The use of video self-modelling and feedback to teach cooking skills to individuals with traumatic brain injury: a pilot study.

OBJECTIVE: To evaluate the effectiveness of video self-modelling plus prompting and feedback to teach a cooking skill to people with traumatic brain injury (TBI) and to examine skill generalization to a novel food item. RESEARCH DESIGN: Multiple probe across participants. METHODS AND PROCEDURES: Four individuals with TBI received instruction in cooking. They watched videotapes of themselves cooking and practiced that skill while receiving prompts and feedback. Treatment effects were evaluated by comparing performance before, during and after training and at a 2 and 4 week follow-up. Additionally, cooking performance on a novel food item was examined. MAIN OUTCOMES AND RESULTS: Three of the four individuals achieved criterion performance within four training sessions. Those individuals also substantially maintained their skills 2 and 4 weeks following training and generalized their skills to a novel food item. CONCLUSIONS: Video self-modelling plus prompting and feedback appears to be an effective treatment for teaching simple cooking skills to individuals with TBI. Further research should examine whether the video alone is sufficient for skill acquisition and evaluate the effectiveness of video self-modelling to teach other skills.

p73.

The discovery of p73 as a family member of p53 has instigated a number of studies in search of its function, regulation, and involvement in tumorigenesis. p73 has been identified as a transcription factor that can regulate p53-dependent transcriptional targets. Similarly to p53, p73 can induce apoptosis in response to various stimuli, including certain types of DNA damage. This evidence suggests that p73 may act as a tumor suppressor with overlapping functions of p53. While mutations of p73 appear rare in human tumors, some leukemias have shown silencing of the gene by hypermethylation. Thus, introduction of p73 into tumor cells possessing inactive p53 may provide a valuable therapeutic approach.

Biological methods for cell-cycle synchronization of mammalian cells.

Understanding the molecular and biochemical basis of cellular growth and division involves the investigation of regulatory events that most often occur in a cell-cycle phase-dependent fashion. Studies examining cell-cycle regulatory mechanisms and progression invariably require cell-cycle synchronization of cell populations. Thus, many methods have been established to synchronize cells at specific phases of the cell cycle. Several of the common methods involve pharmacological agents, which act at various points throughout the cell cycle. Because of adverse cellular perturbations resulting from many of the synchronizing drugs used, other synchrony methods that involve less perturbation of biological systems, such as serum deprivation, contact inhibition, and centrifugal elutriation have a significant advantage. The advantages and disadvantages of these cell synchronization methods are discussed in this review.

Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo.

The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G(1) cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G(1) cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G(0) quiescent cells and becomes hypophosphorylated (approximately 2 mol of PO(4) to 1 mol of pRB) in early G(1) and hyperphosphorylated (approximately 10 mol of PO(4) to 1 mol of pRB) in late G(1) phase. Here, we report that hypophosphorylated pRB, present in early G(1), represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G(0) and hyperphosphorylated pRB in late G(1) fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT-dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G(1) and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G(1). Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16(INK4a) gene, contained hypophosphorylated pRB that was bound to E2Fs in early G(1) and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G(1) cyclin-Cdk complexes.

A common E2F-1 and p73 pathway mediates cell death induced by TCR activation.

Strong stimulation of the T-cell receptor (TCR) on cycling peripheral T cells causes their apoptosis by a process called TCR-activation-induced cell death (TCR-AICD). TCR-AICD occurs from a late G1 phase cell-cycle check point independently of the 'tumour suppressor' protein p53. Disruption of the gene for the E2F-1 transcription factor, an inducer of apoptosis, causes significant increases in T-cell number and splenomegaly. Here we show that T cells undergoing TCR-AICD induce the p53-related gene p73, another mediator of apoptosis, which is hypermethylated in lymphomas. Introducing a dominant-negative E2F-1 protein or a dominant-negative p73 protein into T cells protects them from TCR-mediated apoptosis, whereas dominant-negative E2F-2, E2F-4 or p53 does not. Furthermore, E2F-1-null or p73-null primary T cells do not undergo TCR-mediated apoptosis either. We conclude that TCR-AICD occurs from a late G1 cell-cycle checkpoint that is dependent on both E2F-1 and p73 activities. These observations indicate that, unlike p53, p73 serves to integrate receptor-mediated apoptotic stimuli.

The microtubule binding of Tau and high molecular weight Tau in apoptotic PC12 cells is impaired because of altered phosphorylation.

Although the importance of the microtubule network throughout cell life is well established, the dynamics of microtubules during apoptosis, a regulated cell death process, is unclear. In a previous study (Davis, P. K., and Johnson, G. V. (1999) Biochem. J. 340, 51-58) we demonstrated that the phosphorylation of the microtubule-associated protein tau was increased during neuronal PC12 cell apoptosis. The purpose of this study was to determine whether the increased tau phosphorylation that occurred during apoptosis impaired the microtubule binding capacity of tau. This study is the first demonstration that microtubule-binding by tau and high molecular weight tau is significantly impaired as a result of altered phosphorylation during a naturally occurring process, apoptosis. Furthermore, co-immunofluorescence studies reveal for the first time that tau populations within an apoptotic neuronal PC12 cell exhibit differential phosphorylation. In control PC12 cells, Tau-1 staining (Tau-1 recognizes an unphosphorylated epitope) is evident throughout the entire cell body. In contrast, Tau-1 immunoreactivity in apoptotic PC12 cells is retained in the nuclear/perinuclear region but is significantly decreased in the cytoplasm up to the plasma membrane. The selective distribution of phosphorylated tau in apoptotic PC12 cells indicates that tau likely plays a significant role in the cytoskeletal changes that occur during apoptosis.

Dietary protein or arginine deficiency impairs constitutive and inducible nitric oxide synthesis by young rats.

Effects of dietary protein or arginine deficiency on constitutive and lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis were determined in young rats by quantifying urinary nitrate excretion. In Experiment 1, 30-d-old rats (n = 16) were divided randomly into two groups (n = 8/group) and pair-fed on the basis of body weight semipurified isocaloric diets containing 20 or 5% casein. In Experiment 2, 30-d-old rats (n = 24) were divided randomly into three groups (n = 8) and pair-fed on the basis of body weight purified isonitrogenous and isocaloric diets (composed of amino acids) containing 0.0, 0.3 or 1.0% L-arginine. In both experiments, daily collection of urine was initiated 10 d after the start of pair-feeding. On d 17 after the pair-feeding was initiated, LPS (1 mg/kg body wt) was injected intraperitoneally into rats, and urine was collected daily for an additional 7 d. In Experiments 3 and 4, activities of constitutive and inducible NO synthases were measured in macrophages and various tissues from protein- or arginine-deficient rats (n = 6). Body weight was lower in rats fed the 5% casein diet or the 0.0 and 0.3% arginine diets than in those fed 20% casein or 1% arginine, respectively. Dietary protein or arginine deficiency decreased serum concentrations of arginine and urinary nitrate excretion before and after LPS treatment, indicating impaired constitutive and inducible NO synthesis. Protein malnutrition reduced constitutive and inducible NO synthase activities in brain, heart, jejunum, lung, skeletal muscle and spleen, and inducible NO synthase activity in macrophages. Because NO is a mediator of the immune response and is the endothelium-dependent relaxing factor, impaired NO synthesis may help explain immunodeficiency and cardiovascular dysfunction in protein- or arginine-deficient subjects.

Energy metabolism and protein phosphorylation during apoptosis: a phosphorylation study of tau and high-molecular-weight tau in differentiated PC12 cells.

Apoptosis has been characterized as a regulated, energy-dependent process. Specific protein-phosphorylation events have been demonstrated previously to occur during apoptosis and may play an important role in the regulation of this death process. In this study, energy metabolism and protein phosphorylation during apoptosis of neuronal PC12 cells induced by nerve growth factor and serum deprivation was examined using [32P]Pi-labelling techniques. Although ATP levels were maintained at control levels during apoptosis, [32P]Pi incorporation into ATP was decreased significantly, coinciding with an almost identical decrease in Na+-dependent phosphate uptake. During neuronal PC12-cell apoptosis, increased phosphorylation of tau and high-molecular-weight (HMW) tau was observed within the epitope of Tau-1, a phosphate-dependent tau antibody that only recognizes the unphosphorylated form of its epitope. In addition, based on two-dimensional phosphopeptide mapping, [32P]Pi incorporation into a phosphopeptide of tau and HMW tau from apoptotic cells increased. Whereas [32P]Pi incorporation into total protein decreased to 23% of the control during apoptosis, [32P]Pi incorporation into tau and HMW tau was significantly higher, indicating a preferential phosphorylation of specific proteins during the apoptotic process. This study provides novel information about phosphate uptake, incorporation of [32P]Pi into ATP, and protein phosphorylation events during apoptosis.

A supported relationships intervention to increase the social integration of persons with traumatic brain injuries.

A supported relationships intervention was used to increase the integrated social contacts (ISCs) of 3 persons with traumatic brain injury (TBI) who were each matched with 4 community participants. The intervention consisted of asking participants to meet with their matched counterpart to engage in leisure activities once per week for 4 weeks. Additionally, community participants were provided with a brief training session on TBI, were given specific suggestions on interacting with the persons with TBI with whom they were matched, and received weekly phone calls from the researcher. Frequency of ISCs were analyzed with a multiple baseline design across participants. All 3 participants with TBI increased the frequency of ISCs after implementation of the supported relationships intervention and continued to experience more than baseline levels of ISCs during 8 weeks of follow-up. These data suggest that social integration can be enhanced with a procedure requiring limited staff intervention.

Compartmentation and kinetics of urea cycle enzymes in porcine enterocytes.

We have recently reported the synthesis of urea from ammonia, glutamine and arginine in enterocytes of postweaning pigs. The present study was conducted to determine the compartmentation and kinetics of urea cycle enzymes in these cells. Carbamoyl phosphate synthase I (CPS I) and ornithine carbamoyltransferase (OCT) were located exclusively in mitochondria, whereas argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were found in the cytosol. Arginase isozymes were present in both the cytosol and mitochondria of enterocytes, and differed in their sensitivity to heat inactivation. Except for OCT, Vmax values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their Km values. Because of a low rate of ureagenesis in enterocytes compared with the liver, intestinal urea cycle enzymes may function primarily to synthesize citrulline. The co-localization of CPS I and OCT and a high activity of OCT in enterocyte mitochondria favors the intestinal synthesis of citrulline from ammonia, HCO3- and ornithine. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes. These kinetic properties of intestinal urea cycle enzymes maximize the net synthesis of citrulline from glutamine and explain the release of large amounts of citrulline by the pig small intestine. The two compartmentally separated arginase isozymes in enterocytes may play an important role in regulating the intestinal metabolism of proline, nitric oxide and polyamines.

Select alterations in protein kinases and phosphatases during apoptosis of differentiated PC12 cells.

The involvement of cell cycle-regulatory proteins in apoptosis of neuronally differentiated PC12 cells induced by the removal of nerve growth factor and serum was examined. Three major findings are presented. (1) Cdc2 kinase protein levels increased fivefold in apoptotic PC12 cells by day 3 of serum and nerve growth factor deprivation. Histone H1 kinase activity was increased significantly in p13(suc1) precipitates of apoptotic PC12 cells, which was due to increased activation and/or expression of cdc2 kinase. (2) The protein levels of cyclin-dependent kinase 4, cyclin D, and proliferating cell nuclear antigen that are normally expressed in the cell cycle were increased during neuronal PC12 cell apoptosis. (3) The levels of the catalytic subunit, but not the regulatory subunit of the calcium/calmodulin-dependent protein phosphatase 2B, decreased significantly concomitant with a significant decrease in protein phosphatase 2B activity early in the apoptotic process. Protein phosphatase 2A activity decreased slightly but significantly after 3 days of serum and nerve growth factor deprivation, and no alterations in protein phosphatase 1 were observed during the apoptotic process. These data demonstrate that certain cell cycle-regulatory proteins are inappropriately expressed and that alterations in specific phosphorylation events, as indicated by the increase in histone H1 kinase activity and the decrease in protein phosphatase 2B activity, are most likely occurring during apoptosis of PC12 cells. These observations support the hypothesis that apoptosis may be due in part to a nondividing cell's uncoordinated attempt to reenter and progress through the cell cycle.

Endogenous synthesis of arginine plays an important role in maintaining arginine homeostasis in postweaning growing pigs.

This study was conducted to determine whether endogenous synthesis of arginine plays a role in regulating arginine homeostasis in postweaning pigs. Pigs were fed a sorghum-based diet containing 0. 98% arginine and were used for studies at 75 d of age (28.4 kg body weight). Mitochondria were prepared from the jejunum and other major tissues for measuring the activities of Delta1-pyrroline-5-carboxylate (P5C) synthase and proline oxidase (enzymes catalyzing P5C synthesis from glutamate and proline, respectively) and of ornithine aminotransferase (OAT) (the enzyme catalyzing the interconversion of P5C into ornithine). For metabolic studies, jejunal enterocytes were incubated at 37 degrees C for 30 min in Krebs-Henseleit bicarbonate buffer containing 2 mmol/L L-glutamine, 2 mmol/L L-[U-14C]proline, and 0-200 micromol/L gabaculine (an inhibitor of OAT). The activities of P5C synthase, proline oxidase and OAT were greatest in enterocytes among all of the tissues studied. Incubation of enterocytes with gabaculine resulted in decreases (P < 0.05) in the synthesis of ornithine and citrulline from glutamine and proline. When gabaculine was orally administered to pigs (0.83 mg/kg body weight) to inhibit intestinal synthesis of citrulline from glutamine and proline, plasma concentrations of citrulline (-26%) and arginine (-22%) decreased (P < 0.05), whereas those of alanine (+21%), ornithine (+17%), proline (+107%), taurine (+56%) and branched-chain amino acids (+21-40%) increased (P < 0.05). On the basis of dietary arginine intake and estimated arginine utilization, the endogenous synthesis of arginine in the 28-kg pig provided >/=50.2% of total daily arginine requirement. Taken together, our results suggest an important role for endogenous synthesis of arginine in regulating arginine homeostasis in postweaning growing pigs, as previously shown in neonatal pigs.

Increasing self-determination: teaching people with mental retardation to evaluate residential options.

The community living preferences of 4 institutionalized adults with mild mental retardation were identified using photographs that depicted a variety of residential characteristics. Individuals then were taught to obtain information regarding their preferences during tours of community group homes, to report that information to their social workers, and to evaluate the homes based on the information obtained. A multiple baseline across participants design showed that all 4 participants substantially increased their skills at asking questions, reporting information, and evaluating homes. the results indicate that people with mental retardation can take an active role in major lifestyle decisions that others have typically made for them.

Monoclonal antibody Alz-50 reacts with bovine and human serum albumin.

Alz-50, a monoclonal antibody originally prepared using Alzheimer brain homogenates, reacts with PHF-tau and normal tau on immunoblots, and stains specific neuronal populations in sections from Alzheimer's disease brain. Although the Alz-50 epitope has been mapped to amino acids 2-10 present in all human tau isoforms, minimal Alz-50 immunoreactivity is present in tissue from control brain, suggesting Alz-50 binding may be dependent on tau conformational differences. The absence of conclusive results concerning Alz-50 binding presents the possibility of Alz-50 immunoreactivity with proteins other than tau. The present study demonstrates Alz-50 cross-reactivity with denatured bovine serum albumin (BSA) and human serum albumin (HSA). Using LA-N-5 neuroblastoma cells, BSA from serum-containing media was present in cell homogenates and was found to be Alz-50-reactive on immunoblots. In fact, Alz-50 (0.1 microgram/ml) recognized as little as 78 ng of BSA and 312 ng of HSA. Since Alz-50 does not recognize native BSA, blocking of immunoblots with 3% BSA did not alter Alz-50 reactivity with tau from LA-N-5 cells. On SDS-polyacrylamide gels, HSA (approximately 69 kDa) migrates very closely to the pattern of A68 (PHF-tau) from Alzheimer brain homogenates. Hence, the presence of BSA or other albumins in cell or brain homogenates may be an important concern when using the Alz-50 antibody.

Gas chromatographic determination of p-chloroaniline in a chlorhexidine digluconate-containing alcohol foam surgical scrub product.

A gas chromatographic (GC) method with flame ionization detection was developed to separate and quantitate p-chloroaniline (PCA) from other components in a chlorhexidine digluconate (CHG)-containing alcohol foam surgical scrub product. A simple sample preparation method was developed in which 1-butanol was used to dissolve the foam and precipitate the CHG, which otherwise would interfere with the GC analysis. The method was validated with respect to linear dynamic range, precision, accuracy, selectivity, limit of detection, and limit of quantitation.

A group-oriented contingency to increase leisure activities of adults with traumatic brain injury.

An interdependent group-oriented contingency and graphic feedback were used to increase the activity levels of residents of a group home for persons with traumatic brain injury. Results showed that the intervention was effective for 4 of the 6 subjects. Individual performances must be examined when implementing group contingencies because all subjects may not respond.

"Would I be able to ... "? Teaching clients to assess the availability of their community living life style preferences.

A three-phase program was developed to involve six institutionalized adults with mild mental retardation in their transition to community living. In Phase I, subjects were interviewed to determine their community living life style preferences and were found to be reliable and skillful in stating their preferences. In Phase II, the subjects' 10 strongest preferences were identified. In Phase III, they were taught to obtain preference availability information from group home representatives and report these findings to their social worker. A simultaneous replication design across two component skills, questioning and reporting, revealed that both increased after training and generalized to community group homes. The 5 subjects available for follow-up maintained their posttraining performance. Implications of these results in extending choice and decision-making technology were discussed.

Anoxia-induced changes in extracellular K+ and pH in mammalian central white matter.

In gray matter (GM), anoxia induces prominent extracellular ionic changes that are important in understanding the pathophysiology of this insult. White matter (WM) is also injured by anoxia but the accompanying changes in extracellular ions have not been studied. To provide such information, the time course and magnitude of anoxia-induced changes in extracellular K+ concentration ([K+]o) and extracellular pH (pHo) were measured in the isolated rat optic nerve, a representative central WM tract, using ion-selective microelectrodes. Anoxia produced less extreme changes in [K+]o and pHo in WM than are known to occur in GM; in WM during anoxia, the average maximum [K+]o was 14 +/- 2.9 mM (bath [K+]o = 3 mM) and the average maximum acid shift was 0.31 +/- 0.07 pH unit. The extracellular space volume rapidly decreased by approximately 20% during anoxia. Excitability of the rat optic nerve, monitored as the amplitude of the supramaximal compound action potential, was lost in close temporal association with the increase in [K+]o. Increasing the bath glucose concentration from 10 to 20 mM resulted in a much larger acid shift during anoxia (0.58 +/- 0.08 pH unit) and a smaller average increase in [K]o (9.2 +/- 2.6 mM). The increased extracellular glucose concentration presumably provided more substrate for anaerobic metabolism, resulting in more extracellular lactate accumulation (although not directly measured) and a greater acid shift. Enhanced anaerobic metabolism during anoxia would provide energy for operation of ion pumps, including the sodium pump, that would result in smaller changes in [K+]o. These effects were probably responsible for the observation that the optic nerve showed significantly less damage after 60 min of anoxia in the presence of 20 mM glucose compared to 10 mM glucose. Under normoxic conditions, increasing bath K+ concentration to 30 mM (i.e., well beyond the level shown to occur with anoxia) for 60 min caused abrupt loss of excitability during the period of application but minimal change in the amplitude of the compound action potential following the period of exposure. The anoxia-induced increase in [K+]o, therefore, was not itself directly responsible for irreversible loss of optic nerve function. These observations indicate that major qualitative differences exist between mammalian GM and WM with regard to anoxia-induced extracellular ionic changes.