Kinetic parameters for the vesicular acetylcholine transporter: two protons are exchanged for one acetylcholine

The vesicular acetylcholine transporter (VAChT) mediates ACh storage in synaptic vesicles by exchanging cytoplasmic ACh with vesicular protons. This study sought to determine the stoichiometry of exchange by analysis of ligand binding and transport kinetics. The effects of different pH values inside and outside, external ACh concentrations, and electrical potential gradients on ACh transport by vesicles isolated from the electric organ of Torpedo were determined using a pH-jump protocol. The equilibrium binding of a high-affinity analogue of ACh is inhibited by protonation with a pKa of 7.4 +/- 0.3. A two-proton model fits the transport data much better than a one-proton model does, and uptake increases at more positive internal electrical potential, as expected for the two-proton model. Thus, the results support the two-proton model. The transport cycle begins with binding of external ACh to outwardly oriented site 2 (KACho = 20 mM) and protonation of inwardly oriented site 1 (pKa1 = 4.73 +/- 0.05). Loaded VAChT reorients quickly (73 000 min-1) and releases ACh to the inside (KAChi = 44 000 mM) and the proton to the outside. Unloaded, internally oriented site 2 binds a proton (pKa2 = 7.0), after which VAChT reorients (150 +/- 20 min-1) in the rate-limiting step and releases the proton to the outside to complete the cycle. Rate constants for the reverse direction also were estimated. Two protons provide a thermodynamic driving force beyond that utilized in vivo, which suggests that vesicular filling is regulated. Other phenomena related to VAChT, namely the time required to fill synaptic vesicles, the fractional orientation of the ACh binding site toward cytoplasm, orientational lifetimes, and the rate of nonquantal release of ACh from cholinergic nerve terminals, were computer-simulated, and the results are compared with physiological observations.     

The role of prefrontal cortex in working memory: examining the contents of consciousness

Working memory enables us to hold in our 'mind's eye' the contents of our conscious awareness, even in the absence of sensory input, by maintaining an active representation of information for a brief period of time. In this review we consider the functional organization of the prefrontal cortex and its role in this cognitive process. First, we present evidence from brain-imaging studies that prefrontal cortex shows sustained activity during the delay period of visual working memory tasks, indicating that this cortex maintains on-line representations of stimuli after they are removed from view. We then present evidence for domain specificity within frontal cortex based on the type of information, with object working memory mediated by more ventral frontal regions and spatial working memory mediated by more dorsal frontal regions. We also propose that a second dimension for domain specificity within prefrontal cortex might exist for object working memory on the basis of the type of representation, with analytic representations maintained preferentially in the left hemisphere and image-based representations maintained preferentially in the right hemisphere. Furthermore, we discuss the possibility that there are prefrontal areas brought into play during the monitoring and manipulation of information in working memory in addition to those engaged during the maintenance of this information. Finally, we consider the relationship of prefrontal areas important for working memory, both to posterior visual processing areas and to prefrontal areas associated with long-term memory.     

Flooding in a thin rectangular slit geometry representative of ATR fuel assembly side-plate flow channels

Air-water counter-current flow limit experiments were conducted in thin rectangular channels at atmospheric pressure. The parameters were: narrow channel width, either 1.1 mm or 2.2 mm; inlet water temperature, ranging from 294 K to 330 K; channel surface condition, either clean aluminum, aluminum oxide, or acrylic; location and geometry of the air inlet; method of forcing air through the channel; and liquid head above the channel. Experimental results for each set of parameters can be linearly correlated using the square root of the non-dimensional superficial velocities. Channel surface wetting and location and geometry of the air injection had the greatest effect. Narrow channel width, water temperature, method used to force air through the channel, and liquid head above the channel had little effect on the flooding characteristics.     

Estimation of body fat in caucasian and Polynesian women from anthropometric measurements

Girth measurements provide a simple method for estimation of total body fat that can be used over a wide range of percentage body fat (%BF). Well-established equations for determining %BF from girth measurements were applied to 42 Caucasian and 40 Polynesian women. %BF was determined from total body water measurement using oxygen-18 dilution. The relationship between the criterion measurement of %BF and that calculated from girth measurements differed significantly between the two ethnic groups. Assessment of body fat using girth measurements should use ethnicity-specific equations.     

Recurrence of steroid-resistant nephrotic syndrome in kidney transplants is associated with increased acute renal failure and acute rejection

We performed 73 kidney transplants in 51 patients with steroid-resistant nephrotic syndrome (SRNS) with focal segmental glomerular sclerosis (FSG) ages 18.4 +/- 12.8 (mean +/- SD) years. Recurrence of SRNS, defined by rapid onset of proteinuria, hypoalbuminemia and/or > 95% epithelial cell foot process effacement with or without the presence of FSG, occurred in 26 grafts in 16 patients. Acute renal failure (ARF) occurred in 16 of 26 (61.5%) grafts with recurrence versus 7 of 47 (14.9%) grafts without recurrence (P < 0.0001). ARF occurred in 4 of 9 (44.4%) living-related donor (LRD) recipients with recurrence and 3 of 21 (12.5%) LRD recipients without recurrence (NS). ARF in cadaver donor (CAD) recipients with recurrence was 12 of 17 (70.5%) versus 4 of 23 (17.4%) without recurrence (P < 0.0001). ARF was also higher in LRD or CAD with recurrence than in a control group of non-SRNS patients matched for age, sex and time of transplantation. Graft survival at one year was lower in patients with recurrence and ARF [4 of 16 (25%)] compared to patients with recurrence and no ARF [9 of 11 (82%), P < 0.01]. There was no difference in graft survival in patients without recurrence who did or did not have ARF. One or more acute rejection episodes occurred in all 16 patients with ARF and recurrence, in all 7 patients with ARF without recurrence, and in 7 of 10 patients with recurrence without ARF compared with only 11 of 40 (28%) of patients with neither recurrence nor ARF (P < 0.0001, < 0.001 and < 0.04, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Familial neuroblastoma: report of a kindred with later age at diagnosis

The first reported effective adjuvant combination regimen for patients with operable breast cancer comprised oral cyclophosphamide (C) days 1-14 with intravenous methotrexate (M) and fluorouracil (F) on days 1 and 8, repeated every 28 days ('classical' CMF). These drugs have since been extensively used with or without endocrine therapies and/or other cytotoxics, as well as with radiation therapy to the chest wall yielding conflicting results. Although doses and schedules have varied widely, the combination of these three drugs has been generically referred to as CMF. Evidence exists that reducing the dose and/or altering the schedule of CMF ('modified' CMF) have compromised its efficacy in metastatic breast cancer. Reduction below standard dose of a similar regimen also gave inferior results in the adjuvant setting. In fact, the recently reported improved outcome of adding radiation therapy to CMF was only demonstrated in comparisons with a 'modified' CMF. Furthermore, trials in women with estrogen receptor-positive breast cancer, which did not demonstrate any significant benefit for the addition of adjuvant CMF to tamoxifen compared with tamoxifen alone, also used 'modified' CMF. Therefore, adherence to the 'classical' dose and schedule is recommended when CMF is used in adjuvant therapy.     

HIV-1 infection of monocyte-derived macrophages reduces Fc and complement receptor expression

Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.     

The schistosome granuloma: characterization of lymphocyte migration, activation, and cytokine production

Granuloma formation and its regulation are dependent on lymphocytes. Therefore, we compared the characteristics of lymphocytes derived from the spleens and granulomas of Schistosoma mansoni-infected mice during the course of their disease. We examined lymphocyte cell cycle kinetics, migration, expression of activation Ags (CD69 and IL-2R), cytokine production (IL-2, IL-4, IFN-gamma), and apoptosis. Lymphocytes in the G2/M phase of the cell cycle and high levels of lymphocyte intracellular IL-2 were found in the spleen but not in the granuloma. Cell trafficking experiments showed Ag-specific recruitment of schistosomal egg Ag (SEA)-reactive lymphoblasts into granulomas in vivo, as well as recruitment to, residence within, and egress from granulomas in vitro. Granuloma-derived lymphocytes were more highly activated than splenic lymphocytes based on higher levels of CD69 and IL-2R expression. While the granuloma microenvironment was rich in Th2 cytokines, during peak granuloma formation, the lymphocytes per se from the spleen and granuloma did not exhibit a dominant Th1 or Th2 cytokine profile, producing low but similar levels of IL-4 and IFN-gamma. The discrepancy between high IL-2R expression and low levels of IL-2 protein production by granuloma lymphocytes was associated with increased apoptosis in the granuloma compared with the spleen. These findings support the hypothesis that granulomas may play a role in the regulation of systemic pathology in schistosomiasis by adversely affecting the survival of SEA-reactive, immunopathogenic T lymphocytes.