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The energy requirements of healthy elderly men were investigated by measuring total energy expenditure (TEE), body composition and resting energy expenditure (REE), using 2H2(18)O techniques and indirect calorimetry during ten days when a weight maintaining diet was consumed. Values for TEE/REE (1.75 +/- 0.05, s.e.) were significantly higher than the recommended dietary allowances (RDAs) for energy in this age group, as were previously reported values for TEE/REE in a group of young men in the same experimental protocol. There was a significant negative association between TEE/REE (a physical activity indicator) and body fat mass. Combining data from the elderly men in this study and the young men studied previously, age and TEE/REE together accounted for 73% of the variation in body fat mass between individuals. These results lend additional weight to the suggestion that the current RDAs for energy may significantly under-estimate usual energy requirements. They also indicate that the low levels of energy expenditure suggested by the RDAs may favour unnecessarily high levels of body fat mass and that increased levels of energy expenditure are desirable.

Oct-1 is a transcription factor involved in the cell cycle regulation of histone H2B gene transcription and in the transcription of other cellular housekeeping genes. Oct-1 is hyperphosphorylated as cells enter mitosis, and mitosis-specific phosphorylation is reversed as cells exit mitosis. A mitosis-specific phosphorylation site in the homeodomain of Oct-1 was phosphorylated in vitro by protein kinase A. Phosphorylation of this site correlated with inhibition of Oct-1 DNA binding activity in vivo and in vitro. The inhibition of Oct-1 DNA binding during mitosis suggests a mechanism by which the general inhibition of transcription during mitosis might occur.

A new method for assessing the accuracy of dietary nutrient intakes in metabolic studies is described. The theoretical basis of the method is the comparison of measurements of the urine osmole excretion rate (OER, the product of urine osmolality and urine weight) with values for OER predicted from dietary nitrogen, sodium, and potassium. The method was tested in 34 healthy male and female volunteers aged 18-78 y who made complete 24-h urine collections and consumed a diet over 6 d in metabolic-balance studies involving either overfeeding, weight maintenance, or weight loss. The coefficients of variation for equations relating measured OER to dietary nitrogen, sodium, and potassium intakes ranged from 14.1% for 1-d measurements to 6.94% for 6-d means. These results indicate that it should be possible to use measurements of the urine OER to identify dietary noncompliance in metabolic studies.

Orderly progression through the somatic cell division cycle is accompanied by phase-specific transcription of a variety of different genes. During S phase, transcription of mammalian histone H2B genes requires a specific promoter element and its cognate transcription factor Oct1 (OTF1). A possible mechanism for regulating histone H2B transcription during the cell cycle is direct modulation of Oct1 activity by phase-specific posttranslational modifications. Analysis of Oct1 during progression through the cell cycle revealed a complex temporal program of phosphorylation. A p34cdc2-related protein kinase that is active during mitosis may be responsible for one mitotic phosphorylation of Oct1. However, the temporally controlled appearance of Oct1 phosphopeptides suggests the involvement of multiple kinases and phosphatases. These results support the idea that cell cycle-regulated transcription factors may be direct substrates for phase-specific regulatory enzymes.

The double labeled water technique for determination of the rate of total energy expenditure in human subjects is based on measurement of the differential rates of disappearance of 2H and 18O from body water following oral administration of 2H218O. Two calculation procedures, the "two-point" and "slope-intercept" methods of calculation have previously been validated for use in humans. We describe here a computer program. DLW, that provides options to calculate doubly labeled water data in each of these accepted ways.

Study of the effects of enteral nutrition on neonatal growth and development has been hindered by lack of a suitable model. An isotope dilution method for the measurement of milk intake in suckling rodents was evaluated in 13 hand-fed neonatal guinea pigs, by comparing milk intakes determined using 3H20 with intakes determined by difference in weight before and after feeding. The effects of two experimental durations (1 or 2-3 days) on the accuracy and precision of the method was assessed. Weighted milk intakes were 18 -26 g/day, and body weight changes were -7% - +12% of initial weight. There was no significant difference between milk intakes determined using the two methods for both 1 and 2-3 day comparisons. However, the coefficients of variation for 1 and 2-3 day measurements were 24.4% and 5.5%, respectively. These values are equivalent to precisions of approximately 10% and 2% for estimates of total milk output in a rodent suckling 8 pups. It is concluded that the 3H20 method is no less accurate or precise than the weighing method for 2-3 day measurements of milk intake in individual pups and milk production of dams. These findings strengthen the use of the precocial guinea pig as a model for the study of the effects of early feeding on growth, development and aging.

A new method is described for simultaneously monitoring compliance with prescribed-diet and urine-collection regimens during metabolic balance studies. It involves incorporation of the potassium salt of p-aminobenzoic acid (PABA) into provided foods and measurement of its extent of recovery in urine. A validation study was conducted in 10 healthy adults. During three consecutive 24-h periods, subjects consumed four foods per day that had each been supplemented with 72.0 mg PABA and also made complete urine collections. An additional 24-h urine collection was made by each subject for determination of baseline PABA excretion. The PABA was easily incorporated into the test foods and was not detected by taste. The recovery of the PABA in urine, determined by colorimetric assay, was 98.7 +/- 3.7% (means +/- SD) of intake. Thus, in metabolic studies for greater than or equal to 3 d, PABA can be used to provide a sensitive index of dietary compliance.

A dietary supplement, resulting in a mean net increase in energy intake of over 700 kcal/day, was supplied to all nursing mothers in a West African village for 12 months. The supplement produced a slight initial improvement in maternal body weight and subcutaneous fat stores but did not increase breast-milk output or fat content.

A comparison has been made of protein accretion and growth in premature infants fed banked drip breast milk (BBM) or a preterm formula (PF). Protein accretion was calculated from the difference between dietary nitrogen intake and output in urine and stools, measured in a nutrient balance study. As expected, only the infants fed PF achieved the intrauterine growth rate. However, whilst weight gain in infants fed BBM was 71% of that in the group fed PF, the rate of protein deposition was less than 50%. As a result, the protein concentration in new tissue of infants fed BBM averaged 32% less than in those fed PF and 21% less than the lower end of the range reported to occur in utero. We speculate that infants fed BBM have an abnormally low protein concentration in their lean body mass.

The possibility that lactation increases the efficiency of energy use was investigated by comparing energy expenditure on activity and maintenance in virgin and lactating rats fed either ad libitum or restricted amounts of food. Energy expenditure was determined, by difference, in a nutrient balance study. Lactating rats fed ad libitum expended 34.9% less energy on activity and maintenance than did control animals. Of this change, 82% was specifically associated with lactation, but the remaining 18% was associated with the negative energy balance that occurred during lactation. Lactating rats fed restricted amounts of food expended less energy on activity and maintenance than did virgin rats fed at the same level of restriction, but this change was associated with increased negative energy balance and not specifically with lactation. These results indicate that the efficiency of energy use is increased substantially during lactation in the rat, regardless of the level of energy intake, and that mechanisms associated with negative energy balance play an important role in the change.

We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.

The role of cAMP and cGMP in triggering proliferation of rat myoblasts was evaluated by: (1) measuring effects of mitogens on intracellular cyclic nucleotide levels, and (2) observing effects of agents which altered cyclic nucleotide levels on cell proliferation. Multiplication stimulating activity (MSA, 1 microgram/ml), a member of the somatomedin family, stimulated cell proliferation after 48 hr. It had little effect on cellular cyclic nucleotide levels, measured by radioimmunoassay. Horse serum (HS) and fetal bovine serum (FBS) stimulated cell proliferation approximately equally. Neither affected cAMP levels; FBS reduced cGMP to 33% of control values, but HS had no effect. Thus, there was no simple correlation between mitogenic action and cyclic nucleotide levels at any time from 5 min to 24 hr after addition of a purified mitogen or serum. Furthermore, agents which caused substantial changes in cyclic nucleotide levels had no effect on cell proliferation. Prostaglandin E1 (5 microM) elevated cAMP 440% without affecting cGMP levels or cell growth. A potent phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX, 0.1 mM), when added alone or in the presence of MSA, HS, or FBS, elevated cAMP 200% and cGMP 167%, but it had little effect on their mitogenic action. Lastly, a purified mitogen such as MSA, unlike serum, must be present for extended periods of time in order to stimulate cell proliferation. This makes it unlikely that a trigger mechanism functions in initiating cell division. We conclude that neither cAMP nor cGMP appear to be second messengers for the mitogenic action of MSA or serum on muscle cells.

We investigated the contributions of low energy expenditure and high energy intake to excessive weight gain in infants born to overweight mothers. The subjects were infants of 6 lean and 12 overweight mothers, recruited soon after birth. Total energy expenditure and metabolizable energy intake were measured with a new doubly labeled water method over a period of seven days when the infants were 3 months of age, and the postprandial metabolic rate was measured by indirect calorimetry when the infants were 0.1 and 3 months of age. The results were related to weight gain in the first year of life. No significant difference was observed between infants who became overweight by the age of one year (50 percent of infants born to overweight mothers) and those who did not, with respect to weight, length, skinfold thicknesses, metabolic rate at 0.1 and 3 months of age, and metabolizable energy intake at 3 months. However, total energy expenditure at three months of age was 20.7 percent lower in the infants who became overweight than in the other infants (means +/- SE, 256 +/- 27 and 323 +/- 12 kJ per kilogram of body weight per day; P less than 0.05). This difference could account for the mean difference in weight gain. These data suggest that reduced energy expenditure, particularly on physical activity, was an important factor in the rapid weight gain during the first year of life in infants born to overweight mothers.

A new method is described for measuring the rate of carbon dioxide production, and hence for estimating energy expenditure, in preterm infants receiving assisted ventilation. In a validation study, the mean error in carbon dioxide measurement was 1.9%. Measurements were made, over a 45-min period, on 11 sick, ventilated subjects and carbon dioxide production rate was 5.2 +/- 0.7 (SD) ml/min X kg body weight. We suggest that continuous monitoring of carbon dioxide output will contribute to the clinical assessment of the effects of different ventilator settings on pulmonary gas exchange and that estimated values for energy expenditure will be of value in nutritional studies on sick ventilated infants.

The energy costs of depositing fat and protein in the low-birth-weight infant were determined by multiple-regression analysis from published information on metabolizable-energy intake and fat and protein deposition in groups of subjects fed different dietary regimes. The total energy requirement for deposition was 1.17 kJ/kJ deposited for fat (ie, 1 kJ deposited and 0.17 kJ expended for deposition, and 2.38 kJ/kJ for protein. These values are similar to published determinations for animal species with simple stomachs. The metabolizable-energy requirement for weight gain during infancy was calculated (range, 12.2-25.1 kJ/g, or 2.9-6.0 kcal/g; means, 18.7 kJ/g, or 4.5 kcal/g) from the energy costs of fat and protein deposition and published information on changes in body composition during the first year of life.

An investigation was made of long-term variation in oxygen consumption rate (VO2) in preterm infants. Four subjects (gestational age 27-34 weeks, postnatal age 17-38 days, weight at study 1.1-2.6 kg) were studied for 5 days each using open-circuit, indirect calorimetry. The mean VO2 for each subject (11.0-11.5 litres/kg/day) was within the reported range. However, the between-subject coefficient of variation during the study (2.1%) was smaller than the mean between-measurement coefficient of variation for daily VO2 (3.8%, range 1.7-6.3%). In addition, the between-measurement coefficient of variation was increased further for measurement intervals of less than 24 h (reaching a mean of 8.3% for 1-hour periods), and a relationship between measurement duration and the precision of estimating VO2 over 3 or 5 days is described. Thus, even 24-hour measurements of VO2 in these preterm infants were less representative of the individual's VO2 over 3 days than the group mean estimate. This finding is of relevance to future studies in this area, particularly those in which short-term measurements of energy expenditure are combined with a nutrient balance study to determine the composition of weight gain, because even small errors in the estimate of total energy expenditure can lead to unacceptably large errors in calculated energy deposition.

The effects of dietary supplementation on milk output and maternal body composition were investigated in the lactating rat. The supplement was a cooked homogeneous mixture of eggs and maize oil, and had the same protein-energy: total energy value as the control diet. During 2-12 d post partum rats were fed ad lib., either on the control diet alone or on the control diet plus the supplement. Measurements were made of milk output using an isotope-dilution technique, milk composition, and dam and litter body-composition changes. Compared with the dams receiving only the control diet, dams provided with the supplement consumed 19.7% more energy and protein and produced 31.2% more milk and mobilized less body fat. By 12 d of age, pups in litters of dams receiving the supplement were significantly heavier than those in litters of dams receiving the control diet only, and they contained more protein and more fat. It is concluded that dietary supplementation of lactating rats can enhance lactational performance.

In order to test whether lactational capacity can be improved by dietary interventions, a nutritionally balanced supplement was provided under carefully controlled conditions to 130 nursing mothers in Keneba, The Gambia over 12 months. Maternal mean energy intake (+/- s.e.) increased from 1568 +/- 15 kcal/d (6.56 +/- 0.06 MJ/d) to 2291 +/- 14 kcal/d (9.59 +/- 0.06 MJ/d). Protein intake was in excess of the WHO/FAO recommended intake after supplementation and serious deficits of riboflavin, vitamin A, vitamin C and calcium were rectified. The supplement had no effect on breast-milk volume, compared with retrospective controls, at any stage of lactation or in any season of the year. There was no selective effect on women with poor milk outputs. The average milk protein concentration was slightly improved over the entire period of lactation (+ 6.6 per cent, P less than 0.01), but the total energy content was unchanged since an increase in breast-milk fat concentration (+ 7.9 per cent, n.s.) was offset by a decrease in the milk lactose concentration (- 7.6 per cent, P less than 0.01). Breast milk vitamin content was improved for those vitamins for which the supplement provided a significant proportion of the recommended dietary intake.

The relationship between energy intake and lactational performance in the baboon was investigated by determining energy intake, milk output and energy balance in animals fed ad libitum during a nonreproductive phase and during lactation, or fed 80% or 60% of ad libitum intake during lactation. No evidence was found for an increased efficiency of energy utilization during lactation when feeding was ad libitum, as judged by calculated energy used on activity and maintenance. Restricted feeding did however produce an increase in efficiency, estimated at 17-25%. This change enabled milk output and body nutrient stores to be protected when the restriction was to 80% of the ad libitum intake but at 60% of ad libitum intake, milk output was reduced and body nutrient mobilization was increased. It is proposed that low maternal food intake is associated with impairment of lactational performance when it is also severe enough to increase body nutrient mobilization.

The doubly labeled water method was compared with indirect calorimetry and a nutrient-balance study for simultaneous determination of rates of CO2 production, energy expenditure, and water intake over 5 days in four preterm infants. Additionally, metabolizable energy (ME) intake estimated using the isotope procedure (as energy expenditure plus an estimate for energy deposition based on weight gain), was compared to ME intake measured in the balance study. Compared to values obtained by traditional methods, calculated CO2 production, energy expenditure, and water intake differed by -1.4 +/- 4.8% (SD), +0.3 +/- 2.6%, and +5.7 +/- 1.4%, respectively; the difference in water intake was significant (p less than 0.05). Calculated ME intakes were 5.3 +/- 19.3% less than measured intakes, but the difference was not significant. These findings indicate that the doubly labeled water method can provide accurate information on rates of CO2 production, energy expenditure, and water intake in preterm infants, but individual estimates of ME intake may be subject to substantial error.

In 14 studies, each lasting seven days, on eight bottle fed infants milk intake was measured by deuterium dilution and by weighing the formula consumed and oral losses. Mean (SD) milk intake was 829 (108) g/day when measured by isotope dilution and 837 (98) g/day when measured by direct weighing, giving a mean difference between methods of 1.0 (5.0)%. Thus the stable isotope procedure is a potentially useful method for investigating lactation and nutritional problems.

The role of diet-induced thermogenesis (DIT) in the regulation of energy utilization in preterm infants was investigated by measuring energy expenditure and nutrient deposition in groups of subjects fed either banked drip breast milk (BBM) or a preterm formula (PF). Nutrient deposition was determined by combining a 3-d nutrient balance study and measurement of energy expenditure. Infants fed PF consumed 57 per cent more metabolizable energy (ME) than those fed BBM. There were significant differences between groups in energy expenditure, energy deposition and protein deposition; 76 per cent of the additional ME intake consumed by the PF-fed group was deposited while the remaining 24 per cent was expended. Infants fed PF gained weight 39 per cent faster than those fed BBM (19, compared to 14 g/kg/d), and deposited 128 per cent more fat and 86 per cent more protein. These findings indicate that nutrient deposition, rather than DIT, was the primary use of the extra ME received by the infants fed PF. However, the effect of the high energy intake on weight gain in these subjects was reduced, compared to its effect on nutrient deposition, by increases in the concentrations of fat and protein in new tissue.

The activity of 81 pregnant, lactating and non-pregnant, non-lactating women in the rural subsistence farming village of Keneba, The Gambia, was measured for 12 months using a combination of 24-hour activity recall and activity diaries. During the course of pregnancy women became gradually less active and in the month before giving birth were 25% less active than non-pregnant, non-lactating women and lactating women when women in the month after birth were excluded. There were striking seasonal changes in activity. During the dry season, from January to April, lactating women were active 55% of the 15-hour working day. At the start of the farming season in June and July this figure increased to 92%. Similar changes were observed in the pregnant women whose activity increased from a mean 50% in April to 83% in June. The period of intense activity coincided with a general shortage of food and increased incidence of disease. The intense activity of pregnant women in the farming season coupled with the low food intakes are most probably the main factors responsible for the striking fall in birth weights observed at this time of the year. Reduced lactational performance was apparent in relation to high activity when it necessitated the mother regularly spending much of the day away from her child. FAO/WHO-recommended energy intakes during pregnancy and lactation are inappropriate for this community.

In the doubly-labelled water (2H2(18)O) method for the measurement of carbon dioxide production rate in man, single exponential disappearance curves for 2H2O and H2(18)O in body water are used; the precision with which the slopes and intercepts of the curves are estimated determines the precision of the estimate of carbon dioxide production rate. In studies with infants, and in computer simulations, the effect of different experimental regimes on the overall precision of the carbon dioxide production estimate was investigated. When the number of data points used was progressively reduced by shortening the total observation period from 7 d (about 3 biological half-lives for the isotopes) to 1 d there was a deleterious effect on precision and in the infants there was an upward bias in the values for carbon dioxide production. When the number of data points was reduced by removing points from the middle of the exponential curves with the maintenance of the 7-d experimental period, precision was also reduced but by less than in the former procedure and there were no consistent trends in the average values for carbon dioxide production.

Variations in background 2H and 18O abundances in body water influence the accuracy and precision of the 2H218O method for determination of energy expenditure. To investigate the effect of weaning during infancy on background 2H and 18O abundances, urine samples from 44 breast- or formula-fed infants aged 5-16 wk were analyzed. 2H and 18O abundances were significantly higher (P less than 0.001) in breast- than in formula-fed infants. The relationship between 2H and 18O abundances was linear and independent of diet [slope, 4.16 +/- 0.43 (SE)]. By use of this information, the effect of weaning on the accuracy of the 2H218O method was evaluated, taking into account the effect of 2H-18O abundances in the isotope loading dose. In infants weaned completely from breast milk to formula during the measurement, energy expenditure can be overestimated by 18.0%, even if 2H-18O abundances in the isotope dose equal the ratio of naturally occurring background changes. However, this error can be reduced to less than 3.0% by manipulating the study duration and isotope dose. During gradual weaning, the overestimation of energy expenditure is only 0.3-2.0%.

Rates of cell proliferation essentially equal to those in 10% serum were obtained when Yaffe's L6 myoblasts were incubated in Ham's F-12 medium containing 10(-5) M fetuin, 10(-6) M insulin, and 10(-7) M dexamethasone; we have designated this mixture muscle medium-1 (MM-1). Addition of other growth factors and hormones in various combinations did not increase the proliferation of myoblasts above the rate in MM-1, and neither fetuin nor insulin could be replaced by other growth factors. All glucocorticoids tested (but no other steroid hormones) were active. Fetuins prepared by the rather different procedures of Pedersen, Deutsch, and Spiro were all active, and the active material was heat labile and nondialyzable; this is the first cell culture system in which highly purified Spiro fetuin has been found active. Primary rat myoblasts proliferated more rapidly that fibroblasts in parallel cultures when incubated in MM-1. This simple medium, composed of relatively inexpensive and readily available components, should be useful for the study of muscle cell growth and differentiation.

The (R)- and (S)-isomers of 5-methylhydantoin (5-MH) and of 5-isopropylhydantoin (5-IPH) were synthesized, and incubations of the individual isomers with a rat liver dihydropyrimidinase preparation (100,000g supernatant fraction) were carried out. Only the (R)-isomer of 5-MH or 5-IPH was ring-opened by the enzyme. Reversibility of the enzymatic ring-opening reaction could be demonstrated with only the (R)-isomer of 2-methylhydantoic acid (2-MHA) or 2-isopropylhydantoic acid (2-IPHA). The results of the present investigation show that the replacement in 5-phenylhydantoin of the phenyl group with an alkyl group does not alter the stereospecificity of the hydantoin substrates in the ring-opening reaction. The results are used to form the concept that (R)-dihydrothymine, the optical isomer previously postulated as the natural substrate to the enzyme, may have a different type of binding at the active site of the enzyme.

Physiologically acceptable methods for estimating milk intake in experimental animals area based on the measurement of rates of disappearance of 3H2O from their body-water. This work discusses the problems involved in using methods of this type when steady-states (constant trace inflow and outflow rates, and constant pool-size) do not exist. Theoretically sound techniques are developed and their use justified.

The effect of ambient temperature on the energy requirements of the lactating rat was investigated. Ambient temperature was maintained at 13, 20 or 25 degrees C during lactation, and digestible energy (DE) intake, milk output and energy balance were measured between 4 and 12 d post partum. Ambient temperature did not significantly affect DE intake. Milk output was highest in the rats housed at 13 degrees C and lowest in those kept at 25 degrees C, although the rate of body fat mobilization was lowest in the rats kept at 20 degrees C and highest in the rats kept at 25 degrees C. Energy expenditure on activity and maintenance, determined from the difference between DE intake and body nutrient mobilization plus the energy used for milk production, was significantly higher in the rats housed at 25 degrees C than in those kept at 13 degrees C. This is an opposite trend to that which occurs in nonlactating rats. The results indicate that the gross efficiency of energy utilization during lactation is higher at 13 degrees C than it is at 25 degrees C in the rat.

Transcription of the cell-cycle-regulated human histone genes increases approximately fivefold during S phase. One step toward the elucidation of the biochemical mechanisms that govern cell-cycle-regulated expression of these genes is to purify and characterize the transcription factors that regulate these promoters. Here, we describe the purification of two previously identified factors, H4TF-1 and H4TF-2, which bind the human histone H4 promoter. Purification was achieved through a combination of ion-exchange and oligonucleotide affinity chromatography. On the basis of analysis of purified fractions by SDS-polyacrylamide gels and UV cross-linking, we believe that H4TF-1 is two polypeptides of 105 and 110 kD. This factor binds to a GC-rich DNA sequence required for maximal expression of the H4 gene but does not bind to any Sp1 consensus elements tested. H4TF-2 is a 65-kD protein that binds specifically to sequences within that highly conserved H4 subtype-specific consensus promoter element. Both highly purified factors activated transcription in vitro only from H4 promoters that contained their binding sequences, demonstrating that H4TF-1 and H4TF-2 are H4-specific transcription factors that potentiate expression of this gene.

We have determined the nucleotide sequence of sea urchin (Lytechinus pictus) late stage H3 and H4 histone genes contained on the clone pLpH3H4 -21 and of the early stage H3 gene contained on the plasmid pLpA . Comparison of these differentially regulated histone genes with each other and with other L. pictus late and early stage histone H3 and H4 genes previously sequenced confirms that members of each histone gene family (early and late) are more homologous to each other than they are to members of other histone gene families. The spacer regions between two late H3-H4 gene pairs on the clones pLpH3H4 -19 and pLpH3H4 -21 have diverged to the point where they are no longer homologous. However, comparative analysis of the 5' flanking DNA has identified a sequence 5'C-T-C-A-T-G-T-A-T-T3' upstream of both late H4 genes and another, 5'A-G-A-T-T-C-A3', upstream of both H3 genes. Except for a short conserved sequence near the initiation codon, the transcribed 5' leaders of the late mRNAs differ in length and sequence in the two non-allelic late histone gene pairs. This divergence contrasts with the 95 to 96% conservation found between late histone gene coding sequences. The results suggest that there is intergenic exchange in the germline among members of the late histone gene family and that the unit of exchange is the individual gene rather than the heterotypic dimer which includes the common spacer DNA.

One of each of thirty-five pairs of fresh intact femora were tested to failure in torsion, recording the dynamic torque, the absorbed energy, and the angle of rotation. These results were compared with the results obtained with the contralateral femur, reconstituted after removal of a segment. Intramedullary nails with polymethylmethacrylate cement, strips of titanium mesh with cement, bone plates with and without cement, and multiple Steinmann pins with cement were the reconstituting configurations. Bone plates were the strongest configuration; the failure torques in all cases were limited by the stress concentration effects of the holes in the bone used for screw fixation. The use of cement as an adjunct to single-plate fixation provided some additional strength. The torsional strength of femora fixed with Küntscher and Schneider nails was limited by failure of the cement and bone. The use of titanium mesh with polymethylmethacrylate was less effective, because this composite has a low torsional rigidity. The use of multiple Steinmann pins packed with polymethylmethacrylate in the medullary cavity should be discouraged because severe twisting and fragmentation of the surrounding acrylic will occur at low levels of torque.

The core histone genes (H2A, H2B, H3 and H4) of Caenorhabditis elegans are arranged in approximately 11 dispersed clusters and are not tandemly arrayed in the genome. Three well-characterized genomic clones, which contain histone genes, have one copy of each core histone gene per cluster. One of the clones (lambda Ceh-1) carries one histone cluster surrounded by several thousand base-pairs of non-histone DNA, and another clone (lambda Ceh-3) contains a histone cluster duplication surrounded by non-histone DNA. A third clone (lambda Ceh-2) carries a cluster of core histone genes flanked on one side (12,000 base-pairs away) by a single H2B gene and on the other by non-histone DNA. A fourth cluster (clone BE9) has one copy each of H3 and H4 and two copies each of H2A and H2B. This cluster is also flanked by non-histone DNA. Analysis of cosmid clones which overlap three of the clusters shows that no other histone clusters are closer than 8000 to 60,000 base-pairs, although unidentified non-histone transcription units are present on the flanking regions. Gene order within the histone clusters varies, and histone mRNAs are transcribed from both DNA strands. No H1 sequences are found on these core histone clones. Restriction fragment length polymorphisms between two related nematode strains (Bristol and Bergerac) were used as phenotypic markers in genetic crosses to map one histone cluster to linkage group V and another to linkage group IV. Hybridization of gene-specific probes from sea urchin to C. elegans RNA identifies C. elegans core histone messenger RNAs of sizes similar to sea urchin early stage histone mRNAs (H2A, H2B, H3 and H4). The organization of histone genes in C. elegans resembles the clustering found in most vertebrate organisms and does not resemble the tandem patterns of the early stage histone gene family of sea urchins or the major histone locus of Drosophila.

The oxygen consumption rates of virgin and lactating rats fed ad libitum were measured at temperatures ranging from 1 to 28 degrees C. The oxygen consumption rates of the virgin animals decreased progressively with increasing ambient temperature, but those of the lactating animals decreased slightly between 1 and 8 degrees C, remained approximately constant between 8 and 20 degrees C and fell rapidly above 20 degrees C. It is suggested that the fall in oxygen consumption rate above 20 degrees C in the lactating rats was due to a reduction in milk synthesis rate, and that the thermoneutral zone for normal lactation was 8-20 degrees C; this is a substantially cooler range than that of virgin rats (28-29 degrees C). The net energetic efficiency of milk production was calculated from the energy output in milk and the amount of energy used to produce milk (estimated from the oxygen consumption rates). The value obtained was 57.0%.