A provider-based water planning and management model--WaterSim 4.0--for the Phoenix Metropolitan Area.

Uncertainty in future water supplies for the Phoenix Metropolitan Area (Phoenix) are exacerbated by the near certainty of increased, future water demands; water demand may increase eightfold or more by 2030 for some communities. We developed a provider-based water management and planning model for Phoenix termed WaterSim 4.0. The model combines a FORTRAN library with Microsoft C# to simulate the spatial and temporal dynamics of current and projected future water supply and demand as influenced by population demographics, climatic uncertainty, and groundwater availability. This paper describes model development and rationale. Water providers receive surface water, groundwater, or both depending on their portfolio. Runoff from two riverine systems supplies surface water to Phoenix while three alluvial layers that underlie the area provide groundwater. Water demand was estimated using two approaches. One approach used residential density, population projections, water duties, and acreage. A second approach used per capita water consumption and separate population growth estimates. Simulated estimates of initial groundwater for each provider were obtained as outputs from the Arizona Department of Water Resources (ADWR) Salt River Valley groundwater flow model (GFM). We compared simulated estimates of water storage with empirical estimates for modeled reservoirs as a test of model performance. In simulations we modified runoff by 80%-110% of the historical estimates, in 5% intervals, to examine provider-specific responses to altered surface water availability for 33 large water providers over a 25-year period (2010-2035). Two metrics were used to differentiate their response: (1) we examined groundwater reliance (GWR; that proportion of a providers' portfolio dependent upon groundwater) from the runoff sensitivity analysis, and (2) we used 100% of the historical runoff simulations to examine the cumulative groundwater withdrawals for each provider. Four groups of water providers were identified, and discussed. Water portfolios most reliant on Colorado River water may be most sensitive to potential reductions in surface water supplies. Groundwater depletions were greatest for communities who were either 100% dependent upon groundwater (urban periphery), or nearly so, coupled with high water demand projections. On-going model development includes linking WaterSim 4.0 to the GFM in order to more precisely model provider-specific estimates of groundwater, and provider-based policy options that will enable "what-if" scenarios to examine policy trade-offs and long-term sustainability of water portfolios.

Suppression of cyclooxygenase-2 and inducible nitric oxide synthase expression by conjugated linoleic acid in murine macrophages.

Activated macrophages express inducible isoforms of cyclooxygenase (COX-2) and nitric oxide synthase (iNOS), and produce excessive amounts of prostaglandin E(2) (PGE(2)) and nitric oxide (NO) which play key roles in cancer pathogenesis. Conjugated linoleic acid (CLA) is an anticarcinogen while arachidonic acid (AA) may be a procarcinogen by increased PGE(2) production. This study examined the effects of CLA and AA on PGE(2) and NO synthesis in endotoxin-activated macrophages. RAW264.7 macrophages were incubated in medium containing no added lipid (control), 30 microM AA (AA medium), or 30 microM CLA (CLA medium) for 24 h followed by activation with bacterial endotoxin lipopolysaccharide (LPS; 100 ng/ml) for 9 h. CLA significantly depressed PGE(2) and NO production by 78% (P=0.003) and 57% (P=0.0001) respectively. Northern blot analysis of COX-2 and iNOS showed significant 33% (P=0.01) and 51% (P=0.04) decreases, respectively, paralleling those seen for PGE(2) and NO production. In contrast, AA significantly increased PGE(2) synthesis by 62% (P=0.02) and also suppressed NO production and iNOS expression in the same manner as observed for CLA. These results suggest that the anticarcinogenic effect of CLA in endotoxin-activated macrophages may be related to its ability to decrease both PGE(2) and NO synthesis by suppressing transcription of COX-2 and iNOS.

The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification.

SUMO-1 is an ubiquitin-related protein that is covalently conjugated to a diverse assortment of proteins. The consequences of SUMO-1 modification include the regulation of protein-protein interactions, protein-DNA interactions, and protein subcellular localization. At present, very little is understood about the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that are modified by SUMO-1 interact directly with the SUMO-1 conjugating enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an important role in substrate recognition as well as in substrate modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of conserved residues surrounding the modified lysine residue of most SUMO-1 substrates. This motif conforms to the sequence "PsiKXE," where Psi is a large hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9 binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how SUMO-1 is ultimately transferred to specific lysine residues on these substrates.

Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein.

Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.

Vitamin B-6 deficiency and level of dietary protein affect hepatic tyrosine aminotransferase activity in cats.

Total activity [pyridoxal 5'-phosphate (PLP) added in the assay] of hepatic tyrosine aminotransferase (TAT) measured in cats at 0300, 0900, 1500 and 2100h was 10.3 +/-1.1, 14.0 +/- 0.7, 9.8 +/- 1.3 and 11.0 +/- 0.7 nkat/g liver, indicating little diurnal variation. Activity after 18 h of food deprivation was 10.0 +/- 0.3 nkat/g liver, also not different from cats that were eating ad libitum. These findings support the idea that cats have only limited changes in the activity of hepatic TAT compared with rats. Total TAT activity was measured in cats fed high protein (550 g/kg) and low protein (180 g/kg) diets for 4 wk. Cats fed a high protein diet had activities significantly higher (about twice) than cats fed the low protein diet. Hepatic TAT activity of vitamin B-6-deficient cats (diet without pyridoxine for 9 wk) was compared with cats given the same diet with 8 mg pyridoxine/kg. Total hepatic TAT activity in deficient cats was significantly (P < 0.05) lower per gram soluble or total protein (but not per gram liver) than control cats; holoenzyme activity and percentage of active enzyme in deficient cats were also significantly lower by 75 and 64%, respectively. The apparent Km of TAT from cats for tyrosine (2.1 mmol/L) was similar to that for rats (1.9 mmol/L), but higher for PLP in cats (0.16 micromol/L) than rats (0.034 micromol/L). Part of the reason for the higher plasma tyrosine in vitamin B-6-deficient cats than rats is the higher Km of TAT for PLP in cats than rats.

Dietary protein quality alters ornithine decarboxylase activity but not vitamin B-6 nutritional status in rats.

Weanling male rats were fed diets that varied in protein quality (casein or wheat gluten) and vitamin B-6 (0.0, 0.5, 1.0 and 1.5 mg pyridoxine HCl/kg diet) to test the hypotheses that low protein quality would depress vitamin B-6 nutritional status and that activity of ornithine decarboxylase (ODC) would be a sensitive functional indicator of vitamin B-6 nutritional status. The wheat gluten diet depressed body weight gain approximately 17% at higher vitamin B-6 levels, as expected. However, vitamin B-6 nutritional status was not worse in gluten-fed compared with casein-fed groups, as evidenced by static measures (B-6 vitamer concentrations in plasma and tissues) and a functional indicator (tryptophan load test). The activity of ODC (holo- and total) in liver, kidney and small intestine did not vary significantly at the three higher levels of vitamin B-6 intake. In groups fed casein, total ODC activity in these tissues was two- to fivefold higher in rats fed diets containing 0.0 mg vitamin B-6/kg compared with higher B-6 levels, without corresponding differences in ODC mRNA abundance in liver and kidney. Concentrations of B-6 vitamers (except pyridoxal phosphate in plasma) increased linearly with dietary vitamin B-6 in plasma, liver, kidney and intestine. These data suggest that low quality protein fed as wheat gluten suppresses growth but not vitamin B-6 nutritional status, and that ODC activity is not a sensitive functional indicator of marginal vitamin B-6 status.

Exercise-induced changes in plasma vitamin B-6 concentrations do not vary with exercise intensity.

This study examined the effect of high vs moderate exercise intensity on changes in concentration of plasma pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (4-PA) in human subjects. Eight physically active subjects were tested twice at 60% and 85% maximum oxygen consumption (VO2max) for 30 and 20 min, respectively, on a bicycle ergometer. Blood samples were obtained before and during exercise. Data were adjusted for changes in plasma volume calculated from changes in hematocrit. PLP concentrations significantly increased during exercise (P < 0.0001), with 79% of the rise in PLP concentration occurring within 5 min. 4-PA concentration increased steadily through exercise and was 23% higher at 20 min than at 0 min. Exercise intensity had no effect on the magnitude or rate of either increase for either PLP or 4-PA. PL concentration did not vary with exercise duration, but was significantly higher at 60% compared with 85% VO2max (P = 0.001). No significant differences were observed in glucose concentration. The data do not support hypotheses in the literature that PLP concentration rises during exercise to support exercise-induced shifts in substrate utilization.

Insensitivity of the tryptophan-load test to marginal vitamin B-6 intake in rats.

The tryptophan-load test for vitamin B-6 nutritional status was administered to adult female Long-Evans rats fed graded levels of pyridoxine hydrochloride (PN.HCl) in two experiments, and its sensitivity to marginal vitamin B-6 intake was evaluated. In Experiment 1, rats were 4-h meal-fed an AIN-76A (20% casein) diet devoid of PN.HCl for 3 wk, then repleted (n = 12) for 6 wk with 4-h pair-fed meals of either 0.25, 0.5, 1.0 or 7.0 (control) mg PN.HCl/kg diet. In Experiment 2, rats (n = 16) were pair-fed for 10 wk either 0.0, 0.5, 1.0 or 7.0 (control) mg PN.HCl/kg diet, with 24-h access to food. Vitamin B-6 nutritional status was assessed at the end of each experiment. Except in rats fed 0 mg PN.HCl/kg diet, mean body weights were not significantly different among diet groups of either experiment. Plasma pyridoxal phosphate (PLP), pyridoxal and total vitamin B-6 concentrations, determined by HPLC, were very sensitive to gradations in dietary PN.HCl concentrations (P less than 0.05). Red blood cell endogenous and PLP-stimulated alanine and aspartate aminotransferase activity did not statistically differentiate all levels of dietary vitamin B-6, although the calculated activity coefficient for each enzyme (stimulated/endogenous activity) did. Urinary xanthurenic acid excretion following a tryptophan load [24.5 mumol (5 mg) L-tryptophan/100 g body weight, injected intraperitoneally] was significantly (P less than 0.05) elevated compared with controls only in the group fed 0 mg PN/HCl/kg diet. At the tryptophan dose used here, the tryptophan-load test was not useful in detecting marginal vitamin B-6 intake in rats.

The level of dietary protein affects the vitamin B-6 requirement of cats.

A depletion-repletion model was used to study the effect of the level of dietary protein on the vitamin B-6 requirement of growing kittens. Twenty kittens were given a pyridoxine-free diet for 42 d to deplete vitamin B-6 reserves. They then were divided into four groups of five kittens each. Two groups were fed diets containing 30% casein with either 1.0 (Group 30-1) or 2.0 (Group 30-2) mg pyridoxine/kg diet and two groups were fed 60% casein diets with either 1.0 (Group 60-1) or 2.0 (Group 60-2) mg pyridoxine/kg diet for 44 d. During repletion, body weight gain of kittens from Group 30-2 was higher than that of kittens from the other groups. Body weight gains of kittens from Groups 30-1 and 60-2 were higher than that of kittens from Group 60-1, but there was no difference in body weight gain between Groups 30-1 and 60-2. At 44 d of repletion, kittens from Group 60-1 generally had higher plasma concentrations of free tyrosine and cystathionine, lower plasma B-6 vitamin concentrations and a higher urinary oxalate excretion than did kittens from the other groups. These findings indicate that the vitamin B-6 requirement of growing kittens--as is true of humans, chickens and mice--is positively related to the level of protein in the diet. For a 30% casein diet, the vitamin B-6 requirement was greater than or equal to 1.0 but less than 2.0 mg pyridoxine/kg diet; for a 60% casein diet, the requirement was greater than or equal to 2.0 mg pyridoxine/kg diet.

Response of B-6 vitamers in plasma, erythrocytes and tissues to vitamin B-6 depletion and repletion in the rat.

We determined the response patterns of B-6 vitamers in blood and tissues to vitamin B-6 depletion and repletion. B-6 vitamers were measured in plasma, erythrocytes, liver, muscle, kidney, heart, brain, spleen and lung by reverse-phase high performance liquid chromatography in male rats pair-fed control or vitamin B-6-deficient diets for 2 or 4 wk, or for 4 wk followed by 1 wk of repletion with the control diet (n = 4/group). Food intake (15.6 +/- 0.3 g/d, mean +/- SEM; n = 28) and body weight (190 +/- 2 and 290 +/- 5 g at wk 0 and 5, respectively; n = 28) of control groups were not different from those of deficient groups throughout the study. After 2 wk of vitamin B-6 depletion, tissue concentrations of pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) were about 50% and 10-40% lower, respectively, in the deficient than in the control group (except for spleen PMP); in plasma and erythrocytes, PLP and pyridoxal concentrations were about 90% lower in the deficient group. Differences in vitamer concentrations between control and deficient groups were not larger after 4 wk of depletion than after 2 wk. Vitamer concentrations in plasma, erythrocytes and all tissues returned to control levels after 1 wk of repletion with the control diet. These results demonstrate that B-6 vitamers in blood and tissues of the rat respond quickly and reversibly to changes in dietary vitamin B-6, with larger percentage changes occurring in plasma and erythrocytes than in tissues.

Evaluation of vitamin B-6 status and function of rats fed excess pyridoxine.

We compared the vitamin B-6 status of 12-wk-old rats (n = 12) fed excess (1400 mg/kg diet) or the recommended level (7 mg/kg diet, control) of pyridoxine (PN) hydrochloride to test if excess vitamin B-6 would cause tissue depletion of pyridoxal phosphate (PLP), the active coenzyme form of vitamin B-6. Plasma PLP, tryptophan-load test results, food intake, and tissue and body weights were not different at wk 6. Red blood cell endogenous alanine aminotransferase activity and PLP concentration were elevated (P less than 0.01) in rats fed 1400 mg PN.HCl/kg diet. In contrast, PLP concentration in muscle was significantly lower (P = 0.01) in rats fed excess vitamin B-6 (9.7 +/- 0.8 nmol/g, mean +/- SEM) than in controls (14.9 +/- 1.4). PLP concentration in other tissues, including plasma, was not affected. In rats fed excess vitamin B-6, pyridoxal was increased in all tissues examined (P less than 0.05), and total vitamin B-6 was increased in plasma, red blood cells and kidneys (P less than 0.05). Total glycogen phosphorylase (a + b) activity in the gastrocnemius was not affected, but phosphorylase a activity was increased in rats fed excess vitamin B-6 (P = 0.025). Concentrations of dopamine and metabolites in the caudate nucleus of the basal ganglia were not affected. A transient, but significant, elevation in acoustic startle response, a central nervous system reflex, was observed in rats fed excess vitamin B-6. The depletion in muscle PLP could not hae been predicted by either plasma or red blood cell PLP concentration, although the latter did reflect vitamin B-6 intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin B-6 requirement of growing kittens.

Two experiments were conducted to determine the vitamin B-6 requirement for growing kittens. Ten kittens were divided into two groups and given a purified diet containing 8.0 mg pyridoxine (PN)/kg diet (+PN) or a PN-free diet (-PN) for 11 wk. Daily body weight gain, food intake, weekly plasma free amino acids, plasma B-6 vitamers, urinary oxalate excretion, hemoglobin (Hb) and hematocrit were measured. Kittens fed a -PN diet had depressed body weight gain, food intake, plasma pyridoxal phosphate (PLP) and pyridoxal (PL), Hb and hematocrit, and had elevated urinary oxalate, plasma tyrosine and plasma cystathionine. In a second experiment, 24 kittens were given a -PN diet for 45 d to deplete their body reserves. The kittens were then divided into six groups of four kittens each and given a purified diet containing either 0.5, 1.0, 2.0, 3.0, 4.0 or 8.0 mg PN/kg diet for 46 d. Following supplementation, positive responses in body weight gain, PLP, Hb and hematocrit, and decreased urinary oxalate excretion, plasma tyrosine and plasma cystathionine occurred in all groups except those fed 0.5 mg PN/kg diet. At the end of the repletion period, kittens fed 1.0 mg PN/kg diet had lower body weight gain, higher plasma tyrosine and cystathionine, slower rate of decrease in urinary oxalate, and lower values for Hb, hematocrit and PLP than did the kittens from groups fed 2.0-8.0 mg PN/kg diet. These findings indicate that the dietary requirement for PN is greater than 1.0 mg/kg diet, but 2.0 mg PN/kg diet is adequate for growing kittens given a 35% casein diet.

B6 vitamer content of rat tissues measured by isotope tracer and chromatographic methods.

Feeding [14C]pyridoxine to growing rats for 146 days produced uniform labelling of the total vitamin B6 pool, thus permitting the radioactivity to be used as an absolute standard for evaluating the accuracy of vitamin B6 analyses. The results demonstrated that trichloroacetic acid extraction followed by cation exchange chromatography accurately measures the B6 vitamers. It is essential to homogenize tissues in a protein-denaturing agent in order to avoid shifts in the vitamer content, particularly in liver. In rats approximately 80% of the radioactivity was found in carcass and 8-9% each in liver and skin. Pyridoxamine phosphate equalled or exceeded the concentration of pyridoxal phosphate in heart, brain and kidney. The total vitamin B6 pool in weanling and adult rats averaged about 16 nmol/g body wt. Meta-phosphoric acid extraction followed by reverse phase chromatography gave good agreement with the cation exchange method in rat liver but with cat plasma yielded pyridoxal phosphate values below those of the cation exchange or enzymatic methods. The discrepancies encountered between different homogenization techniques and chromatographic methods emphasize the need for constant vigilance and continual verification of results by independent methods.

Dietary protein quality, protein quantity and food intake: effects on lactation and on protein synthesis and tissue composition in mammary tissue and liver in rats.

Rate of protein synthesis in mammary tissue and liver as well as pup weight and milk yield were measured at d 15 of lactation in rats fed throughout gestation and lactation diets that varied in protein quality (wheat, wheat supplemented with lysine and threonine or casein supplemented with methionine), protein quantity [11 or 21% (wt/wt)] and feeding level (ad libitum or pair-fed to 100 or 85% of the consumption of dams fed wheat ad libitum). Improvement in dietary protein quality or quantity at the ad libitum feeding level produced large increases in mammary protein synthesis and pup weight; pair-feeding at 100 or 85% of the ad libitum wheat consumption level reduced or eliminated these increases. Increases in protein synthesis and pup weight associated with protein quality improvement were of similar magnitude to those associated with nearly doubling protein content of maternal diets. The responses of liver protein synthesis to dietary manipulation were qualitatively similar to those seen in mammary tissue. These data demonstrate that maternal dietary protein quality, protein quantity and feeding level all affect mammary protein synthesis and pup weight, and that, considered across all diets, changes in mammary protein synthesis are highly correlated with changes in pup weight and protein nutritional value.

Free amino acid levels during lactation in rats: effects of protein quality and protein quantity.

Free amino acid levels were determined in serum, mammary gland, liver and muscle during lactation in rats fed diets varying in protein. Using a 3 X 2 factorial design, after conception female rats were fed purified diets varying in protein quality (wheat, wheat + lysine + threonine or casein + methionine) and in protein level (11.6% vs. 22.3%). On d 15 of lactation dams were decapitated; blood was collected, and the serum was quickly frozen. Samples of mammary gland, liver and calf muscle were also removed and quickly frozen. After deproteinization, free amino acids were determined on an LKB 4400 Amino Acid Analyzer. As the protein nutritional value of the diet increased, milk yield increased threefold and the level of free lysine in the mammary gland increased six- to eightfold. With protein quality or quantity improvement, the level of free methionine increased two- to threefold, and no consistent changes were seen in the levels of free threonine, aromatic and branched chain amino acids in mammary glands. Free lysine concentrations in mammary gland, liver and muscle were highly correlated with free lysine in serum (r2 = 0.94, 0.96, 0.93, respectively). Under the conditions of this experiment, a direct relationship between dietary lysine, free lysine in the mammary gland and milk secretion was observed.

The effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat.

Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of [3-3H]phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis.

Protein synthesis in rat tissues during lactation. No effect of diethyl ether anaesthesia.

Rates of protein synthesis were measured in mammary gland, liver, intestinal mucosa and muscle of lactating rats by using a flooding dose of [3H]phenylalanine that was injected into diethyl ether-anaesthetized dams via a lateral vein or into undisturbed dams via a jugular cannula. Ether anaesthesia did not alter rates of protein synthesis significantly in any tissue.

Measurement of milk yield in the lactating rat from pup weight and weight gain.

A new method was developed that enables milk yield to be estimated from pup weight and pup weight gain in well-nourished rats. Daily milk yield, body weight, and daily weight gain were measured over 4-5-day periods in 38 suckling pups aged 3-13 days postpartum. Each 24-h period was divided into consecutive 6-h cycles, during which dams and pups were separated for 4 h and then reunited for 2-h suckling intervals. Daily milk yield per pup was calculated from pup weight gain during the four suckling intervals for each 24-h period, and corrected for insensible weight loss of the pups. The following equation, relating pup milk yield to pup weight and weight gain, was obtained by stepwise forward multiple regression analysis: yield = 0.0322 + 0.0667 (weight) + 0.877 (gain); where yield is daily yield per pup (g/pup/day), weight is pup weight (g), and gain is pup weight gain per day (g/day) (n = 174; F = 552.3; p less than 0.001; r2 = 0.866). Per litter milk yield can be obtained by multiplying per pup yield by litter size. Using this equation, dams fed a stock diet and nursing eight pups were estimated to have produced 29.5 ml of milk on day 10 of lactation. This value is similar to yields measured by more laborious methods. SEs of yield estimates calculated with this method represent 1-3% of predicted yield values. The new method gives useful estimates of rat milk yield during the first 2 weeks of lactation from measurements that are more easily obtained than is the case with other methods.

Protein synthesis during lactation: no circadian variation in mammary gland and liver of rats fed diets varying in protein quality and level of intake.

Circadian changes in protein synthesis were studied in lactating rats fed diets varying in protein quality and feeding level. At parturition, rats previously fed a stock diet were assigned to semipurified diets supplying 23% protein. Two groups were fed wheat gluten (WG) or casein (C) ad libitum; a third group was pair-fed casein to consumption of the WG group (C-PF). At four different times on day 15 of lactation, protein synthesis was measured in vivo in mammary gland, liver and calf muscle with a large dose of L-[4-3H]phenylalanine. Pup weights at day 15 for dams fed WG, C and C-PF were 19.3 +/- 0.3, 33.9 +/- 0.5 and 27.2 +/- 0.5 g, respectively. No significant circadian variation in fractional synthesis rate (FSR) was observed in either mammary gland or liver. Higher variability in muscle FSRs precludes conclusions in regard to this tissue. However, protein quality and level of feeding both affected FSR. For rats fed WG, C and C-PF, FSRs were, respectively: 77 +/- 4, 116 +/- 6 and 64 +/- 3%/day in mammary gland; 67 +/- 4, 78 +/- 5 and 74 +/- 4%/day in liver; and 3.4 +/- 0.5, 6.7 +/- 1.0 and 4.0 +/- 0.8%/day in muscle. Absolute synthesis rates were, respectively: 584 +/- 60, 1743 +/- 148 and 663 +/- 58 mg/day in mammary gland; and 469 +/- 46, 970 +/- 72 and 702 +/- 62 mg/day in liver. The results confirm that dietary protein quality and feeding level affect protein synthesis rates in lactating rats and demonstrate that there is no circadian variation in protein synthesis in mammary gland and liver of lactating rats.

A method for isolation of undegraded free and membrane-bound ribosomes from rat lactating mammary gland.

Mammary gland polysomes are difficult to isolate from the lactating rat using methods developed for other species and tissues, most likely due to high calcium-stimulated ribonuclease activity in that tissue. A new method, utilizing ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) to bind calcium, yields highly aggregated polysomes from lactating rat mammary gland. Fresh mammary tissue is pulverized under liquid nitrogen. Free and membrane-bound polysomes are isolated by differential centrifugation in solutions containing 100 mM KCl, 100 mM MgCl2, 75 mM EGTA, 500 micrograms/ml heparin and 50 mM Tris buffer, pH 8.2 at 5 degrees C. Bound polysomes are released from the endoplasmic reticulum using Triton X-100 and deoxycholate. Polysome profiles are obtained on linear sucrose gradients and scanned at 254 nm. The method gives quantitative recovery of homogenate total RNA. To demonstrate that the method can be used to study nutritional effects on mammary gland polysome aggregation, lactating rats were fasted 22-66 h and then refed a stock diet for 71-95 h. Refeeding increased the percentage of polysomes (trimers or larger) in the bound fraction from 84 +/- 1 to 93 +/- 1% (P less than 0.001) and in the free fraction from 42 +/- 2 to 55 +/- 3% (P less than 0.001).