In yeast, ubiquitin plays a central role in proteolysis of a multitude of proteins and serves also as a signal for endocytosis of many plasma membrane proteins. We showed previously that ubiquitination of the general amino acid permease (Gap1) is essential to its endocytosis followed by vacuolar degradation. These processes occur when NH(4)(+), a preferential source of nitrogen, is added to cells growing on proline or urea, i.e. less favored nitrogen sources. In this study, we show that Gap1 is ubiquitinated on two lysine residues in the cytosolic N terminus (positions 9 and 16). A mutant Gap1 in which both lysines are mutated (Gap1(K9K16)) remains fully stable at the plasma membrane after NH(4)(+) addition. Furthermore, each of the two lysines harbors a poly-ubiquitin chain in which ubiquitin is linked to the lysine 63 of the preceding ubiquitin. The Gap1(K9) and Gap1(K16) mutants, in which a single lysine is mutated, are down-regulated in response to NH(4)(+) although more slowly. In proline-grown cells lacking Npr1, a protein kinase involved in the control of Gap1 trafficking, newly synthesized Gap1 is sorted from the Golgi to the vacuole without passing through the plasma membrane (accompanying article, De Craene, J.-O., Soetens, O., and André, B. (2001) J. Biol. Chem. 276, 43939-43948). We show here that ubiquitination of Gap1 is also required for this direct sorting to the vacuole. In an npr1Delta mutant, neosynthesized Gap1(K9K16) is rerouted to and accumulates at the plasma membrane. Finally, Bul1 and Bul2, two proteins interacting with Npi1/Rsp5, are essential to ubiquitination and down-regulation of cell-surface Gap1, as well as to sorting of neosynthesized Gap1 to the vacuole, as occurs in an npr1Delta mutant. Our results reveal a novel role of ubiquitin in the control of Gap1 trafficking, i.e. direct sorting from the late secretory pathway to the vacuole. This result reinforces the growing evidence that ubiquitin plays an important role not only in internalization of plasma membrane proteins but also in their sorting in the endosomes and/or trans-Golgi.

Membrane trafficking of the general amino acid permease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation. In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH 4 ؉ , a preferential nitrogen source, Gap1 is endocytosed and targeted to the vacuole, where it is degraded. This down-regulation requires ubiquitination of the permease, and this ubiquitination is dependent on the essential Npi1/Rsp5 ubiquitin ligase. In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking. We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1 ts mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH 4 ؉ is added to wildtype cells. The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1⌬ mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH 4 ؉ . Finally, Gap1 is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other permeases. We propose that these kinases also function as regulators of permease trafficking.

Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (P ‫؍‬ 0.036).Cytomegalovirus (CMV) infection during pregnancy can be transmitted to the fetus, resulting in a congenital infection. Congenital CMV infection (cCMV) occurs in 0.2 to 2.2% of live-born infants. An incidence of 0.62% is found in the Brussels, Belgium, region (11).The standard diagnosis of cCMV relies on the isolation of the virus from urine or saliva collected in the first 2 weeks of life. The diagnosis of cCMV infection in children older than 2 weeks cannot be made or excluded on the basis of viral isolation. Blood is routinely collected from neonates in the first 2 weeks of life and is stored as dried blood spots (DBSs) on Guthrie cards. The detection of CMV DNA on these stored DBSs could be an opportunity to diagnose cCMV during later life, when symptoms suggestive of cCMV develop (3).The aim of this study was to test the clinical sensitivity of CMV DNA PCR with DBSs from consecutive cases of neonates with cCMV infections. We evaluated two methods for the extraction of CMV DNA and two methods for the amplification of CMV DNA from DBSs on Guthrie cards for the retrospective diagnosis of cCMV. MATERIALS AND METHODSThe study protocol was approved by the Committee of Medical Ethics of the UZ Brussel. cCMV infections. From 1996 to 2006 a neonatal screening program (11) detected cCMV in 76 newborns at the Universitair Ziekenhuis Brussels. The diagnosis of cCMV infection was made by isolation of the virus from urine within 7 days of birth. Blood from the first consecutive 57 cases and from the last 4 cases was retrieved and placed on Guthrie cards (no. 903 ...

A variety of microorganisms were isolated with frequent mixed infection. NTHi was one of the major pathogens found, especially in association with recurrent CAP, possibly because of improved detection with the FOB with BAL procedure. This suggests that the burden of pediatric CAP could be reduced by addressing NTHi as a major causative pathogen.

Carbapenem-resistant Acinetobacter baumannii isolates were obtained from 17 patients between September 2004 and August 2005 at the Academisch Ziekenhuis Vrije Universiteit Brussel, Brussels, Belgium. These multidrug-resistant isolates, which belonged to a single clone, remained susceptible to colistin and tigecycline only and produced the carbapenem-hydrolyzing oxacillinase OXA-58. This study highlights the importance of the intercountry spread of this ␤-lactamase-mediated resistance mechanism and its epidemic evolution.Imipenem and meropenem are among the drugs of choice for the treatment of nosocomial infections due to multidrugresistant (MDR) Acinetobacter baumannii (2). However, their efficacies are increasingly being compromised and carbapenem-resistant isolates are becoming widespread in several regions of the world (7, 16). A large variety of molecular mechanisms for resistance to carbapenems have been reported in A. baumannii, i.e., not only the acquisition of carbapenemhydrolyzing ␤-lactamases of molecular Ambler class B (metalloenzymes) and the acquisition of mostly class D enzymes (oxacillinases) (19, 24) but also rare mutations in genes coding for penicillin-binding proteins (9) and decreased outer membrane permeability (5). Whereas the class B enzymes found in A. baumannii are of the IMP and VIM types (24), the acquired carbapenem-hydrolyzing class D ␤-lactamases (CHDLs) are members of three subgroups of enzymes: the OXA-23, 18). OXA-58 shares less than 50% amino acid identity with the members of two other CHDL groups, which share 60% amino acid sequence identity. Recently, a fourth subgroup of OXA ␤-lactamases sharing less than 63% amino