Constraints on CD4 recovery postchemotherapy in adults: thymic insufficiency and apoptotic decline of expanded peripheral CD4 cells

To examine the mechanisms of CD4 reconstitution in an adult population, lymphocyte repopulation was assessed following dose-intense chemotherapy in 25 breast cancer patients, ages 33 to 69 years. Chemotherapy resulted in a greater than 60% reduction in total CD4 T cells and, in particular, a greater than 90% loss of the CD45RA+ CD4 cells. CD4 recovery was protracted, achieving less than 50% of pretreatment levels after 12 to 14 months. Two facets of the CD4 recovery were notable. First, generation of CD45RA+ CD4 cells played only a minor role in the first year, suggesting that thymic production was not the main route of CD4 regeneration. Indeed, recovery of CD45RA+ CD4 cell levels remained limited in half of the patients even after 2 years. Second, expansion of the mature peripheral CD4 cells (CD45RO+) remaining after chemotherapy was the main source of early CD4 repopulation, peaking at 3 to 6 months postchemotherapy. This expansion was limited in duration, however, and was followed by a secondary decline, such that the total CD45RO+ CD4 levels at 9 to 12 months were lower than at 6 months. When stimulated by mitogens, an increased susceptibility to apoptosis was observed in postchemotherapy CD4 cells as compared with those from normal donors. The elevated expression of markers such as HLA-DR during chemotherapy and for several months postchemotherapy is consistent with the presence of an activated T-cell population. CD4 apoptotic frequency correlated with the frequency of HLA-DR expression on T cells. Thus, CD4 recovery is constrained in adults by a limited thymic regenerative capacity and by an increased susceptibility to apoptosis within the expanding peripheral CD4 population.     

Sugar transport by the marine chitinolytic bacterium Vibrio furnissii. Molecular cloning and analysis of the glucose and N-acetylglucosamine permeases

Chitin catabolism by the marine bacterium Vibrio furnissii involves chemotaxis to and transport of N-acetyl-D-glucosamine (GlcNAc) and D-glucose. We report the properties of the respective permeases that complemented E. coli Glc- Man- mutants. Although the V. furnissii Glc-specific permease (55,941 Da) shares 38% identity with E. coli IIGlc (ptsG), it is 67% identical to MalX of the E. coli maltose operon (Reidl, J., and Boos, W. (1991) J. Bacteriol. 173, 4862-4876). An adjacent open reading frame encodes a protein with 52% identity to E. coli MalY. Glc phosphorylation requires only V. furnissii MalX and the accessory phosphoenolpyruvate:glycose phosphotransferase system proteins. The V. furnissii equivalent of IIGlc was not found in the 25,000 transformants screened. The GlcNAc/Glc-specific permease (52,894 Da) shares 47% identity with the N-terminal, hydrophobic domain of E. coli IINag, but is unique among IINag proteins in that it lacks the C-terminal domain and thus requires IIIGlc for sugar fermentation in vivo and phosphorylation in vitro. While there are similarities between the phosphoenolpyruvate:glycose phosphotransferase system of V. furnissii and enteric bacteria, the differences may be important for survival of V. furnissii in the marine environment.     

Separations of derivatized amino acid enantiomers by cyclodextrin-modified capillary electrophoresis: mechanistic and molecular modeling studies

The enantiomers of 5-dimethylamino-1-naphthalene sulfonyl (DNS)-derivatives of selected amino acids were successfully separated using capillary electrophoresis (CE) employing cyclodextrins (CD) as enantio-selective running buffer additives. A previously described model for retention and chiral recognition in CD-modified CE is shown to adapt well in this application. Resolution of the isomers is strongly influenced by the type and concentration of cyclodextrin employed, as predicted by the model. Although data indicates differences in the electrophoretic mobilities for some of the completely complexed enantiomer pairs, selectivity generally requires exploiting differences in the amino acid-CD complexation constants for enantiomer pairs. In this work, the D-enantiomers exhibit larger formation constants and are complexed to a greater degree (elute first) at moderate CD concentration. When mixtures of amino acids are analyzed, the effects of separation conditions on general elution behavior must be considered or separated enantiomer pairs will co-elute with other enantiomers. Preliminary results aimed at predicting the strength of DNS-amino acid enantiomer-CD interactions based on molecular modeling studies are presented. A statistical mechanical approach to treating computationally derived enantiomer-CD interaction energies is shown to provide reasonable correlation with separation performance.     

Design of a high-pressure ebulliometer,with vapor-liquid equilibrium results for the systems CHF2 Cl + CF3 CH3 and CF3 CH2 F + CH2F2

We describe the design and operation of a new high-pressure metal ebulliometer which can operate at pressures to at least 3 MPa in the range 220–400 K. Infinite-dilution activity coefficients are presented for the system CHF₂Cl + CF₃-CH, at 275 K and for the system CF₃-CH₂F + CH₂F₂, at 260, 230, and 300 K. The Wilson activity coellicient model and a virial coefficient model are applied to these systems, and the phase equilibrium conditions are calculated. The results are shown to agree well with predicted and with published measured values. The excess enthalpy is calculated and compared with results from a Peng Robinson equation of state. Vapor densities on the dew curves are given.     

Plasma Asp13-Ki-ras oncoprotein expression in vinyl chloride monomer workers in Taiwan

Vinyl chloride (VC) workers are known to be at risk for development of liver angiosarcoma, a rare tumor. Previously, more than 80% of VC workers with liver angiosarcoma have been found to have an Asp-13 c-Ki-ras oncogene mutation, and more than 50% of VC-exposed workers without liver tumors were found to have Asp13-Ki-ras oncoprotein in their plasma. Some workers in Taiwan had also been exposed to VC, and some have contracted liver tumors. In this study, we used enhanced chemiluminescence Western blotting to detect Asp13-p21-Ki-ras in the sera of VC-exposed workers in Taiwan. There were 14 of 113 (12.4%) VC workers positive for the Asp13-Ki-ras oncoprotein in plasma, but 0 of 18 controls were positive. There were 10 of 69 (14.5%) plasma-positives among the more highly exposed (> 1000 ppm-months) workers and 4 of 48 (9.1%) plasma-positives among the lesser exposed (< or = 1000 ppm-months). Compared with the unexposed controls, the odds ratios (and 95% confidence intervals [CI]) for plasma-positivity were 4.11 (95% CI = 0.21, 80.4) in the lower-exposed workers and 6.53 (95% CI = 0.37, 116.9) in the higher-exposed workers, and there was a linear trend between exposure and plasma-positivity (P = 0.073). After adjusting for age and drinking status, the odds ratios (and 95% CIs) were 1.64 (95% CI = 0.17, 15.8), and 2.65 (95% CI = 0.42, 16.8), respectively, and there was a significant linear trend between exposure and plasma-positivity (P = 0.048). In summary, Asp13-Ki-ras oncoprotein can be found in the plasma of VC workers in Taiwan, and a significant dose-response relationship exists between plasma oncoprotein expression and VC exposure.     

BID: a novel BH3 domain-only death agonist

The BCL-2 family of proteins consists of both antagonists (e.g., BCL-2) and agonists (e.g., BAX) that regulate apoptosis and compete through dimerization. The BH1 and BH2 domains of BCL-2 are required to heterodimerize with BAX and to repress cell death; conversely, the BH3 domain of BAX is required to heterodimerize with BCL-2 and to promote cell death. To extend this pathway, we used interactive cloning to identify Bid, which encodes a novel death agonist that heterodimerizes with either agonists (BAX) or antagonists (BCL-2). BID possesses only the BH3 domain, lacks a carboxy-terminal signal-anchor segment, and is found in both cytosolic and membrane locations. BID counters the protective effect of BCL-2. Moreover, expression of BID, without another death stimulus, induces ICE-like proteases and apoptosis. Mutagenesis revealed that an intact BH3 domain of BID was required to bind the BH1 domain of either BCL-2 or BAX. A BH3 mutant of BID that still heterodimerized with BCL-2 failed to promote apoptosis, dissociating these activities. In contrast, the only BID BH3 mutant that retained death promoting activity interacted with BAX, but not BCL-2. This BH3-only molecule supports BH3 as a death domain and favors a model in which BID represents a death ligand for the membrane-bound receptor BAX.     

Aldosterone and dexamethasone stimulate calcineurin activity through a transcription-independent mechanism involving steroid receptor-associated heat shock proteins

1. PD 81,723 has been shown to enhance binding of adenosine to A1 receptors by stabilizing G protein-receptor coupling ('allosteric enhancement'). Evidence has been provided that in the perfused hearts and isolated atria PD 81,723 causes a sensitization to adenosine via this mechanism. 2. We have studied the effect of PD 81,723 in guinea-pig isolated atrial myocytes by use of whole-cell measurement of the muscarinic K+ current (I[K(ACh)]) activated by different Gi-coupled receptors (A1, M2, sphingolipid). PD 81,273 caused inhibition of I[K(ACh)] (IC50 approximately 5 microM) activated by either of the three receptors. Receptor-independent I[K(ACh)] in cells loaded with GTP-gamma-S and background I[K(ACh)], which contributes to the resting conductance of atrial myocytes, were equally sensitive to PD 81,723. At no combination of concentrations of adenosine and PD 81,723 could an enhancing effect be detected. 3. The compound was active from the outside only. Loading of the cells with PD 81,723 (50 microM) via the patch pipette did not affect either I[K(ACh)] or its sensitivity to adenosine. We suggest that PD 81,723 acts as an inhibitor of inward rectifying K+ channels; this is supported by the finding that ventricular I(K1), which shares a large degree of homology with the proteins (GIRK1/GIRK4) forming I[K(ACh)] but is not G protein-gated, was also blocked by this compound. 4. It is concluded that the functional effects of PD 81,723 described in the literature are not mediated by the A1 adenosine receptor-Gi-I[K(ACh)] pathway.