Bcl-XL and Bcl-2 repress a common pathway of cell death.

The effect of Bcl-xL upon the developmental death of T cells was assessed by generating transgenic mice that expressed Bcl-xL within all thymocyte subsets. Bcl-xL protected thymocytes from a variety of apoptotic stimuli, including gamma irradiation, glucocorticoids, and anti-CD3 treatment. Bcl-xL altered thymocyte maturation, resulting in increased numbers of CD3int/hi and CD4-8+ thymocytes. Overall, the phenotype of Bcl-xL transgenics was essentially indistinguishable from a Bcl-2 transgenic model. Overexpression of Bcl-xL or Bcl-2 resulted in the down-regulation of the other molecule, providing further evidence of their reciprocal regulation. In a genetic test of redundancy, the Bcl-xL transgene rescued mature T cells in Bcl-2 null mice. Immunoprecipitation indicated that Bcl-xL, like Bcl-2, heterodimerized with the death-promoting molecule Bax in thymocytes. This in vivo model argues that Bcl-xL, like Bcl-2, functions in a common pathway to repress cell death.

The protein tyrosine phosphatase SHP-1 regulates integrin-mediated adhesion of macrophages.

The Src homology 2 domain phosphatase-1 (SHP-1) is a tyrosine phosphatase containing two amino-terminal SH2 domains and is expressed primarily by hematopoietic-derived cells [1]. The viable motheaten (Hcphme-v) mutant mice (mev) suffer from progressive inflammation due to a deficiency of SHP-1 enzyme activity [2,3] and die at 3-4 months of age from macrophage and neutrophil accumulation in the lung [4]. The mechanism by which SHP-1 deficiency leads to inflammation is unknown. We found that macrophages from mev mice adhered and spread to a greater extent than normal macrophages through alpha m beta 2 integrin-mediated contacts. Whereas macrophages deficient in the transmembrane tyrosine phosphatase CD45 (CD45-/-) spontaneously detached from alpha m beta 2 integrin contacts [5], cells deficient in both CD45 and SHP-1 did not. In SHP-1 deficient macrophages there was a 10-15-fold increase in D-3 phospholipid products of phosphatidylinositol (PI) 3-kinase. Concomitantly, there was a 2-5-fold increase in membrane-associated PI 3-kinase activity in mev macrophages relative to normal macrophages. Treatment of macrophages with the PI 3-kinase inhibitors wortmannin or LY294002 resulted in a dramatic detachment of cells, indicating that PI 3-kinase activity is required for adhesion. These data demonstrate that SHP-1 is necessary for detachment from alpha m beta 2 integrin-mediated contacts in primary macrophages and suggest that a defect in this pathway may contribute to inflammatory disease.

Expression of the p56(Lck) Y505F mutation in CD45-deficient mice rescues thymocyte development.

Mice deficient in the transmembrane protein tyrosine phosphatase CD45 exhibit a block in thymocyte development. To determine whether the block in thymocyte development was due to the inability to dephosphorylate the inhibitory phosphorylation site (Y505) in p56(lck) (Lck), we generated CD45-deficient mice that express transgenes for the Lck Y505F mutation and the DO11.10 T-cell antigen receptor (TCR). CD4 single-positive T cells developed and accumulated in the periphery. Treatment with antigen resulted in thymocyte apoptosis and the loss of transgenic-TCR-bearing cells. Peripheral CD45-deficient T cells from the mice expressing both transgenes responded to antigen by increasing CD69 expression, interleukin-2 production, and proliferation. These results indicate that thymocyte development requires the dephosphorylation of the inhibitory site in Lck by CD45.

Absence of apparent phenotype in mice lacking Cdc25C protein phosphatase.

The Cdc25 family of protein phosphatases positively regulate the cell division cycle by activating cyclin-dependent protein kinases. In humans and rodents, three Cdc25 family members denoted Cdc25A, -B, and -C have been identified. The murine forms of Cdc25 exhibit distinct patterns of expression both during development and in adult mouse tissues. In order to determine unique contributions made by the Cdc25C protein phosphatase to embryonic and adult cell cycles, mice lacking Cdc25C were generated. We report that Cdc25C(-/-) mice are viable and do not display any obvious abnormalities. Among adult tissues in which Cdc25C is detected, its transcripts are most abundant in testis, followed by thymus, ovary, spleen, and intestine. Mice lacking Cdc25C were fertile, indicating that Cdc25C does not contribute an essential function during spermatogenesis or oogenesis in the mouse. T- and B-cell development was also found to be normal in Cdc25C(-/-) mice, and Cdc25C(-/-) mouse splenic T and B cells exhibited normal proliferative responses in vitro. Finally, the phosphorylation status of Cdc2, the timing of entry into mitosis, and the cellular response to DNA damage were unperturbed in mouse embryo fibroblasts lacking Cdc25C. These findings indicate that Cdc25A and/or Cdc25B may compensate for loss of Cdc25C in the mouse.

Immune system dysfunction and autoimmune disease in mice lacking Emk (Par-1) protein kinase.

Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption. Emk(-/-) mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4(+)T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. As Emk(-/-) animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals.

Glucose metabolism and effect of acetate in ovine adipocytes.

Isolated ovine adipocytes were incubated in vitro with specifically labeled 14C-glucose in the presence or absence of acetate. The flux patterns of glucose carbon through major metabolic pathways were estimated. When glucose was added as the sole substrate, approximately equal portions of glucose carbon (10%) were oxidized to CO2 in the pentose phosphate pathway, in the pyruvate dehydrogenase reaction and in the citrate cycle. Fifteen percent of the glucose carbon was incorporated into fatty acids and 43% was released as lactate and pyruvate. Addition of acetate to the medium increased glucose carbon uptake by 1.5-fold. Most of this increase was accounted for by a sevenfold increase in the activity of the pentose phosphate pathway. Acetate increased glucose carbon fluxes via pentose phosphate pathway to triose phosphates, from triose phosphate to pyruvate, into glyceride glycerol, into lactate and pyruvate and into pyruvate dehydrogenase and citrate cycle CO2. Glucose carbon incorporated into fatty acids was decreased 50% by acetate while, carbon fluxes through the phosphofructokinase-aldolase reactions were not significantly increased. Results of this study suggest that, when glucose is the sole substrate, the conversion of glucose to fatty acids in ovine adipocytes may not be limited by the maximum capacity of hexokinase, the pentose phosphate pathway or enzymes involved in the conversion of triose phosphates to pyruvate and of pyruvate to fatty acid. Acetate increased glucose utilization apparently by increasing activity of the pentose phosphate pathway as a result of enhanced NADPH utilization for fatty acid synthesis.

A model program: stress management unit--a clinic run by army nurses.

People suffering from stress and stress-related disorders are a great challenge to our already depleted military health care system. Early identification and separation of soldiers not able to adjust, immediate intervention for temporarily stressed soldiers, and stress management for dependents and retirees help decrease visits to military hospitals and clinics. Immediate intervention for salvageable soldiers also helps improve work performance and productivity. A Stress Management Unit has been open for two years at Brooke Army Medical Center (BAMC), Texas. This article identifies the need for stress management clinics in the military and briefly describes the nurse-run program at BAMC.

Synthetic lethality of Chk1 inhibition combined with p53 and/or p21 loss during a DNA damage response in normal and tumor cells.

Cell cycle checkpoints ensure genome integrity and are frequently compromised in human cancers. A therapeutic strategy being explored takes advantage of checkpoint defects in p53-deficient tumors in order to sensitize them to DNA-damaging agents by eliminating Chk1-mediated checkpoint responses. Using mouse models, we demonstrated that p21 is a key determinant of how cells respond to the combination of DNA damage and Chk1 inhibition (combination therapy) in normal cells as well as in tumors. Loss of p21 sensitized normal cells to the combination therapy much more than did p53 loss and the enhanced lethality was partially blocked by CDK inhibition. In addition, basal pools of p21 (p53 independent) provided p53 null cells with protection from the combination therapy. Our results uncover a novel p53-independent function for p21 in protecting cells from the lethal effects of DNA damage followed by Chk1 inhibition. As p21 levels are low in a significant fraction of colorectal tumors, they are predicted to be particularly sensitive to the combination therapy. Results reported in this study support this prediction.

Lactate metabolism and cytosolic NADH reducing equivalents in ovine adipocytes.

1. Isolated ovine adipocytes, unlike rat adipose tissue, could utilize lactate at a high rate. 2. When the rate of fatty acid synthesis was attenuated with 5-(tetradecyloxy)-2-furoic acid, a fatty acid synthesis inhibitor, there was a good positive correlation between the rates of lactate oxidation to CO2 and lactate incorporation into fatty acids. 3. Addition of 2,4-dinitrophenol enhanced lactate oxidation to CO2 independent of fatty acid synthesis. Under this condition, estimated cytosolic NADH formation from lactate dehydrogenation exceeded the need of NADH for cytosolic oxaloacetate reduction and for glyceride glycerol formation. 4. Mitochondria isolated from ovine adipocytes oxidized added NADH rapidly in a reconstituted alpha-glycerophosphate shuttle system. 5. It is possible that the ability of ovine adipocytes to utilize lactate may be related to the active alpha-glycerophosphate shuttle for cytosolic NADH reoxidation.

Shorter length of stay for simple cholecystectomy: cost-effectiveness of alternative strategies.

Patients undergoing simple cholecystectomy during a 12-month period at a Boston teaching hospital were retrospectively divided into two groups: short stay surgery (SSS) patients had a hospital length of stay (LOS) of 5 or 6 days; conventional stay surgery (CSS) patients were hospitalized for more than 6 days. After matching for sex, age, preoperative LOS, and secondary diagnoses, 18 pairs were identified. None of the patients had major complications after the hospital discharge. One to 2 years after surgery, health status was identical in the two groups. Patients in the SSS group reported a larger number of minor complications after hospital discharge and less satisfaction with the adequacy of their length of stay. Shortened length of hospital stay for a subgroup of simple cholecystectomy patients is a promising means of reducing cost with minimal, if any, sacrifice in quality of care and deserves further evaluation.

Role of leukemia inhibitory factor and its receptor in mouse primordial germ cell growth.

The pleiotropic cytokine leukemia inhibitory factor (LIF) is able to promote the growth of mouse primordial germ cells (PGCs) in culture. It is unclear whether LIF acts directly on PGCs or indirectly via feeder cells or embryonic somatic cells. To understand the role of LIF in PGC growth, we have carried out molecular and cell culture analyses to investigate the role of both the LIF ligand and its receptor in PGC development. LIF is able to stimulate PGC growth independently of the presence of feeder cells supporting the hypothesis that LIF acts directly on PGCs to promote their growth. We show here that transcripts for the low-affinity LIF receptor (LIFR), an integral component of the functional LIF receptor complex, are expressed in the developing gonad. Fluorescence-activated cell sorter (FACS) analysis, using an anti-LIFR antiserum, demonstrates that LIFR is present on the surface of PGCs, suggesting that PGCs are likely to be a direct target of LIF action in culture. Signalling via LIFR is essential for PGC growth in culture since the anti-LIFR antiserum, which blocks LIF binding to its receptor, abolishes PGC survival in culture. Two LIF-related cytokines, namely oncostatin M and ciliary neurotrophic factor, can also promote PGC growth in culture in addition to LIF. Thus one or more of these LIFR-dependent cytokines may play an important role in PGC development in mice.

Dual-energy X-ray absorptiometric densitometry in osteoarthritis of the hip. Influence of secondary bone remodeling of the femoral neck.

PURPOSE: The aim of this study was to evaluate the influence of buttressing on bone densitometry measurements in the femoral neck, in Ward's triangle, and in the greater trochanter. In addition, we attempted to establish the length of the femoral axis (FAL) and the true length of the femoral neck (FNL) as potential correlates with osteoarthritis (OA) or with buttressing. MATERIAL AND METHODS: Our study comprised 101 hips in 68 adult patients. Conventional radiographs of the hip joints were obtained in order to assess the presence and extent of OA by means of the 6-step grading system introduced in 1990 by CROFT et al., and in order to measure the cortical thickness at the medial aspect of the femoral neck. In addition, FAL and FNL were measured. All patients underwent dual energy x-ray absorptiometry so that bone density could be assessed in the femoral neck, in Ward's triangle, and in the greater trochanter. The Spearman rank correlation was used to compare the measurements. RESULTS: Statistical analysis showed a significant positive correlation between cortical thickness and bone density in the femoral neck and in Ward's triangle. No correlation was found between cortical thickness and bone density in the greater trochanter, nor between cortical thickness and OA, FNL, and FAL, nor between OA and bone density, FNL, and FAL. CONCLUSION: Buttressing influenced our bone density measurements in the femoral neck and in Ward's triangle. It did not affect the region of the greater trochanter which may therefore be the best region of interest for a long-term follow-up of bone density in patients with OA.

Cutting edge: the tyrosine phosphatase SHP-1 regulates thymocyte positive selection.

The binding kinetics of the TCR for its interacting ligand and the nature of the resulting signal transduction event determine the fate of a developing thymocyte. The intracellular tyrosine phosphatase SHP-1 is a potential regulator of the TCR signal transduction cascade and may affect thymocyte development. To assess the role of SHP-1 in thymocyte development, we generated T cell-transgenic mice that express a putative dominant negative form of SHP-1, in which a critical cysteine is mutated to serine (SHP-1 C453S). SHP-1 C453S mice that express the 3.L2 TCR transgene are increased in CD4 single positive cells in the thymus and are increased in cells that express the clonotypic TCR. These data suggest that the expression of SHP-1 C453S results in increased positive selection in 3.L2 TCR-transgenic mice and support a role for SHP-1 thymocyte development.

CD45-associated protein is not essential for the regulation of antigen receptor-mediated signal transduction.

CD45 is a transmembrane protein tyrosine phosphatase required for signaling through the T-and B-cell antigen receptors. In lymphocytes, CD45 interacts with CD45-associated protein (CD45AP), a 32 000 Mr phosphoprotein, through their respective transmembrane domains. To determine whether CD45AP affects the ability of CD45 to regulate antigen receptor signaling, CD45AP-deficient mice were generated. Thymocyte development was grossly normal. Moreover, the cellularity of the thymus and spleens were normal. CD45 expression on thymocytes and splenocytes, ascertained by flow cytometry, was comparable between CD45AP-deficient mice and littermate controls. In contrast to a previous report (Matsuda et al., J. Exp. Med. 1998 187: 1863 - 1870). CD45AP-deficient and normal thymocytes and splenocytes proliferated similarly in response to various mitogens or antigen receptor cross-linking. Furthermore, thymocyte CD45-associated p56(lck) kinase activity was similar between CD45AP-deficient and normal cells. We conclude that CD45AP is not essential for the regulation of Src-family kinase activity by CD45.