Minimalist style boot improves running but not walking economy in trained men.

This study examined movement economy under load with 1000 g minimalist (MIN) vs. 1600 g traditional (TRD) style boots. Fourteen trained, male participants completed a VO2peak test (46.6 ± 7.3 ml/kg/min) while wearing a 16 kg external load. Treadmill speeds for the running economy (RE) trials were determined by the slowest pace in which participants completed a full stage with a running gait pattern during the VO2peak test. Walking economy (WE) pace was 1.6 km/h slower than RE pace. During the second session, participants completed 5-min exercise bouts at WE and RE pace under load wearing MIN and TRD. There were no differences for any measured variables during WE trials. In contrast, RE (MIN = 2.95 ± 0.28 vs. TRD = 3.04 ± 0.30 L/min; p = .003: Cohen's d = 0.32), respiratory exchange ratio (p < .001), and perceptual measures (p < .05) were all improved while wearing MIN. Practitioner summary: In trained men, 1000 g/pair minimalist style boots (MIN) resulted in improvements of approximately 3% and 5% for running economy and respiratory exchange ratio versus 1600 g/pair traditional boots while wearing a 16 kg kit. Perceptual responses, including comfort, also favoured MIN. These effects were not found at walking pace. Abbreviations: MIN: minimalist style boots; TRD: traditional style boots; RE: running economy; WE: walking economy; ES: effect size; RER: respiratory exchange ratio; HR: heart rate.

Sweat gland density and response during high-intensity exercise in athletes with spinal cord injuries.

Sweat production is crucial for thermoregulation. However, sweating can be problematic for individuals with spinal cord injuries (SCI), as they display a blunting of sudomotor and vasomotor responses below the level of the injury. Sweat gland density and eccrine gland metabolism in SCI are not well understood. Consequently, this study examined sweat lactate (S-LA) (reflective of sweat gland metabolism), active sweat gland density (SGD), and sweat output per gland (S/G) in 7 SCI athletes and 8 able-bodied (AB) controls matched for arm ergometry VO2peak. A sweat collection device was positioned on the upper scapular and medial calf of each subject just prior to the beginning of the trial, with iodine sweat gland density patches positioned on the upper scapular and medial calf. Participants were tested on a ramp protocol (7 min per stage, 20 W increase per stage) in a common exercise environment (21±1°C, 45-65% relative humidity). An independent t-test revealed lower (p<0.05) SGD (upper scapular) for SCI (22.3 ±14.8 glands · cm(-2)) vs. AB. (41.0 ± 8.1 glands · cm(-2)). However, there was no significant difference for S/G between groups. S-LA was significantly greater (p<0.05) during the second exercise stage for SCI (11.5±10.9 mmol · l(-1)) vs. AB (26.8±11.07 mmol · l(-1)). These findings suggest that SCI athletes had less active sweat glands compared to the AB group, but the sweat response was similar (SLA, S/G) between AB and SCI athletes. The results suggest similar interglandular metabolic activity irrespective of overall sweat rate.

Multidimensional Treatment Foster Care for Adolescents in English care: randomised trial and observational cohort evaluation.

BACKGROUND: Children in care often have poor outcomes. There is a lack of evaluative research into intervention options. AIMS: To examine the efficacy of Multidimensional Treatment Foster Care for Adolescents (MTFC-A) compared with usual care for young people at risk in foster care in England. METHOD: A two-arm single (assessor) blinded randomised controlled trial (RCT) embedded within an observational quasi-experimental case-control study involving 219 young people aged 11-16 years (trial registration: ISRCTN 68038570). The primary outcome was the Child Global Assessment Scale (CGAS). Secondary outcomes were ratings of educational attendance, achievement and rate of offending. RESULTS: The MTFC-A group showed a non-significant improvement in CGAS outcome in both the randomised cohort (n = 34, adjusted mean difference 1.3, 95% CI -7.1 to 9.7, P = 0.75) and in the trimmed observational cohort (n = 185, adjusted mean difference 0.95, 95% CI -2.38 to 4.29, P = 0.57). No significant effects were seen in secondary outcomes. There was a possible differential effect of the intervention according to antisocial behaviour. CONCLUSIONS: There was no evidence that the use of MTFC-A resulted in better outcomes than usual care. The intervention may be more beneficial for young people with antisocial behaviour but less beneficial than usual treatment for those without.

Effects of caffeine on session ratings of perceived exertion.

This study examined effects of caffeine on session ratings of perceived exertion (RPE) following 30 min constant-load cycling. Individuals (n = 15) of varying aerobic fitness completed a [Formula: see text] max trial and two 30 min cycling bouts (double-blind, counterbalanced) following ingestion of 6 mL/kg of caffeine or matched placebo. RPE overall, legs and breathing were estimated every 5 min and session RPE was estimated 30 min post-exercise using the OMNI pictorial scale. Session RPE for caffeine and placebo trails were compared using paired t test. Between-trial comparisons of HR, RPE overall, RPE legs and RPE breathing were analyzed using an independent 2 (trial) × 6 (time point) repeated measures analysis of variance (ANOVA) for each dependent variable. Caffeine resulted in a significantly lower session RPE (p < 0.05) for caffeine (6.1 ± 2.2) versus placebo (6.8 ± 2.1). Acute perceptual responses were significantly lower for caffeine for RPE overall (15, 20, 25, and 30 min), RPE breathing (15, 20, 25, and 30 min) and RPE legs (20 and 30 min). Survey responses post-exercise revealed greater feelings of nervousness, tremors, restlessness and stomach distress following caffeine versus placebo. Blunted acute RPE and survey responses suggest participants responded to caffeine ingestion. Caffeine decreased acute RPE during exercise which could partially account for lower session RPE responses. However, decreased session RPE could also reveal a latent analgesic affect of caffeine extending into recovery. Extending the understanding of session RPE could benefit coaches in avoiding overtraining when adjusting training programs.

Limited role of CD28-mediated signals in T helper subset differentiation.

The role of CD28-mediated signals in T helper cell maturation is not fully understood. We tested the requirement for costimulation through CD28 in several systems of CD4+, T cell differentiation. In vivo priming of mice with genetic disruption of CD28 (CD28-/-) yielded normal levels of antigen-specific interferon gamma production but markedly diminished levels of interleukin 4 (IL-4) after in vitro restimulation. In response to the pathogenic microbe, Leishman a major, C57BL6 CD28-/- mice were fully capable of controlling infection and exhibited a normal T helper 1 response. BALB/c CD28-/- mice unexpectedly exhibited normal susceptibility to L. major. BALB/c CD28-/- mice developed high levels of IL-4 mRNA and protein induction in the draining lymph nodes. In addition, susceptibility of BALB/c CD28-/- mice was reversed by neutralization of IL-4 in vivo. We also activated transgenic CD28-bearing T cells from the BALB and C57BL background in vitro in the presence of CTLA4Ig. BALB cells had greater IL-4 producing capacity than C57BL cells in the absence of costimulation. Diverse factors including costimulatory signals, genetic polymorphism, and the nature of the immunogen all influence T helper phenotype commitment, but these results provide evidence that CD28 is not an absolute requirement for generating either Th1 or Th2 responses.

CD43 is a murine T cell costimulatory receptor that functions independently of CD28.

Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.

Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.

Evaluation of artificial sweat in athletes with spinal cord injuries.

Athletes with spinal cord injury often experience high heat storage due to reduced sweating capacity below the spinal injury. Spray bottle (SB) may be used to apply mist for evaporative cooling during breaks in competitions. This study examined the efficacy of SB during rest breaks. Seven participants, four female and three males, (mean +/- SD age 24 +/- 4.1 year, weight 56.2 +/- 7.0 kg, upper-body VO(2) peak 2.4 +/- 0.6 l/min) volunteered for the study. Participants were paraplegic athletes (T3-T12/L1) with both complete and incomplete lesions. Participants arm-cranked using a ramp protocol in an environment of 21 +/- 1.5 degrees C and 55 +/- 3% rh once using a SB during 1-min rest between 7-min stages of increasing intensity and once without the SB (CON). Mean total work was similar (p = 0.86) for the SB and CON (2495.7 +/- 914.6 vs. 2407.1 +/- 982.3 kJ, respectively). Likewise, the mean work times were similar between trials (27 +/- 6 and 26 +/- 7 min for SB and CON, respectively). Furthermore, there were no significant differences detected between trials for skin temperature, rectal temperature, esophageal temperature (p > 0.05). There were no statistically significant differences detected between trials for RPE (p > 0.05). In conclusion, the application of artificial sweat via SB was ineffective in attenuating the onset of uncompensable heat strain during high-intensity arm exercise in a comfortable environment.

Effect of gender on fatigue and recovery following maximal intensity repeated sprint performance.

AIM: This study investigated the effects of gender on repeated, maximal-intensity intermittent sprint exercise following variable day-to-day recovery periods. METHODS: Sixteen volunteers (8 men, 8 women) performed four trials of high-intensity intermittent sprint exercise consisting of three bouts of eight 30 m sprints (total of 24 sprints). Following completion of the baseline trial, in repeated-measures design, participants were assigned, in counter-balanced order, variable recovery periods of 24, 48, and 72 h whereupon they repeated an identical exercise trial. RESULTS: Results from a series of 4 (trial) x 3 (bout) repeated measures ANOVAs revealed men produced significantly (P < 0.01) faster times throughout all bouts and trials of repeated sprint exercise. Additionally, women exhibited significantly lower (P < 0.05) blood lactate concentration and significantly lower (P < 0.05) decrement in performance, indicating increased resistance to fatigue during repeated exercise sessions. There were no significant differences (P > 0.05) between genders for heart rate or rating of perceived exertion during or following trials. There were no significant differences for overall sprint performance within either gender among trials. CONCLUSION: These results indicate men, while able to produce higher absolute power outputs (i.e., lower sprint time), demonstrate higher decrement scores within a trial compared to women, thus suggesting women may recover faster and fatigue less. Also, gender differences affecting recovery within in a trial were observed to be diminished between trials (i.e., day-to-day recovery) of maximal intermittent sprint work evidenced by the observed stability of performance between trials following various recovery durations.

Role for a glycan phosphoinositol anchor in Fc gamma receptor synergy.

While many cell types express receptors for the Fc domain of IgG (Fc gamma R), only primate polymorphonuclear neutrophils (PMN) express an Fc gamma R linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked Fc gamma R (Fc gamma RIIIB) cooperates with the transmembrane Fc gamma R (Fc gamma RIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fc gamma receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fc gamma receptors. Jurkat T cells were stably transfected with cDNA encoding Fc gamma RIIA and/or Fc gamma RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by stimulation of either Fc gamma RIIA or Fc gamma RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking Fc gamma RIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca2+]i rise, as did crosslinking CD59 with Fc gamma RIIA on PMN, suggesting that interactions between the extracellular domains of the two Fc gamma receptors are not required for synergy. Replacement of the GPI anchor of Fc gamma RIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the Fc gamma RIIA cytoplasmic tail abolished synergy. While the ITAM of Fc gamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc gamma RIIA was diminished when cocrosslinked with Fc gamma RIIIB. These data demonstrate that Fc gamma RIIA association with GPI-linked proteins facilitates Fc gamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc gamma R of human PMN.

Role of cholesterol in formation and function of a signaling complex involving alphavbeta3, integrin-associated protein (CD47), and heterotrimeric G proteins.

Integrin-associated protein (CD47) is a multiply membrane spanning member of the immunoglobulin superfamily that regulates some adhesion-dependent cell functions through formation of a complex with alphavbeta3 integrin and trimeric G proteins. Cholesterol is critical for the association of the three protein components of the supramolecular complex and for its signaling. The multiply membrane spanning domain of IAP is required for complex formation because it binds cholesterol. The supramolecular complex forms preferentially in glycosphingolipid-enriched membrane domains. Binding of mAb 10G2 to the IAP Ig domain, previously shown to be required for association with alphavbeta3, is affected by both the multiply membrane spanning domain and cholesterol. These data demonstrate that cholesterol is an essential component of the alphavbeta3/IAP/G protein signaling complex, presumably acting through an effect on IAP conformation.

Physiological and comfort effects of a commercial "cooling cap" worn under protective helmets.

Ballistic protective helmets can impair heat dissipation. A cooling device in the helmet (cooling pad, CP) could help prevent heat problems in military personnel and potentially enhance comfort. This study examined the effects of CP on rectal and skin temperatures, heart rate, percent change in plasma volume, urine specific gravity, rating of perceived exertion, and other subjective measures while performing light work in a hot environment. It was hypothesized that the CP would act as an insulator to the head, which would not positively affect any physiological variable but could positively affect wearer subjective comfort or temperature. Participants performed a work protocol for approximately 2 hr. A ballistic vest, slacks, short-sleeved button-up shirt, and a ballistic helmet (one trial with CP and one trial without) were worn. Repeated measures analysis of variance (ANOVA) showed no differences (p > 0.05) between wearing and not wearing the CP for any physiological parameter. However, participants perceived the CP as cooler (p = 0.002). Other trends in perceptual data such as thermal strain and helmet comfort indicated the CP felt cooler. However, based on forehead temperature and participant comments, the CP lost its cooling ability relatively quickly (within approximately 30 min).

Cloning of the gene encoding peptide-binding protein 74 shows that it is a new member of the heat shock protein 70 family.

We have previously described peptide-binding proteins of 72 and 74 kDa (PBP72/74), which have been implicated as playing a role in antigen processing and are serologically related to the 70-kDa heat shock protein (hsp70) family. Here we report the cloning and sequencing of the cDNA encoding PBP74 in mice and in humans, accomplished by using amino acid sequence information obtained from the purified protein. We show that PBP74 is highly homologous to members of the hsp70 family but, significantly, is not identical to any known member of this family. Inspection of the cDNA nucleotide sequence indicates that it encodes a 46-residue N-terminal peptide which is not present in the mature protein. Transcription and translation in vitro of the PBP74 cDNA verified that it encodes a form of PBP74 which is larger than the mature protein. The presequence does not conform to known motifs for organelle-targeting sequences, and at present, its function is not known. By confocal microscopy, PBP74 was localized to cytoplasmic vesicles but not to the nucleus, mitochondria, or plasma membrane by using antibodies specific for the N-terminal 16 residues of PBP74. By RNA filter hybridization analysis, PBP74 mRNAs are detected in all cell types tested. Exposure of cells to heat shock does not result in an increase in the mRNA levels of PBP74, unlike the dramatic increase observed for the stress-inducible hsp70 mRNA. Thus, PBP74 appears to be a constitutive, new member of the hsp70 family.

The amphiregulin gene encodes a novel epidermal growth factor-related protein with tumor-inhibitory activity.

We have isolated the gene for a novel growth regulator, amphiregulin (AR), that is evolutionarily related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). AR is a bifunctional growth modulator: it interacts with the EGF/TGF-alpha receptor to promote the growth of normal epithelial cells and inhibits the growth of certain aggressive carcinoma cell lines. The 84-amino-acid mature protein is embedded within a 252-amino-acid transmembrane precursor, an organization similar to that of the TGF-alpha precursor. Human placenta and ovaries were found to express significant amounts of the 1.4-kilobase AR transcript, implicating AR in the regulation of normal cell growth. In addition, the AR gene was localized to chromosomal region 4q13-4q21, a common breakpoint for acute lymphoblastic leukemia.

Effect of re-implanted particles from intramedullary reaming on mechanical properties and callus formation. A laboratory study.

This study investigated the quality and quantity of healing of a bone defect following intramedullary reaming undertaken by two fundamentally different systems; conventional, using non-irrigated, multiple passes; or suction/irrigation, using one pass. The result of a measured re-implantation of the product of reaming was examined in one additional group. We used 24 Swiss mountain sheep with a mean tibial medullary canal diameter between 8 mm and 9 mm. An 8 mm 'napkin ring' defect was created at the mid-diaphysis. The wound was either surgically closed or occluded. The medullary cavity was then reamed to 11 mm. The Reamer/Irrigator/Aspirator (RIA) System was used for the reaming procedure in groups A (RIA and autofilling) and B (RIA, collected reamings filled up), whereas reaming in group C (Synream and autofilling) was performed with the Synream System. The defect was allowed to auto-fill with reamings in groups A and C, but in group B, the defect was surgically filled with collected reamings. The tibia was then stabilised with a solid locking Unreamed Humerus Nail (UHN), 9.5 mm in diameter. The animals were killed after six weeks. After the implants were removed, measurements were taken to assess the stiffness, strength and callus formation at the site of the defect. There was no significant difference between healing after conventional reaming or suction/irrigation reaming. A significant improvement in the quality of the callus was demonstrated by surgically placing captured reamings into the defect using a graft harvesting system attached to the aspirator device. This was confirmed by biomechanical testing of stiffness and strength. This study suggests it could be beneficial to fill cortical defects with reaming particles in clinical practice, if feasible.

PBP74, a new member of the mammalian 70-kDa heat shock protein family, is a mitochondrial protein.

The cloning of a cDNA encoding a new member of the highly conserved mammalian 70-kDa heat shock protein (hsp 70) family termed PBP74 was recently reported. Critical to an understanding of the function of this new hsp 70 is delineating its subcellular localization. Here we use a variety of immunological and biochemical approaches both in vitro and in vivo to demonstrate that PBP74 is imported into and resides in mitochondria. By confocal immunofluorescence microscopy PBP74 is detected in mitochondria, colocalizing with the mitochondrial 60-kDa heat shock protein. To address the inherent problem of serological cross-reactivity among the hsp70 family members, an influenza virus hemagglutinin epitope tag was introduced into the PBP74 cDNA. The epitope-tagged PBP74 protein transiently expressed in L cells localized to mitochondria. Moreover, deletion of the N-terminal 46-amino acid presequence results in a cytosolic localization of the epitope-tagged protein. Cell fractionation studies demonstrated PBP74 in purified mitochondria in a protease-protected location. After coupled transcription-translation the precursor of PBP74 is imported into isolated yeast mitochondria, where it becomes processed to the mature protein. According to a subfractionation of the mitochondria, the imported protein was found to be localized in the matrix space. Import in vitro is time- and temperature-dependent, requires matrix ATP, and is abolished upon depletion of the membrane potential across the mitochondrial inner membrane. Similarly, in mammalian cells PBP74 is synthesized as a pre-protein that requires membrane potential-dependent import into mitochondria for its maturation. Taken together, our data demonstrate that PBP74 is a mammalian mitochondrial hsp70.

RPE drift during cycling in 18 degrees C vs 30 degrees C wet bulb globe temperature.

AIM: The potential influence of a hotter vs cooler environment on ratings of perceived exertion (RPE) estimations during longer duration exercise is not well-understood. This study compared overall and differentiated RPEs during cycling in 18 degrees C vs 30 degrees C wet bulb globe temperature (WBGT). METHODS: Male volunteers (n=16) completed a maximal cycling trial (60 rev . min(-1), 25 Watts . min(-1)) to determine VO(2) max and ventilatory threshold (VT) before completing 2 (counterbalanced) longer duration cycling trials. At 30 degrees C WBGT (30C) and 18 degrees C WBGT (18C), subjects cycled 60 min (60 rev . min(-1), 90% individualized VT). Heart rate (HR, b . min(-1)) and rectal temperature (Tre, degrees C) were recorded every 5 min with corresponding RPE-overall (RPE-O), RPE-legs (RPE-L) and RPE-chest (RPE-C) estimations. RESULTS: HR was not significantly different at 5 min but was greater (P<0.05) for 30C at all other time points. During 30C, Tre was significantly greater (25, 30, 35, 40, 45, 50, 55 and 60 min), RPE-O was significantly greater (5, 40, 45, 50, 55 and 60 min), RPE-L was significantly greater (55 and 60 min) and RPE-C was significantly greater (35, 40, 45, 50, 55 and 60 min). CONCLUSIONS: Greater cardiovascular (HR) and thermal (Tre) strain partially explain greater perceptual ratings during 30C. Discernible RPE differences resulted mid-way through 60 min cycling with minimal differences initially. Results suggest RPEs are magnified in a 30 degrees C (vs 18 degrees C) environment beyond 30 min duration. Additionally, a 30 degrees C environment resulted in a less pronounced impact on RPE-L (vs RPE-C and RPE-O).

Influence of aerobic fitness on ratings of perceived exertion during graded and extended duration cycling.

AIM: Ratings of perceived exertion (RPE) have been shown similar across subjects of varying fitness when estimations are made at relative physiological criteria. Because few studies have investigated the influence of fitness during longer duration bouts, the current investigation compared overall exertion (RPE-O), leg exertion (RPE-L) and breathing/chest exertion (RPE-C) between aerobically fit and unfit subjects. METHODS: Aerobically fit (61.6+/-2.5 mL . kg . min(-1)) (n=7) and unfit (41.8+/-6.3 mL . kg . min(-1)) (n=6) males completed a maximal bike test and then cycled for 60 min at approximately 90% of individualized ventilatory threshold (VT) (V(E)/VO(2) vs V(E)/VCO(2)). Heart rate (HR, b . min(-1)), rectal temperature (Tre, degrees C) and RPE estimations were collected during graded testing every 2 min and every 10 min during 60 min bouts. RESULTS: During graded testing, RPE estimations at VT were not significantly different between groups. During 60 min cycling, HR and Tre were not significantly different between groups. Also, there were no significant differences for HR increase (HR 60 min HR 5 min) or Tre increase (Tre 60 min Tre 5 min). Interactions between groups were; RPE-O (P=0.09), RPE-L (P=0.06) and RPE-C (P=0.19). Analyses suggest groups experienced similar relative cardiovascular (HR) and thermal (Tre) strain. CONCLUSIONS: Although RPE responses between groups were similar at 10, 20 and 30 min, RPE drift was magnified in aerobically unfit subjects (vs aerobically fit subjects) beyond the 30 min point. Contrary to previous studies suggesting aerobic fitness does not influence RPE, current results show lower aerobic fitness magnifies RPE at individualized relative intensities when cycling extends beyond 30 min.

Escherichia coli aminodeoxychorismate synthase: analysis of pabB mutations affecting catalysis and subunit association.

p-Aminobenzoic acid (PABA), an essential component of the vitamin folic acid, is derived from the aromatic branch-point precursor chorismate in two steps. 4-Amino-4-deoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate, and is composed of two subunits, PabA and PabB. While various experiments have suggested that PabA and PabB act as a complex, attempts to isolate the intact complex have failed. We report here the first successful copurification of PabA and PabB by gel filtration chromatography. The association of PabA and PabB is greatly enhanced by the presence of 5 mM glutamine, and by preincubation at 37 degrees C. Conversely, the association is greatly reduced at cold temperatures. We also report the isolation and characterization of both chemically induced and site-directed mutations in PabB. Mutated PabB enzymes fall into three categories according to their properties: deficiency of chorismate amination coupled with failure to associate with PabA, deficiency of chorismate amination coupled with retention of PabA association, and competency of chorismate amination with failure of PabA association.

Mitochondria control of cell death induced by anti-HLA-DR antibodies.

We have previously reported that crosslinking HLA-DR directly induces programmed cell death of malignant B cells. The present study further characterizes the biochemical mechanism for HLA-DR-mediated programmed cell death of tumor cells. Phosphatidylserine exposure on the plasma membrane and propidium iodide incorporation occur with very rapid kinetics and are observed as early as 10 min after the induction of cell death with anti-HLA-DR. In striking contrast to anti-CD95, we observe no activation of caspase-3, -8, or -9 upon anti-HLA-DR addition. Furthermore, the irreversible caspase inhibitor Z-VAD.fmk also failed to inhibit anti-HLA-DR-mediated cell death, further supporting the conclusion that HLA-DR induces cell death via a caspase-independent mechanism. We demonstrate that anti-HLA-DR-induced cell death is instead associated with a rapid disruption of the inner mitochondrial transmembrane potential, DeltaPsi(m), a process that is significantly inhibited by Bcl-2 overexpression. Furthermore, we find that DeltaPsi(m) disruption results in the selective release of apoptosis-inducing factor (AIF) from the mitochondria. We propose that AIF is acting to initiate the morphological and biochemical changes observed in HLA-DR-mediated cell death.