPN were grown in 500-mL flasks, containing 100 and 200 mL of PY sucrose medium. PHB production and its molecular mass were analyzed at the end of the culture. The molecular mass (MM) was significantly influenced by the aeration conditions and strain used. A polymer with a higher molecular weight was produced under low aeration conditions for both strains. A maximal molecular mass of 2,026 kDa (equivalent to 3,670 kDa measured by GPC) was obtained with strain OPN cultured under low-aeration conditions, reaching a value two-fold higher than that obtained from the parental strain OP (MM?=?1,013 kDa) grown under the same conditions. Aeration conditions and the ptsN mutation influence the molecular mass of the PHB produced by A. vinelandii affecting in turn its physico-chemical properties.     

Insights into heterogeneous phosphodiester hydrolysis using a simple hydrogel-based copper(II)-imidazole catalyst

A family of copolymer hydrogels containing different mass percentages of vinylimidazole, acrylamide and N,N′-methylenebisacrylamide were used to bind copper(II) ions. The resultant copper-loaded gels demonstrated spectroscopic features that indicated copper was bound in a distorted square planar geometry. The hydrolysis activity of these the most active of these systems towards bis(3-nitrophenyl)phosphate at pH 8 was 2.78 × 10⁻⁶ s⁻¹, five orders of magnitude greater than the uncatalyzed reaction. While these systems obey Michaelis-Menten kinetics, they also subject both competitive and non-competitive inhibition from excess substrate and excess hydroxide due to constraints based in the coordination geometry of the copper(II) active sites.     

Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models

Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity (or antimutator) polymerase variant, C483Y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. To further investigate effects of position 483 on RNA-dependent RNA-polymerase (RdRp) fidelity, we substituted every amino acid at this position. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity.     

Interaction of fluoroaniline with cytochrome P-450d mutants: Difference absorption spectral studies

By site-directed mutagenesis near the axial ligand, Cys456, of cytochrome P-450_d (P-450_d), we obtained five stable P-450_d mutants, i.e. mutant (B), Gly450Ser; mutant (E), Lys453Glu; mutant (G), Arg455Gly; mutant (L), Glu459Ala and mutant (M), Ile460Ser. Bindings of aniline and fluoroaniline to the wild type P-450_d and these five P-450_d mutants were studied with difference absorption spectra at the Soret region. The following results were obtained: (i) binding constants (K_b) of fluoroaniline to wild P-450_d were higher than those to cytochrome P-450_scc by more than one order; (ii) K_b value of para-fluoroaniline to the wild P-450_d was higher than those of aniline, ortho- or meta-fluoroaniline by two orders; (iii) K_b values of para-fluoroaniline to five mutants were lower than that to the wild type by more than one order; (iv) K_b values of para-fluoroaniline to the mutants (E) and (G) were the lowest among those to the five mutants and were lower than that to the wild type by two orders; (v) K_b values of aniline, ortho- and meta-fluoroaniline to mutant (B) were one order higher than those to the wild and other four mutants. These results indicate that the binding of the external axial ligand to the heme iron of P-450_d is remarkably influenced by mutations at the proximal site possibly due to change in the polarity of the distal site and/or alteration of the secondary protein structure at the distal site.     

Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells

We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10^?8M and was found to be completely blocked by indomethacin. Adding back 10^?8M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.     

Influence of carbon source on growth,biochemical composition and pigmentation of Ankistrodesmus convolutus

The unicellular chlorophyte Ankistrodesmus convolutus Corda was grown in the light using inorganic medium (Bold's Basal Medium, BBM) and BBM enriched with 0.1% w/v of glucose, sodium acetate, sodium citrate or sodium bicarbonate. Glucose supported the highest specific growth rate (μ = 0.93 d^-1) and gave the highest biomass (453 mg dry weight L^-1) at the time of harvest. Of four glucose concentrations (0.05, 0.1, 0.25, 0.5% w/v), best growth was attained at 0.1% w/v. At 0.5% w/v glucose, the cells had high carbohydrates but low lipids and proteins. The relative amounts of 16:0, 18:0, 18:1 and 18:2 increased at the expense of 18:3(n-3) in the carbon-supplemented cultures and at glucose concentrations higher than 0.1% w/v. Cultures grown on glucose had less chlorophyll and carotenoid contents than cultures grown on other carbon sources. Chlorophyll and carotenoid contents decreased with increasing glucose concentrations in the medium.     

The Effect of Insulin on the Distribution of Glucose between the Blood Plasma and the Liver

In normal fasted rats whole liver tissue contains as much glucose as the blood plasma, i.e., the ratio of the concentrations is about unity. The concentration of glucose in hepatic intracellular water is about 1.2 times higher than in plasma water. In rats injected with insulin the concentration of glucose in the liver falls to a lesser extent than in the plasma: resulting in a ratio of concentrations higher than unity. If insulin hypoglycemia is prevented by the ingestion of glucose the concentration ratio is less than in hypoglycemic rats but still significantly above unity. In normal rats the specific activities of plasma and hepatic glucose do not differ significantly at 7.5, 15, and 30 minutes after the intravenous injection of C¹⁴-labeled glucose. In rats injected with insulin the specific activity of glucose is higher in the plasma than in the liver at 7.5 and 15 minutes, but not at 30 minutes following the injection of tracer. In insulin-treated hypoglycemic rats considerably higher concentrations of labeled glucose are found in hepatic intracellular than in plasma water. The penetration of C¹⁴-glucose from plasma into hepatic intracellular water is found to be fast. Excess insulin causes an accumulation of glucose within the liver cells by retaining newly formed glucose and by the taking up of glucose from the plasma against an existing concentration gradient.