Lysosome proteins are redistributed during expression of a GTP-hydrolysis-defective rab5a.

The functioning of the endocytic pathway is influenced by a distinct set of rab GTPases, including rab5a, which regulates homotypic fusion of early endosomes. Expression of a dominant active, GTPase-defective rab5a accelerates endosome fusion, causing the formation of a greatly enlarged endocytic compartment. Here we present evidence that rab5a also regulates trafficking between endosomes and lysosomes and may play a role in lysosome biogenesis. The GTPase defective rab5aQ79L mutant was inducibly expressed as an EGFP fusion in HEK293 cells, and the distribution of lysosome proteins and endocytic markers then assessed by deconvolution fluorescence microscopy. During expression of EGFP-rab5aQ79L, the lysosome proteins LAMP-1, LAMP-2 and cathepsin D were found in dilated EGFP-rab5aQ79L-positive vesicles, which also rapidly labeled with transferrin Texas Red. Exogenous tracers that normally traffic to lysosomes after prolonged chase (dextran Texas Red and DiI-LDL) also accumulated in these vesicles. Dextran Texas Red preloaded into lysosomes localized with subsequently expressed EGFP-rab5a Q79L, suggesting the existence of lysosome to endosome traffic. Cells expressing EGFP-rab5a wt or the dominant negative EGFP-rab5aS34N did not exhibit these abnormalities. Despite the dramatic alterations in lysosome protein distribution caused by expression of EGFP-rab5a Q79L, there was little change in the endocytosis or recycling of a cell-surface receptor (beta2-adrenergic receptor). However, there was a deficiency of dense beta-hexosaminidase-containing lysosomes in cells expressing EGFP-rab5aQ79L, as assessed by Percoll gradient fractionation. These results suggest that expression of a GTPase-defective rab5a affects lysosome biogenesis by alteration of traffic between lysosomes and endosomes.

Specific changes in beta2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation.

Agonist-activated beta2-adrenoceptors rapidly internalize and then recycle to the cell surface, however chronic agonist eventually causes receptor downregulation. To characterize beta2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation, human embryonic kidney cells expressing epitope-tagged receptors were examined by radioligand binding with (+/-)-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole- 2-on hydrochloride ([3H]CGP12177) and immunofluorescence microscopy. The first-order receptor recycling rate constant declined after 18 h of agonist compared with 15 min (0.05 min(-1) vs. 0.12 min(-1)), thus increasing the intracellular transit time (20.0 min vs. 8.3 min). There was also a reduction in the rate of receptor endocytosis and a decline in the total number of receptors. Although the intracellular receptor fraction did not increase between 15 min and 18 h of agonist, some receptors moved irreversibly into a protease-containing compartment while retaining radioligand binding activity. Our results indicate that beta2-adrenoceptor downregulation is associated principally with an increased intracellular transit time during recycling. This could promote the diversion of receptors into protease-containing compartments, where there is an irreversible commitment to downregulation prior to loss of radioligand binding activity.

Upstairs downstairs: vertical integration of a pediatric service.

BACKGROUND: The combined effects of recent changes in health care financing and training priorities have compelled academic medical centers to develop innovative structures to maintain service commitments yet conform to health care marketplace demands. In 1992, a municipal hospital in the Bronx, New York, affiliated with a major academic medical center reorganized its pediatric service into a vertically integrated system of four interdependent practice teams that provided comprehensive care in the ambulatory as well as inpatient settings. One of the goals of the new system was to conserve inpatient resources. OBJECTIVE: To describe the development of a new vertically integrated pediatric service at an inner-city municipal hospital and to test whether its adoption was associated with the use of fewer inpatient resources. DESIGN: A descriptive analysis of the rationale, goals, implementation strategies, and structure of the vertically integrated pediatric service combined with a before-and-after comparison of in-hospital resource consumption. METHODS: A before-and-after comparison was conducted for two periods: the period before vertical integration, from January 1989 to December 1991, and the period after the adoption of vertical integration, from July 1992 to December 1994. Four measures of inpatient resource use were compared after adjustment for case mix index: mean certified length of stay per case, mean number of radiologic tests per case, mean number of ancillary tests per case, and mean number of laboratory tests per case. Difference-in-differences-in-differences estimators were used to control for institution-wide trends throughout the time period and regional trends in inpatient pediatric practice occurring across institutions. Results. In 1992, the Department of Pediatrics at the Albert Einstein College of Medicine reorganized the pediatric service at Jacobi Medical Center, one of its principal municipal hospital affiliates, into a vertically integrated pediatric service that combines ambulatory and inpatient activities into four interdependent practice teams composed of attending pediatricians, allied health professionals, house officers, and social workers. The new vertically integrated service was designed to improve continuity of care for patients, provide a model of practice for professional trainees, conserve scarce resources, and create a clinical research infrastructure. The vertically integrated pediatric service augmented the role of attending pediatricians, extended the use of allied health professionals from the ambulatory to the inpatient sites, established interdisciplinary practice teams that unified the care of pediatric patients and their families, and used less inpatient resources. Controlling for trends within the study institution and trends in the practice of pediatrics across institutions throughout the time period, the vertical integration was associated with a decline in 0.6 days per case, the use of 0.62 fewer radiologic tests per case, 0.21 fewer ancillary tests per case, and 2.68 fewer laboratory tests per case. CONCLUSIONS: We conclude that vertical integration of a pediatric service at an inner-city municipal hospital is achievable; conveys advantages of improved continuity of care, enhanced opportunities for primary care training, and increased participation of senior clinicians; and has the potential to conserve significant amounts of inpatient resources.

Adenosine aminohydrolase from monkey brain: partial purification and some kinetic properties.

Adenosine aminohydrolase from monkey brain was purified ten fold. In pH 7.3 phosphate buffer at 37 degrees, this enzyme preparation deaminated adenosine and arabinosyladenine with apparent values for the Michaelis constant of 32 muM and 370 muM respectively. The products of both deamination reactions, i.e., inosine and arabinosylhypoxanthine, were competitive inhibitors with Ki equal to 220 muM and 1,000 muM, respectively. N6-benzyladenosine and 9-(1-hydroxy-2-octyl)adenine were competitive inhibitors and were more effective in inhibiting deamination of arabinosyladenine than of adenosine. It is suggested that these compounds might potentiate arabinosyladenine chemotherapy of neoplasms of the central nervous system.

Purine and pyrimidine inhibitors of arginase.

1. Adenosine, inosine, adenine and uric acid are competitive inhibitors and cytidine and cytosine noncompetitive inhibitors of bovine liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1). 2. The affinity of the enzyme for these inhibitors was 10--100 times as great as for substrate in terms of Ki versus Km. 3. These nucleic acid metabolites may thus function in vivo to regulate the urea cycle. 4. Several naturally occuring competitive and noncompetitive inhibitors of arginase of unknown structure have been isolated from plant and animal tissue. From their properties and methods of isolation, they may be the purines and pyrimidines herein described. 5. These purines and pyrimidines have no effect on tryptic hydrolysis.