Physical interaction of the HIV-1 Nef protein with beta-COP, a component of non-clathrin-coated vesicles essential for membrane traffic.

Nef is a 27-kDa myristylated protein conserved in most human immunodeficiency virus (HIV)-1, HIV-2, and simian immunodeficiency virus isolates. Simian immunodeficiency virus Nef is required in macaques for both high viral load and full pathological effects. Nef down-regulates the cell surface expression of CD4 by a post-translational mechanism that is not yet fully elucidated. We have used the yeast two-hybrid system to identify cellular proteins that interact with Nef. A cDNA was isolated which encodes a COOH-terminal fragment of human beta-COP, a major coat component of non-clathrin-coated vesicles. Nef and beta-COP interacted in vitro and were found to be physically associated in HIV-1-infected cells by co-immunoprecipitation. These observations suggest that beta-COP might be one of the cellular mediators of Nef function in HIV-1-infected cells.

P56lck: a transducing protein that binds to SH2 containing proteins and to phosphotyrosine containing proteins.

In T lymphocytes, several proteins are rapidly phosphorylated on tyrosine after stimulation. In this study we examine the ability of tyrosine phosphorylated proteins from Jurkat T cells stimulated by CD2 or T cell receptor (TcR)-CD3 to interact with the src homology 2 (SH2) domains from p56lck (Lck). Our data show that the patterns are different depending on the stimulation. The specificity of the interactions was assessed by blocking experiments with high affinity phosphotyrosine [Y(P)] peptides. Phosphorylation experiments suggest that one or several kinases are able to interact with the SH2 from Lck. On the other hand, full length Lck overexpressed in Sf9 cells, which is tyrosine-phosphorylated at least on two sites, can interact in vitro with the SH2 from Lck, phospholipase C (PLC)-gamma 1, p85 (the regulatory subunit of phosphatidyl-inositol-3 kinase (PI3K)) and Nck and with the full length Grb2. These data give additional support to the idea that Lck is an important signal transducing molecule in lymphocytes.

Effect of long- and short-term antidiuretic hormone availability on internephron heterogeneity in the adult rat.

We have previously shown that certain aspects of internephron heterogeneity are reduced or absent in Brattleboro rats with hereditary diabetes insipidus (DI) lacking ADH and can be restored by long-term ADH administration started before complete kidney maturation. In the present study, the effects of long- and short-term availability of ADH in adulthood were studied in Brattleboro DI rats. Single nephron glomerular filtration rate (SNGFR), glomerular volume (GV), and proximal tubular length (PTL) were measured in superficial and juxtamedullary nephrons using the ferrocyanide and microdissection techniques. ADH administration for 6 wk in adult DI rats (group A) restored normal nephron heterogeneity of SNGFR, GV, and PTL by increasing the filtration and size of deep nephrons. Acute changes in ADH availability induced either by 1-h ADH infusion in DI rats (group C) or by ADH discontinuation for 2 days in treated DI rats (group D) did not significantly change the anatomical parameters and only moderately affected SNGFR compared with the preexisting states (groups B and A, respectively). These results suggest that the influence of ADH on internephron heterogeneity is initiated by an increase in deep nephron SNGFR. Based on recent findings concerning the effects of ADH on the medullary (M) part of the thick ascending limbs (TAL), we suggest that the increase in deep nephron SNGFR after ADH may be due to a change in the tubuloglomerular feedback signal at the macula densa resulting from ADH-induced stimulation of the solute reabsorption in the MTAL. Superficial nephrons would be less sensitive to this change due to their long cortical TAL, which removes the macula densa further from the MTAL and provides additional sites for solute reabsorption.

Effects of osmolality and antidiuretic hormone on prostaglandin synthesis by renal papilla. Study in Brattleboro rats with diabetes insipidus.

Prostaglandin (PG) production by the kidney is known to be reduced both in vivo and in vitro in rats with hereditary diabetes insipidus (DI), totally lacking ADH. Exogenous ADH restores normal PG excretion in these rats. On the other hand, osmolality in vitro, and urine flow rate in vivo have been shown to influence PG synthesis rate. In order to determine whether the decreased PG synthesis of DI rats is due to the lack of antidiuretic hormone itself or to low tissue osmolality, we studied in vivo and in vitro PG production in DI rats in which urine osmolality had been raised either with ADH (infused by Alzet minipumps), or without ADH (by dehydratation) and in control DI rats. PGE2 and PGF2 alpha were measured by radioimmunoassay in the urines and in supernatants of papillary homogenates incubated at 37 degrees C for 15-120 min. ADH administration and dehydration led to similar urine osmolalities (congruent to 900-1,000 mosmol/kg H2O versus 150 in controls). However, only ADH administration but not dehydration increased PG urinary excretion (X 5, P less than 0.001) and subsequent in vitro papillary synthesis (X 1.6, P less than 0.01). These results show that antidiuretic hormone increases PG-synthesis of the renal papilla directly and not through its effects on papillary osmolality.