Supplementing an energy adequate, higher protein diet with protein does not enhance fat-free mass restoration after short-term severe negative energy balance.

Negative energy balance during military operations can be severe and result in significant reductions in fat-free mass (FFM). Consuming supplemental high-quality protein following such military operations may accelerate restoration of FFM. Body composition (dual-energy X-ray absorptiometry) and whole body protein turnover (single-pool [15N]alanine method) were determined before (PRE) and after 7 days (POST) of severe negative energy balance during military training in 63 male US Marines (means ± SD, 25 ± 3 yr, 84 ± 9 kg). After POST measures were collected, volunteers were randomized to receive higher protein (HIGH: 1,103 kcal/day, 133 g protein/day), moderate protein (MOD: 974 kcal/day, 84 g protein/day), or carbohydrate-based low protein control (CON: 1,042 kcal/day, 7 g protein/day) supplements, in addition to a self-selected, ad libitum diet, for the 27-day intervention (REFED). Measurements were repeated POST-REFED. POST total body mass (TBM; -5.8 ± 1.0 kg, -7.0%), FFM (-3.1 ± 1.6 kg, -4.7%), and net protein balance (-1.7 ± 1.1 g protein·kg-1·day-1) were lower and proteolysis (1.1 ± 1.9 g protein·kg-1·day-1) was higher compared with PRE (P < 0.05). Self-selected, ad libitum dietary intake during REFED was similar between groups (3,507 ± 730 kcal/day, 2.0 ± 0.5 g protein·kg-1·day-1). However, diets differed by protein intake due to supplementation (CON: 2.0 ± 0.4, MOD: 3.2 ± 0.7, and HIGH: 3.5 ± 0.7 g·kg-1·day-1; P < 0.05) but not total energy (4,498 ± 725 kcal/day). All volunteers, independent of group assignment, achieved positive net protein balance (0.4 ± 1.0 g protein·kg-1·day-1) and gained TBM (5.9 ± 1.7 kg, 7.8%) and FFM (3.6 ± 1.8 kg, 5.7%) POST-REFED compared with POST (P < 0.05). Supplementing ad libitum, energy-adequate, higher protein diets with additional protein may not be necessary to restore FFM after short-term severe negative energy balance.NEW & NOTEWORTHY This article demonstrates 1) the majority of physiological decrements incurred during military training (e.g., total and fat-free mass loss), with the exception of net protein balance, resolve and return to pretraining values after 27 days and 2) protein supplementation, in addition to an ad libitum, higher protein (~2.0 g·kg-1·day-1), energy adequate diet, is not necessary to restore fat-free mass following short-term severe negative energy balance.

Shank vessel injuries: the forgotten vascular injuries.

INTRODUCTION: Vascular injuries in branch vessels of the popliteal artery, such as the tibioperoneal trunk, and shank vessels, such as anterior, posterior tibial, and peroneal vessels, occur in both blunt and penetrating trauma. Their management has evolved significantly in the past few decades. While their incidence is variable, limb loss and morbidity remain significant. MATERIAL AND METHODS: Physical examination, along with measuring an Ankle-Brachial Index (ABI), is still sometimes all that is required for diagnosis and can expeditiously triage those that require urgent operation. Despite our technological advancements and newer algorithms for lower extremity vascular trauma, operative intervention and exposure still remain difficult and pose a great challenge for surgeons that normally do not operate on this area. CONCLUSIONS: Shank vessel injuries still comprise a significant proportion of combat and civilian vascular injuries, and modern advances have led to a dramatic decrease in amputation rates.

Subclavian vessel injuries: difficult anatomy and difficult territory.

INTRODUCTION: Thoracic and thoracic related vascular injuries represent complex challenges to the trauma surgeon. Subclavian vessel injuries, in particular, are uncommon and highly lethal. Regardless of the mechanism, such injuries can result in significant morbidity and mortality. MATERIALS AND METHODS: Systematic review of the literature, with emphasis on the diagnosis, treatment and outcomes of these injuries, incorporating the authors' experience. CONCLUSIONS: These injuries are associated with significant morbidity and mortality. Patients who survive transport are subject to potentially debilitating injury and possibly death. Management of these injuries varies, depending on hemodynamic stability, mechanism of injury, and associated injuries. Despite significant advancements, mortality due to subclavian vessel injury remains high.

Operative exposure and management of axillary vessel injuries.

INTRODUCTION: Axillary vessel injuries are uncommon and challenging injuries encountered by trauma surgeons. Proximity of this vessel to other adjacent veins including the axillary vein, brachial plexus and the osseous structures of the shoulder and upper arm account for a large number of associated injuries. MATERIALS AND METHODS: Systematic review of the literature, with emphasis on the diagnosis, treatment and outcomes of these injuries, incorporating the authors' experience. CONCLUSIONS: Although uncommon, axillary arterial injuries can result in significant morbidity, limb loss and mortality. Early diagnosis and timely repair of the artery leads to good outcomes.

RADIOSENSITIVITY TO HIGH ENERGY IRON IONS IS INFLUENCED BY HETEROZYGOSITY for ATM, RAD9 and BRCA1.

Loss of function of DNA repair genes has been implicated in the development of many types of cancer. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced, for example by ionizing radiation. Since the effect of heterozygosity for one gene is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes critical to pathways that control DNA damage signaling, repair or apoptosis. We investigated the role of heterozygosity for Atm, Rad9 and Brca1 on cell oncogenic transformation and cell survival induced by 1GeV/n 56Fe ions. Our results show that cells heterozygous for both Atm and Rad9 or Atm and Brca1 have high survival rates and are more sensitive to transformation by high energy Iron ions when compared with wild-type controls or cells haploinsufficient for only one of these proteins. Since mutations or polymorphisms for similar genes exist in a small percentage of the human population, we have identified a radiosensitive sub-population. This finding has several implications. First, the existence of a radiosensitive sub-population may distort the shape of the dose-response relationship. Second, it would not be ethical to put exceptionally radiosensitive individuals into a setting where they may potentially be exposed to substantial doses of radiation.

The radiation-induced bystander effect for clonogenic survival.

It has long been accepted that the radiation-induced heritable effects in mammalian cells are the result of direct DNA damage. Recent evidence, however, suggests that when a cell population is exposed to a low dose of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. Experiments involving the Columbia University microbeam, which allows a known fraction of cells to be traversed by a defined number of alpha particles, have demonstrated a bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. When 1 to 16 alpha particles were passed through the nuclei of 10% of a C3H 10T(1/2) cell population, more cells were unable to form colonies than were actually traversed by alpha particles. Both hit and non-hit cells contributed to the outcome of the experiments. The present work was undertaken to assess the bystander effect of radiation in only non-hit cells. For this purpose, Chinese hamster V79 cells transfected with hygromycin- or neomycin-resistance genes were used. V79 cells stably transfected with a hygromycin resistance gene and stained with a nuclear dye were irradiated with the charged-particle microbeam in the presence of neomycin-resistant cells. The biological effect was studied in the neomycin-resistant V79 cells after selective removal of the hit cells with geneticin treatment.

The effects of ginseng, ephedrine, and caffeine on cognitive performance, mood and energy.

A variety of claims regarding the purported energy-enhancing properties of nutritional supplements and food constituents have recently been made. It appears that the supplements most frequently associated with such assertions are ginseng, ephedrine, and caffeine. Claims of increased energy are difficult to evaluate objectively because their meaning is not usually defined or specified. Often it is not clear whether the claims refer to physical or mental energy or both. Furthermore, an agreed upon scientific definition of either physical or mental energy enhancement does not exist. In spite of obvious differences in what the term physical energy, as opposed to mental energy implies, there is no clear scientific consensus on whether there is a difference between the two types of energy. Because the substances in question have been anecdotally associated with improvements in both physical and mental performance, their effects on both functions will be discussed, but with an emphasis placed on cognitive function and mood. Of the three substances discussed, caffeine's effects on cognitive and physical function, mood, and energy are best understood. It is clear that this food/drug enhances these functions when administered in moderate doses. Ephedrine may also enhance certain physical and mental functions related to "energy," but the evidence that ginseng has such properties is exceedingly weak.

ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation.

ATM (ataxia-telangiectasia-mutated) is a Ser/Thr kinase involved in cell cycle checkpoints and DNA repair. Human Rad9 (hRad9) is the homologue of Schizosaccharomyces pombe Rad9 protein that plays a critical role in cell cycle checkpoint control. To examine the potential signaling pathway linking ATM and hRad9, we investigated the modification of hRad9 in response to DNA damage. Here we show that hRad9 protein is constitutively phosphorylated in undamaged cells and undergoes hyperphosphorylation upon treatment with ionizing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU). Interestingly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM. Ser(272) of hRad9 is phosphorylated directly by ATM in vitro. Furthermore, hRad9 is phosphorylated on Ser(272) in response to IR in vivo, and this modification is delayed in ATM-deficient cells. Expression of hRad9 S272A mutant protein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S checkpoint activation and increased cellular sensitivity to IR. Together, our results suggest that the ATM-mediated phosphorylation of hRad9 is required for IR-induced checkpoint activation.

Tyrosine improves behavioral and neurochemical deficits caused by cold exposure.

The effects of acute cold stress were assessed behaviorally and neurochemically. The norepinephrine (NE) precursor, tyrosine (TYR), the catecholamine-releasing compound, amphetamine (AMPH), and the adrenoceptor agonist, phenylpropanolamine (PPA), were administered systemically either alone or in conjunction with TYR 30 min prior to cold exposure. All three sympathomimetic treatments dose-dependently improved performance in a forced swim test following hypothermia (T(c)=30 degrees C). AMPH/TYR or PPA/TYR combinations further improved performance vs. either agent given alone. Microdialysis showed elevated hippocampal NE concentrations in response to hypothermia. TYR further elevated NE concentration in cold/restrained rats vs. saline (SAL)-treated controls. These results suggest that sympathomimetic agents, including the nutrient TYR, which enhance noradrenergic function, improve performance in animals acutely stressed by hypothermia.

Mutant alleles of Schizosaccharomyces pombe rad9(+) alter hydroxyurea resistance, radioresistance and checkpoint control.

Schizosaccharomyces pombe rad9 mutations can render cells sensitive to hydroxyurea (HU), gamma-rays and UV light and eliminate associated checkpoint controls. In vitro mutagenesis was performed on S.pombe rad9 and altered alleles were transplaced into the genome to ascertain the functional significance of five groups of evolutionarily conserved amino acids. Most targeted regions were changed to alanines, whereas rad9-S3 encodes a protein devoid of 22 amino acids normally present in yeast but absent from mammalian Rad9 proteins. We examined whether these rad9 alleles confer radiation and HU sensitivity and whether the sensitivities correlate with checkpoint control deficiencies. One rad9 mutant allele was fully active, whereas four others demonstrated partial loss of function. rad9-S1, which contains alterations in a BH3-like domain, conferred HU resistance but increased sensitivity to gamma-rays and UV light, without affecting checkpoint controls. rad9-S2 reduced gamma-ray sensitivity marginally, without altering other phenotypes. Two alleles, rad9-S4 and rad9-S5, reduced HU sensitivity, radiosensitivity and caused aberrant checkpoint function. HU-induced checkpoint control could not be uncoupled from drug resistance. These results establish unique as well as overlapping functional domains within Rad9p and provide evidence that requirements of the protein for promoting resistance to radiation and HU are not identical.

PCNA interacts with hHus1/hRad9 in response to DNA damage and replication inhibition.

The hHus1 and several hRad proteins are involved in the control of DNA integrity checkpoints, although the mechanisms underlying these processes are unknown. Using a yeast two-hybrid system to detect protein-protein interactions, we found that human proliferating cell nuclear antigen (PCNA), a protein known to function in both DNA replication and repair, interacts with the human checkpoint-related protein Hus1 (hHus1). In human skin fibroblast cells, exposure to ionizing radiation of hydroxyurea triggers translocation of hHus1 from the cytosol to the nucleus, where it associates with PCNA as well as another checkpoint protein, hRad9. This nuclear translocation and the complex formation or hHus1 with PCNA and hRad9 correlate closely with changes in cell cycle distribution in response to radiation exposure. These results suggest that this multi-protein complex may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.

Schizosaccharomyces pombe Rad9 contains a BH3-like region and interacts with the anti-apoptotic protein Bcl-2.

Here we report that the Schizosaccharomyces pombe Rad9 (SpRad9) protein contains a group of amino acids with similarity to the Bcl-2 homology 3 death domain, which is required for SpRad9 interaction with human Bcl-2 and apoptosis induction in human cells. Overexpression of Bcl-2 in S. pombe inhibits cell growth independently of rad9, but enhances resistance of rad9-null cells to methyl methanesulfonate, ultraviolet and ionizing radiation. These observations suggest that SpRad9 may represent the first member of the Bcl-2 protein family identified in yeast, though the cell death pathways in S. pombe may differ from those found in mammals.

Antisense oligonucleotide sequences targeting the muscarinic type 2 acetylcholine receptor enhance performance in the Morris water maze.

Blocking autoinhibitory muscarinic type 2 (m2) acetylcholine receptors in the central nervous system may increase the release of acetylcholine and improve learning and memory. Antisense oligonucleotides (OGNs) complementary to m2 receptor mRNA were synthesized and evaluated for their efficacy at decreasing receptor number and reversing deficits in a cognitive task. Three antisense OGNs, which decreased m2 receptor binding in NG108-15 cells, were continuously infused into the lateral cerebral ventricle of rats for 6 days at a rate of 0.5 micro1/h and a daily dose of 72 microg. Performance in the Morris water maze was compared to groups receiving control OGNs or vehicle alone. Decrements induced by 0.2 mg/kg of scopolamine i.p. were significantly reversed by 2 of the 3 antisense OGNs. Use of antisense OGNs targeting the m2 receptor may be a new strategy to increase cholinergic neurotransmission and improve learning and memory.

The effects of choline supplementation on physical performance.

It has been reported that plasma choline levels decrease following certain types of strenuous exercise. Preliminary findings also suggest that a drop in plasma choline may limit physical performance, while choline supplementation may delay fatigue during prolonged efforts. A double-blind crossover design was used to determine the relationship between plasma choline and performance during and after 4 hr of strenuous exercise. Volunteers (N = 14) received either a placebo or treatment beverage (8.425 g choline citrate) prior to and midway through a 4-hr load carriage treadmill exercise (3% grade at 5.6 km/h 3 20 km) carrying a total load of 34.1 kg. Following the treadmill test, run time-to-exhaustion and squat tests were performed, and perceived exertion, plasma choline, glycerophosphocholine, and phosphatidylcholine were measured. Plasma choline levels increased 128% after the run-to-exhaustion with the choline supplemented beverage but remained unchanged with the placebo beverage. No significant effects were seen with choline supplementation on any outcome performance measure. Consequently, soldiers conditioned to carry heavy loads over long distances do not deplete plasma choline as a result of a prolonged exhaustive exercise under a placebo beverage, nor do they benefit from choline supplementation to delay fatigue under the same conditions.

Physical interactions among human checkpoint control proteins HUS1p, RAD1p, and RAD9p, and implications for the regulation of cell cycle progression.

Schizosaccharomyces pombe hus1 promotes radioresistance and hydroxyurea resistance, as well as S and G2 phase checkpoint control. We isolated a human cDNA homologous to hus1, called HUS1. The major focus of this report is on a detailed analysis of the physical interactions of the HUS1-encoded protein and two other checkpoint control proteins, RAD1p and RAD9p, implicated in the cellular response to DNA damage. We found that HUS1p interacts with itself and the N-terminal region of RAD1p. In contrast, the C-terminal portion of the checkpoint protein RAD9p is essential for interacting with HUS1p and the C-terminal region of RAD1p. Since the N-terminal portion of RAD9p was previously demonstrated to participate in apoptosis, this protein likely has at least two functional domains, one that regulates programmed cell death and another that regulates cell cycle checkpoint control. Truncated versions of HUS1p are unable to bind RAD1p, RAD9p, or another HUS1p molecule. RAD1p-RAD1p and RAD9p-RAD9p interactions can be demonstrated by coimmunoprecipitation, but not by two-hybrid analysis, suggesting that the proteins associate as part of a complex but do not interact directly. Northern blot analysis indicates that HUS1 is expressed in different tissues, but the mRNA is most predominant in testis where high levels of RAD1 and RAD9 message have been detected. These studies suggest that HUS1p, RAD9p, and RAD1p form a complex in human cells and may function in a meiotic checkpoint in addition to the cell cycle delays induced by incomplete DNA replication or DNA damage.

Disorder and twinning in molecular crystals: impurity-induced effects in adipic acid.

The variation in physical properties of crystals grown in the presence of additives or impurities have previously been attributed to lattice disorder developed during crystallization. Adipic acid crystallized in the presence of a variety of stereochemically related impurities typifies such behavior with disorder manifest in variations of dissolution rates and enthalpies of solution and fusion. In this case the most extreme habit, produced by the presence of added monoalkanoic acids, is a rounded dumbbell that was suggested previously to be a twinned crystal. In this contribution such crystals are fully characterized both through their external morphology and by means of single crystal X-ray diffraction. These techniques show that these particles are not twinned but rather are disordered single crystals comprising a small number of slightly misaligned domains. The interaction between additive and substrate is modeled and new additives selected that induce the formation of true mechanical twins in adipic acid.

Ionizing radiation activates the ATM kinase throughout the cell cycle.

The ATM protein kinase is a critical intermediate in a number of cellular responses to ionizing irradiation (IR) and possibly other stresses. ATM dysfunction results in abnormal checkpoint responses in multiple phases of the cell cycle, including G1, S and G2. Though downstream targets of the ATM kinase are still being elucidated, it has been demonstrated that ATM acts upstream of p53 in a signal transduction pathway initiated by IR and can phosphorylate p53 at serine 15. The cell cycle stage-specificity of ATM activation and p53Ser15 phosphorylation was investigated in normal lymphoblastoid cell line (GM536). Ionizing radiation was found to enhance the kinase activity of ATM in all phases of the cell cycle. This enhanced activity was apparent immediately after treatment of cells with IR, but was not accompanied by a change in the abundance of the ATM protein. Since IR activates the ATM kinase in all phases of the cell cycle, DNA replication-dependent strand breaks are not required for this activation. Further, since p53 protein is not directly required for IR-induced S and G2-phase checkpoints, the ATM kinase likely has different functional targets in different phases of the cell cycle. These observations indicate that the ATM kinase is necessary primarily for the immediate response to DNA damage incurred in all phases of the cell cycle.

Influence of a carbohydrate drink on nutritional status, body composition and mood during desert training.

BACKGROUND: Nutritional intake by military personnel is typically inadequate during field exercises, potentially compromising health and performance. HYPOTHESIS: Drinking a supplemental carbohydrate (CHO) beverage will increase total caloric intake and maintain nutritional status during military training in the desert. METHODS: A total of 63 volunteers were randomly assigned to one of two groups to receive either a CHO or placebo beverage with military rations during an 11-d desert field exercise. Fluid intake was ad libitum and adequate rations were provided. Blood samples were collected twice to assess nutritional status, and nutrient intake was determined with consumption data. Mood state was examined by questionnaire. RESULTS: Energy intake was significantly higher in the CHO group (3050 kcal x d(-1) vs. 2631 kcal x d(-1)), with additional CHO from the beverage providing energy with some compensation by reduced fat and protein intake. Intakes of energy, folacin, calcium, magnesium, iron, and zinc in both groups were inadequate, with intakes significantly lower (p<0.05) for calcium, magnesium, and zinc in the CHO beverage group. Blood parameters of nutritional status remained within normal ranges with no differences between groups, but significant decreases were seen in pre-albumin. No changes in mood were seen during the training, nor after exposure to desert conditions. CONCLUSIONS: The operational ration supplemented with a CHO beverage significantly increases CHO and energy intakes compared with standard rations and maintains nutritional status for short exercises. Fortification with micronutrients most at risk for deficient intake from foods may be needed for longer deployments.

Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis.

DNA damage induces apoptosis through a signalling pathway that can be suppressed by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown. Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that RAD9, a human protein involved in the control of a cell-cycle checkpoint, interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-x L, but not with the pro-apoptotic BAX and BAD. When overexpressed in mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-x L. Conversely, antisense RAD9 RNA suppresses cell death induced by methyl methanesulphonate. These findings indicate that RAD9 may have a new role in regulating apoptosis after DNA damage, in addition to its previously described checkpoint-control and other radioresistance-promoting functions.

Pilot study on the effect of hyperimmune egg protein on elevated cholesterol levels and cardiovascular risk factors.

Coronary heart disease is the leading cause of death in the United States, accounting for almost half of all deaths. Animal studies have suggested that the daily addition of "hyperimmune egg" to one's diet might reduce the risk of cardiovascular disease. Military personnel with initial total cholesterol levels higher than 180 mg/dl were randomly enrolled in a 26-week double-blind study of a drink containing a hyperimmune egg protein. Subjects were randomly assigned to three groups: control (no drink); placebo (drink without egg); and active (drink combined with hyperimmune egg). Throughout the study this physically fit group maintained a program of strenuous exercise and participated in a dietary education program intended to reduce fat and cholesterol intake. At the end of the trial, total cholesterol levels of the control and placebo groups had increased, whereas the group that consumed the drink with hyperimmune egg showed no significant change in total cholesterol. The ratio of total cholesterol to high-density lipoproteins and the apolipoprotein B level increased in both control and placebo groups but remained essentially unchanged in the group consuming hyperimmune egg. Triglyceride and apolipoprotein A-I values did not change significantly in any of the groups. These findings suggest that hyperimmune egg may beneficially modify the regulation of serum lipoprotein levels and thereby reduce the possibility of cardiovascular diseases.