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A few countries permit women to serve in combat roles, but their long term performance in these positions has not been reported. The incidences of overuse injuries and attrition of 85 male and 235 female recruits in a light infantry brigade was followed in a three-year prospective study. Females were shorter (162 cm, CI 161–163 cm) than males (174 cm, CI 173–176), had more body fat (18.9 kg, CI 18.2–19.6 kg) than males (12.6 kg, 11.3–13.8 kg), had lowerV˙O2max (36.8 mL·min−1·kg−1, CI 35.8–37.78 mL·min−1·kg−1) than males (50.48 mL·min−1·kg−1, CI 48.4 to 52.48 mL·min−1·kg−1), had more stress fractures (21.0%, 95% CI 16.2–26.5%) than males (2.3%, CI 0.3–8.2%), and had more anterior knee pain (41.2%, CI 34.9–47.7%) than males (24.7%, CI 16.0–35.2%). Three-year attrition was 28% CI 22–34% for females and 37% CI 26–48% for males. The females in this study successfully served as light infantry soldiers. Their lower fitness and high incidence of overuse injuries might impede service as regular infantry soldiers.

Physiological systems like bone respond to many genetic and environmental factors by adjusting traits in a highly coordinated, compensatory manner to establish organ-level function. To be mechanically functional, a bone should be sufficiently stiff and strong to support physiological loads. Factors impairing this process are expected to compromise strength and increase fracture risk. We tested the hypotheses that individuals with reduced stiffness relative to body size will show an increased risk of fracturing and that reduced strength arises from the acquisition of biologically distinct sets of traits (ie, different combinations of morphological and tissue-level mechanical properties). We assessed tibial functionality retrospectively for 336 young adult women and men engaged in military training, and calculated robustness (total area/bone length), cortical area (Ct.Ar), and tissue-mineral density (TMD). These three traits explained 69% to 72% of the variation in tibial stiffness (p

Objective Stress fracture injuries sustained during military basic combat training (BT) are a significant problem and occur at a higher rate in female recruits than male recruits. Insulin-like growth factor-I (IGF-I) is an easily measured biomarker that is involved in bone formation and positively correlated with bone mineral density, especially in women. This study examined the response of the IGF-I system between female soldiers that sustained a stress fracture (SFX, n = 13) during BT and female soldiers who did not (NSFX, n = 49). Design Female soldiers (n = 62, 18.8 ± 0.6 yr) from 2 companies of a gender-integrated combat battalion in the Israeli Defense Forces participated in this study. Height, weight and blood draws were taken upon entry to BT (preBT) and after a four-month BT program (postBT). Stress fractures were diagnosed by bone scan. Serum was analyzed for total IGF-I, free IGF-I, IGF binding proteins (IGFBP)1–6, BAP, calcium, CTx, IL1β, IL6, PINP, PTH, TNFα, TRAP, and 25(OH)D. Statistical differences between SFX and NSFX groups and time points were assessed by RM ANOVA with Fisher post-hoc (p ≤ 0.05). Results The SFX group was significantly taller and had lower BMI than NSFX (p ≤ 0.05). Serum concentrations of total IGF-I, bioavailable IGF-I, other bone biomarkers, and cytokines were not significantly different between SFX and NSFX preBT. Serum IGFBP-2 and IGFBP-5 were significantly higher in the SFX compared to the NSFX preBT (p ≤ 0.05). In both groups, total IGF-I increased pre to postBT (p ≤ 0.05). Additionally, a significant difference was observed in the bioavailable IGF-I response pre to postBT for both groups. The SFX group demonstrated a significant decrease in bioavailable IGF-I pre to postBT (preBT: 0.58 ± 0.58 ng/mL; postBT 0.39 ± 0.48; p ≤ 0.05) whereas the NSFX group demonstrated a significant increase in bioavailable IGF-I pre to postBT (preBT: 0.53 ± 0.37 ng/mL; postBT: 0.63 ± 0.45; p ≤ 0.05). Conclusions Our study demonstrated that serum IGF-I changes during basic training and that women sustaining stress fractures during BT significantly decreased bioavailable IGF-I, whereas their uninjured counter parts increased bioavailable IGF-I. These results suggest that stress fracture susceptibility may be related to differential IGF-I system concentrations and response to physical training.

Submariners taking part in prolonged missions are exposed to environmental factors that may adversely affect bone health. Among these, relatively high levels of CO(2), lack of sunlight exposure affecting vitamin D metabolism, limited physical activity, and altered dietary habits. The aims of this study were to examine the effect of a prolonged submersion (30 days) on changes in bone strength using quantitative bone speed of sound and in markers of bone metabolism that include bone turnover (BAP, PINP, TRAP5b, and CTx) and endocrine regulators (serum calcium, PTH, and 25[OH]D) in a group of 32 young healthy male submariners. The prolonged submersion led to increases in body weight and BMI and to a decrease in fitness level. There was a significant decrease in bone strength following the submersion. Speed of sound exhibited continued decline at 4 weeks after return to shore and returned to baseline levels at the 6-month follow-up. There was a significant increase in circulating calcium level. PTH and 25(OH)D levels decreased significantly. Significant decreases were observed in both TRAP5b and CTx levels, markers of bone resorption, as well as in N-terminal propeptide of type I collagen (PINP), a bone formation marker. Prolonged submersion led to a significant decrease in bone strength, accompanied by an overall decrease in bone metabolism. Bone strength was regained only 6 months after return to shore. Prevention and/or rehabilitation programs should be developed following periods of relative disuse even for young submariners. The effects of repeated prolonged submersions on bone health are yet to be determined.

The purpose of this controlled trial was to determine whether subtle changes in mineralization and geometry of the tibia were evident following short term exercise interventions.Fifty-seven female volunteers (age 20.1±1.6) were randomized to one of four, 13-week training groups: sedentary control, resistance training, aerobic training, or combined aerobic-resistance. A pQCT image analysis software was developed and used to analyze images taken at sites 4%, 38% and 66% from the distal tibia at baseline and at completion of training. Parameters of bone mineral density, geometry and strength were determined for the entire scan cross-section and for each of six 60° polar sectors. Repeated-measures ANOVA and Fisher's LSD post hoc tests analyzed the effects of training over time.Trabecular density (TrDn) at the 4% site increased from 279.8±37.1 to 283.1±36.0 mg/cm(3) in the aerobic group, and from 285.1±24.6 to 287.5±22.9 mg/cm(3) in the combined group over the study period (P≤0.001). Regional sector analyses revealed that impact exercises resulted in localized changes to the medial aspect of the tibia. Small increases in total bone area were observed in the diaphysis (38% site) (P0.05).Subtle, regional increases in trabecular density may be an early measurable manifestation of bone quality changes.

Having a better understanding of how complex systems like bone compensate for the natural variation in bone width to establish mechanical function will benefit efforts to identify traits contributing to fracture risk. Using a collection of pQCT images of the tibial diaphysis from 696 young adult women and men, we tested the hypothesis that bone cells cannot surmount the nonlinear relationship between bone width and whole bone stiffness to establish functional equivalence across a healthy population. Intrinsic cellular constraints limited the degree of compensation, leading to functional inequivalence relative to robustness, with slender tibias being as much as two to three times less stiff relative to body size compared with robust tibias. Using Path Analysis, we identified a network of compensatory trait interactions that explained 79% of the variation in whole-bone bending stiffness. Although slender tibias had significantly less cortical area relative to body size compared with robust tibias, it was the limited range in tissue modulus that was largely responsible for the functional inequivalence. Bone cells coordinately modulated mineralization as well as the cortical porosity associated with internal bone multicellular units (BMU)-based remodeling to adjust tissue modulus to compensate for robustness. Although anecdotal evidence suggests that functional inequivalence is tolerated under normal loading conditions, our concern is that the functional deficit of slender tibias may contribute to fracture susceptibility under extreme loading conditions, such as intense exercise during military training or falls in the elderly. Thus, we show the natural variation in bone robustness was associated with predictable functional deficits that were attributable to cellular constraints limiting the amount of compensation permissible in human long bone. Whether these cellular constraints can be circumvented prophylactically to better equilibrate function among individuals remains to be determined.

Background: The training of elite infantry recruits takes a year or more. Stress fractures are known to be endemic in their basic training and the clinical presentation of tibial, femoral, and metatarsal stress fractures are different. Stress fracture incidence during the subsequent progressively more demanding training is not known. The study hypothesis was that after an adaptation period, the incidence of stress fractures during the course of 1 year of elite infantry training would fall in spite of the increasingly demanding training. Materials and Methods: Seventy-six male elite infantry recruits were followed for the development of stress fractures during a progressively more difficult training program composed of basic training (1 to 14 weeks), advanced training (14 to 26 weeks), and unit training (26 to 52 weeks). Subjects were reviewed regularly and those with clinical suspicion of stress fracture were assessed using bone scan and X-rays. Results: The incidence of stress fractures was 20% during basic training, 14% during advanced training and 23% during unit training. There was a statistically significant difference in the incidence of tibial and femoral stress fractures versus metatarsal stress fractures before and after the completion of phase II training at week 26 ( p = 0.0001). Seventy-eight percent of the stress fractures during phases I and II training were either tibial or femoral, while 91% of the stress fractures in phase III training were metatarsal. Prior participation in ball sports ( p = 0.02) and greater tibial length ( p = 0.05) were protective factors for stress fracture. Conclusion: The study hypothesis that after a period of soldier adaptation, the incidence of stress fractures would decrease in spite of the increasingly demanding elite infantry training was found to be true for tibial and femoral fractures after 6 months of training but not for metatarsal stress fractures. Further studies are required to understand the mechanism of this difference but physicians and others treating stress fractures should be aware of this pattern. Level of Evidence: IV, Case Series

The effect of several hormones known to alter intracellular free Ca2+ on rates of O2 uptake in periportal and pericentral regions of the liver lobule was studied in the perfused liver. Regional O2 uptake was measured by stopping the flow and monitoring the decrease in O2 concentration. When perfusion was in the anterograde direction, basal rates of O2 uptake were two to three times higher in periportal than in pericentral regions, and phosphorylase alpha activity, which increases as a function of intracellular free Ca2+ levels, was higher in periportal regions. In contrast, when perfusion was in the retrograde direction, rates of O2 uptake were two to three times greater in pericentral regions. Infusion of epinephrine (0.1 microM) or angiotensin II (5 nM) increased the rate of O2 uptake nearly exclusively in downstream areas of the lobule where O2 tension was low. When perfusions were in the anterograde direction, epinephrine increased phosphorylase alpha activity significantly only in pericentral regions. Stimulation of O2 uptake by epinephrine was blocked by the alpha-adrenergic receptor blocker phentolamine (1 microM) but not by the beta-receptor blocker propranolol. Thus hormones that increase intracellular calcium stimulate O2 uptake predominantly in regions of the liver lobule where O2 tension is lowest, supporting the hypothesis that oxygen tension regulates O2 uptake in the liver via mechanisms involving intracellular free Ca2+.

Prescribing exercise based on intensity, frequency, and duration of loading may maximize osteogenic responses in bone, but a model of the osteogenic potential of exercise has not been established in humans. In rodents, an osteogenic index (OI) has been used to predict the osteogenic potential of exercise. The current study sought to determine whether aerobic, resistance, or combined aerobic and resistance exercise programs conducted over eight weeks and compared to a control group could produce changes in biochemical markers of bone turnover indicative of bone formation. We further sought to determine whether an OI could be calculated for each of these programs that would reflect observed biochemical changes. We collected serum biomarkers [bone-specific alkaline phosphatase (BAP), osteocalcin, tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide fragment of type I collagen (CTx), deoxypyridinoline (DPD), 25-hydroxy vitamin D (25(OH)D), and parathyroid hormone (PTH)] in 56 women (20.3+/-1.8 years) before, during and after eight weeks of training. We also measured bone mineral density (BMD) at regional areas of interest using DXA and pQCT. Biomarkers of bone formation (BAP and osteocalcin) increased in the Resistance and Combined groups (p

The two inositol phosphate-binding proteins, the Ins(1,4,5)P3 (InsP3) and Ins(1,3,4,5)P4 (InsP4) receptors, and the two particulate InsP3-metabolizing enzymes, InsP3 5-phosphatase and InsP3 3-kinase, were solubilized with detergent from rat cerebellar membranes. These four activities are shown to be distinct molecular species by separation using a variety of protein chromatographic steps. The pharmacology of the partially purified InsP4-binding site indicates that the binding has a high affinity and selectivity for InsP4 over InsP3. These results suggest the existence of a distinct specific InsP4-binding protein which may represent the receptor for this putative second messenger.

This study documented the incidence of acute injuries related to the Army Physical Fitness Test (APFT).A questionnaire was administered to 1,532 soldiers after they completed a biannual APFT. Self-reported injury responses determined the rates of injuries resulting from the push-up, sit-up, and 2-mile run events and were classified into three categories, i.e., all injuries, performance-limiting injuries, and time-loss injuries.A total of 117 soldiers (injury rate, 7.6%) reported sustaining an injury (all injuries), with 11% attributed to the push-up event, 56% to the sit-up event, and 32% to the run event. Forty-six of these injuries reportedly limited performance (injury rate, 3.0%), and 11 soldiers received a duty-limiting profile (time-loss injury rate, 0.7%), which did not differ among events. Injury rates were not significantly associated with the number of sit-ups performed per week or the number of days per week a soldier participated in physical training and were not greater for soldiers who trained specifically for the APFT. History of previous injury was a significant risk factor for injury.The push-up, sit-up, and run events of the APFT do not pose a considerable acute injury risk to active duty soldiers.

The purpose of this study was to compare bone densitometry to quantitative ultrasound (QUS) and to assess gender differences in a young healthy population. Seventy-five active, aged-matched (^ 29 yrs) men (N = 29) and women (N = 46) were assessed for bone mineral density (BMD, g/sq cm) and broadband ultrasound attenuation (BUA, db/MHz). BMD of the total body, regional and lumbar spine was measured via dual-energy X-ray absorptiometry (DXA), and BUA was measured using the QUS-2TM ultrasonometer. Data analysis included Pearson product-moment to compare DXA to QUS, and receiver operating characteristic (ROC) curves and Kappa coefficients for discrimination of the two procedures. There was no significant gender difference in lumbar spine (LS) BMD. Regional and total body BMD and BUA were greater for men than women. Total body BMD (mean +/- SD) for men was 1.29 +/- 0.09 g/sq cm and for women was 1.22 +/- 0.07 g/sq cm. BUA (mean +/- SD) was 107.0 +/- 16.7 db/MHz vs. 97.8 +/- 11.6 db/MHz for men and women, respectively. All BMD sites except LS and pelvis were significantly correlated to BUA for men (r = 0.46-0.48), but only LS (r = 0.32) and arms (r = 0.30) were significantly correlated to BUA for women. Kappa coefficients ranged from - 0.09 to 0.17 for men, none of which were significant, and the range for women was 0.09 to 0.25 with pelvis and legs attaining significance. There was low percent agreement between QUS and DXA (28%-45% for men and 41%-50% for women), and the area under the curve (AUC) derived from ROC analysis resulted in little discrimination (AUC = 0.60-0.64). Our results showed little agreement between DXA and QUS; therefore, QUS would not be a surrogate indicator for the measurement of BMD by DXA. These results indicate that QUS measures different aspects of bone health and may complement the use of DXA.

The stability of highly purified supercoiled plasmid DNA formulated in simple phosphate or Tris-buffered saline solutions has been characterized to establish the overall degradation processes that occur during storage in aqueous solution. Plasmid DNA stability was monitored during accelerated stability studies (at 50 degrees C) by measurements of supercoiled, open-circle, and linear DNA content, as well as the accumulation of apurinic sites and 8-hydroxydeoxyguanosine residues over time. The effects of formulation pH, demetalation, metal ion chelators, and ethanol (hydroxyl radical scavenger) on the supercoiled content of plasmid DNA during storage at 50 degrees C were also determined. The results indicate that free radical oxidation may be a major degradative process for plasmid DNA in pharmaceutical formulations unless specific measures are taken to control it by the addition of free radical scavengers, specific metal ion chelators, or both. The generation of hydroxyl radicals in phosphate-buffered saline was confirmed by examining the hydroxylation of phenylalanine over time by reverse phase high-performance liquid chromatography. Ethanol was found to enhance plasmid DNA stability and to inhibit the hydroxylation of phenylalanine; both observations are consistent with the known ability of ethanol to serve as a hydroxyl radical scavenger. Moreover, the combination of ethylenediamine tetraacetic acid (EDTA) and ethanol had a synergistic enhancing effect on DNA stability. However, the metal ion chelator diethylenetriaminepentaacetic acid (DTPA) was as potent as the combination of EDTA and ethanol for enhancing the stability of plasmid DNA. By controlling free radical oxidation with EDTA and ethanol, the rate constants of plasmid DNA degradation by means of depurination and beta-elimination were then determined, allowing accurate predictions of DNA storage stability as a function of formulation pH and temperature. The ability to predict plasmid DNA storage stability in the absence of free radical oxidation should prove to be a valuable tool for the design of stable pharmaceutical formulations of plasmid DNA.

The deoxyuridine triphosphatase gene of vaccinia virus, encoded by the open reading frame F2L, was cloned into Escherichia coli and expressed under the control of a bacteriophage T7 promoter. After induction of T7 RNA polymerase by isopropyl beta-D-thiogalactopyranoside, a 16.5-kDa peptide accumulated to high levels. This 16.5-kDa protein was purified to homogeneity and characterized. Gel filtration of the purified protein revealed a trimeric native structure. Biochemical analysis revealed the enzyme to be a metalloenzyme; enzymatic activity is inhibited by EDTA. This inhibition was reversed by the addition of Mg2+, Mn2+, or Zn2+. While the enzyme activity was highly specific for dUTP with an apparent Km of 0.94 microM, inhibition studies show that 8-azido-ATP acted as a competitive inhibitor of dUTP with a Ki of approximately 173 microM. Also, protection studies demonstrated that nucleotide competitors inhibit photoincorporation of the photoaffinity analogues [gamma-32P]5-azido-dUTP and [gamma-32P]8-azido-ATP. This suggests that while catalytic activity is limited to dUTP, other nucleotides can bind the active site.

Background Appropriate and sufficient dietary intake is one of the main requirements for maintaining fitness and health. Inadequate energy intake may have a negative impact on physical performance which may result in injuries among physically active populations. The purpose of this research was to evaluate a possible relationship between dietary intake and stress fracture occurrence among combat recruits during basic training (BT). Methods Data was collected from 74 combat recruits (18.2 ± 0.6 yrs) in the Israeli Defense Forces. Data analyses included changes in anthropometric measures, dietary intake, blood iron and calcium levels. Measurements were taken on entry to 4-month BT and at the end of BT. The occurrence of stress reaction injury was followed prospectively during the entire 6-month training period. Results Twelve recruits were diagnosed with stress fracture in the tibia or femur (SF group). Sixty two recruits completed BT without stress fractures (NSF). Calcium and vitamin D intakes reported on induction day were lower in the SF group compared to the NSF group-38.9% for calcium (589 ± 92 and 964 ± 373 mg·d-1, respectively, p < 0.001), and-25.1% for vitamin D (117.9 ± 34.3 and 157.4 ± 93.3 IU·d-1, respectively, p < 0.001). During BT calcium and vitamin D intake continued to be at the same low values for the SF group but decreased for the NSF group and no significant differences were found between these two groups. Conclusions The development of stress fractures in young recruits during combat BT was associated with dietary deficiency before induction and during BT of mainly vitamin D and calcium. For the purpose of intervention, the fact that the main deficiency is before induction will need special consideration.

A highly photosensitive analogue of thymidine, 5-azidodeoxyuridine 5'-triphosphate, has been incorporated into 61-base pair (bp) DNA fragments corresponding to the central region of Xenopus somatic-type 5 S RNA genes such that 5-azidodeoxyuridine replaces some or all T residues in either the coding or noncoding strand of the TFIIIA binding site. Photolysis of TFIIIA.DNA complexes formed with these probes results in efficient, sequence-specific cross-linking to the Zn-finger protein providing direct evidence that this class of proteins have contacts in the major groove of their target sequence. Of the 20 T residues present in the 61-bp probes, greater than 90% of the cross-linking occurs from two sites in the 5 S RNA gene corresponding to T residues at positions 84 and 88 in the noncoding and coding strands, respectively. Digestion by V8 protease of the complex formed with the noncoding strand probe releases peptides not bound to the DNA. Amino acid sequence analysis of the remaining, cross-linked peptides indicates the region including zinc-finger 2 plus the finger 2-3 linker is in contact with position 84. The linker region between fingers 5 and 6 is also in close proximity to the major groove somewhere upstream from position 84.

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.

A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.

Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [3H]leucine into trichloroacetic acid-precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation-reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within 3h in vitro. Ganglion explants prepared by cutting the tissue into 300-pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α-ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.

We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP) and used it to synthesize light-sensitive DNA by enzymatic incorporation. In the absence of ultraviolet light, 5-N3dUTP is a substrate for Escherichia coli DNA polymerase I. In in vitro DNA synthesis reactions using bacteriophage M13 single-stranded DNA as the template and 5-N3dUTP in place of dTTP, a photoactive complementary strand was synthesized by DNA polymerase I. The complementary strand was not synthesized when the 5-N3dUTP was substituted for dCTP or when it was exposed to ultraviolet light prior to the addition of DNA polymerase I. Using a synthetic lac operator template of 26 bases and a 15-base primer, we generated a photoactive 26-base-pair lac operator by enzymatically incorporating 5-N3dUMP with DNA polymerase I. Crosslinking of this photoactive DNA fragment to lac repressor was totally dependent on the presence of UV light and was reduced 78% by 150 microM isopropyl beta-D-thiogalactoside. Under the same conditions no crosslinking to lac repressor was observed using a nonphotoactive 26-base-pair lac operator. Photoactivatable deoxyuridine analogs have potential application as reagents to crosslink DNA binding proteins to 5-azidouracil-containing DNA and as active-site-directed photoaffinity labelling reagents.

The effect of a number of nucleotides and metabolic intermediates was tested on the activity of purified NADPH-cytochrome c reductase. ATP, ADP, GTP, GDP, α-ketoglutarate and oxalacetate were found to inhibit the enzyme. Adenine nucleotides were inhibitory at concentrations noted in liver; however, the other metabolites were only effective at supra-physiological concentrations. Inhibition was observed with two artificial electron acceptors as well as with cytochrome c. Inhibition by adenine and guanine nucleotides was competitive with NADPH, whereas the α-ketoacids showed competitive inhibition with cytochrome c.

Mixed-function oxidation of p-nitroanisole in isolated perfused livers from fasted rats was studied in the presence and absence of 2-bromooctanoate, an inhibitor of the beta-oxidation of acyl CoA compounds. These experiments were designed to test the hypothesis that reducing equivalents from fatty acid oxidation may play a role in drug metabolism in the fasted state. Complete inhibition of ketogenesis, as indicated by beta-hydroxybutyrate and acetoacetate production, was achieved with the addition of 0.6 mM 2-bromooctanoate to the perfusate. This concentration of 2-bromooctanoate had no effect on p-nitroanisole O-demethylation measured in isolated microsomes but diminished maximal rates of p-nitroanisole O-demethylation by 78% in perfused livers of fasted, phenobarbital-treated rats. No inhibition of p-nitroanisole O-demethylation was seen in perfused livers from fed rats. Intracellular concentrations of ATP, NADH, glutamate, alpha-ketoglutarate and isocitrate decreased significantly in the presence of 2-bromooctanoate; oxygen uptake also decreased by about 50%. These data indicate that by inhibiting endogenous acyl CoA oxidation, 2-bromooctanoate diminishes beta-oxidation of fatty acids, thereby decreasing the supply of NADH for the generation of ATP. This decreases the synthesis of NADPH, an obligatory cofactor for drug metabolism. 2-Bromooctanoate also decreased the concentration of intermediates which act through substrate shuttle mechanisms to move reducing equivalents from the mitochondrial into the extramitochondrial space. It is concluded that the decrease in mixed-function oxidation observed when beta-oxidation of fatty acids is inhibited with 2-bromooctanoate is due to diminished availability of mitochondrial reducing equivalents for the generation of cytosolic NADPH. 24CA-23080

Conjugation of p-nitrophenol in perfused livers from control and phenobarbital-treated rats was measured spectrally by determining influent minus effluent concentration differences of the free drug. Rates of conjugation (1.0 to 2.0 mu moles/g/h) were essentially the same in livers from fasted control or fasted phenobarbital-treated rats. In control fed livers, rates of conjugation did not differ from the fasted state. However, rates of conjugation in livers from carbohydrate-rich, phenobarbital-treated rats were 6- to 10-fold greater than rates in livers from fasted phenobarbital-treated animals. Differences in rates of conjugation in phenobarbital-treated rats in different nutritional states were more closely related to carbohydrate reserves as indicated by glycogen levels than to activities of p-nitrophenyl UDP-glucuronyl transferase.

Blood levels of 5-hydroxytryptamine (5-HT) and epinephrine are increased in conditions such as severe hypoxia, and hypovolemic or septic shock. The effect of these agents, which influence the blood coagulation system, were studied in vitro on platelets from newborns. 5-HT was determined in platelets from 18 newborns and 28 adults. The 5-HT content was 0.305 +/- 0.144 microgram/10(9) platelets in newborns and 0.675 +/- 0.212 microgram/10(9) platelets in the adult controls, a difference which is statistically significant (P less than 0.001). The 5-HT content of newborn platelets proved to be higher than that reported in the literature. Further studies on platelet aggregation due to 5-HT and epinephrine were then carried out. These demonstrated first-phase aggregation in 40% of cases when both 5-HT and epinephrine were applied simultaneously, while in the majority of the newborns no reaction was encountered when each of these substances was applied separately. However, in 2 cases, simultaneous application of 5-HT and epinephrine produced both first- and second-phase platelet aggregation. The reduced response of the platelets in the newborn to these aggregating agents can be considered as a protection against disseminated intravascular coagulation (DIC), particularly in high-risk situations.

A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.

Since September 1961, a prospective study of premedical and science students has been conducted at the University of British Columbia. On completion of their sophomore year, after a year of changes from one group to another or withdrawal from either group, only 82 students existed in the diminished Premedical cohort while 137 students existed in the augmented Science cohort. These two groups have now become similar demographically, and their academic potential, as indicated by achievement and ability tests, has also become similar in terms of their mean test scores. In academic performance the present Premedical cohort has displayed some superiority over the Science cohort in high school, freshman and sophomore years. The sophomore premedical mean grade was 66.0% compared with 63.7% for the Science cohort. The hypothesis is developed that these findings reflect a difference in motivation, and therefore that perception of ultimate career goals will motivate and affect academic performance of students in their undergraduate years.

All applicants and those who subsequently enrolled for the 1964-65 session in the Western medical schools were studied with the hope that it would encourage a national registration of applicants. Seven hundred and sixty-four applicants completed 865 applications for 288 places in four schools. Although the principal factor in selecting medical students in all Western schools is pre-medical performance, 49 "good-quality" (academically of good standing and under 30 years of age) resident applicants were not accepted in their own provincial school, and 49 places were filled with "poor-quality" students.The loss of good applicants to the Western medical schools and the 20% overlap of each school's applicant pool with that of other schools suggests that objective standards of quality must be developed, and that a regular annual national assessment of applicants should be conducted by the Association of Canadian Medical Colleges.

Background Standard tibial motor nerve conduction measures are established with recording from the abductor hallucis. This technique is often technically challenging and clinicians have difficulty interpreting the information particularly in the short segment needed to assess focal tibial nerve entrapment at the medial ankle as occurs in posterior tarsal tunnel syndrome. The flexor hallucis brevis (FHB) has been described as an alternative site for recording tibial nerve function in those with posterior tarsal tunnel syndrome. Normative data has not been established for this technique. This pilot study describes the technique in detail. In addition we provide reference values for medial and lateral plantar orthodromic sensory measures and assessed intrarater reliability for all measures. Methods Eighty healthy female participants took part, and 39 returned for serial testing at 4 time points. Mean values ± SD were recorded for nerve conduction measures, and coefficient of variation as well as intraclass correlation coefficients (ICC) were calculated. Results Motor latency, amplitude and velocity values for the FHB were 4.1 ± 0.9 msec, 8.0 ± 3.0 mV and 45.6 ± 3.4 m/s, respectively. Sensory latencies, amplitudes, and velocities, respectively, were 2.8 ± 0.3 msec, 26.7 ± 10.1 μV, and 41.4 ± 3.5 m/s for the medial plantar nerve and 3.2 ± 0.5 msec, 13.3 ± 4.7 μV, and 44.3 ± 4.0 msec for the lateral plantar nerve. All values demonstrated significant ICC values (P ≤ 0.007). Conclusion Motor recording from the FHB provides technically clear waveforms that allow for an improved ability to assess tibial nerve function in the short segments used to assess tarsal tunnel syndrome. The reported means will begin to establish normal values for this technique.

A highly photosensitive analogue of thymidine, 5-azidodeoxyuridine 5'-triphosphate, has been incorporated into 61-base pair (bp) DNA fragments corresponding to the central region of Xenopus somatic-type 5 S RNA genes such that 5-azidodeoxyuridine replaces some or all T residues in either the coding or noncoding strand of the TFIIIA binding site. Photolysis of TFIIIA.DNA complexes formed with these probes results in efficient, sequence-specific cross-linking to the Zn-finger protein providing direct evidence that this class of proteins have contacts in the major groove of their target sequence. Of the 20 T residues present in the 61-bp probes, greater than 90% of the cross-linking occurs from two sites in the 5 S RNA gene corresponding to T residues at positions 84 and 88 in the noncoding and coding strands, respectively. Digestion by V8 protease of the complex formed with the noncoding strand probe releases peptides not bound to the DNA. Amino acid sequence analysis of the remaining, cross-linked peptides indicates the region including zinc-finger 2 plus the finger 2-3 linker is in contact with position 84. The linker region between fingers 5 and 6 is also in close proximity to the major groove somewhere upstream from position 84.

(1978). STEEL TRANSFORMATION DIAGRAMS: atlas of continuous cooling transformation diagrams for engineering steels. Metal Science: Vol. 12, No. 3, pp. 166-166.