Antiviral activity and cross-resistance profile of P-1946, a novel human immunodeficiency virus type 1 protease inhibitor.

The HIV protease inhibitor P-1946 is a member of a novel family of l-Lysine derivatives. The compound is a specific HIV-1 protease inhibitor that has potent and selective in vitro antiviral activity (EC50 152 nM) against a range of isolates resistant to commercially available protease inhibitors. The presence of at least four primary and four secondary drug resistance mutations is required to achieve greater than four-fold resistance to P-1946. P-1946's favorable resistance profile makes it a good lead for the development of new agents active against existing PI-resistant virus in treatment-experienced patient.

The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo.

Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost alpha-1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins. This processing allows the binding and release of monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endoplasmic reticulum. We have isolated two cDNA isoforms of the human alpha subunit (alpha1 and alpha2) differing by a 66 bp stretch, and a cDNA for the corresponding beta subunit. The alpha1 and alpha2 forms have distinct mobilities on SDS-PAGE and are expressed in most of the cell lines we have tested, but were absent from the glucosidase II-deficient cell line PHA(R) 2.7. Using COS7 cells, the coexpression of the beta subunit with the catalytic alpha subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the alpha/beta complex. Transfected cell extracts expressing either alpha1 or alpha2 forms with the beta subunit showed similar activities, while mutating( )the nucleophile (D542N) predicted from the glycoside hydrolase Family 31 active site consensus sequence abolished enzymatic activity. In order to compare the kinetic parameters of both alpha1/beta and alpha2/beta forms of human glucosidase II the protein was expressed with the baculovirus expression system. Expression of the human alpha or beta subunit alone led to the formation of active human/insect heteroenzymes, demonstrating functional complementation by the endogenous insect glucosidase II subunits. The activity of both forms of recombinant human glucosidase II was examined with a p-nitrophenyl alpha-D-glucopyranoside substrate, and a two binding site kinetic model for this substrate was shown. The K(M1-2) values and apparent K(i1-2 )for deoxynojirimycin and castanospermine were determined and found to be identical for both isoforms suggesting they have similar catalysis and inhibition characteristics. The substrate specificities of both isoforms using the physiological oligosaccharides were assessed and found to be similar.

Treatment of chromoblastomycosis due to Fonsecaea pedrosoi with low-dose terbinafine.

Chromoblastomycosis, or chromomycosis, is a chronic fungal infection of the skin and subcutaneous tissues caused by a species of dematiaceous fungi. We present a patient with chromoblastomycosis due to Fonsecaea pedrosoi, who was treated with 8 months of terbinafine 250 mg by mouth daily with histologic and mycologic cure.

Periorbital edema as the presenting sign of juvenile dermatomyositis.

We report a case of juvenile dermatomyositis that presented with periorbital edema. Dermatomyositis is an autoimmune disorder with cutaneous manifestations including heliotrope patches, Gottron's papules, periungual telangiectasisas, and subcutaneous calcifications. Periorbital edema may accompany the classic heliotrope rash and, as in this case, may be the only presenting sign of juvenile dermatomyositis.

Structure of the gene coding for the mouse TRiC-P5 subunit of the cytosolic chaperonin TRiC.

TRiC is a cytoplasmic chaperonin involved in actin and tubulin folding. It is formed by six to nine different but related proteins of 52 to 65 kDa arranged in two hetero-oligomeric rings. We have cloned the gene coding for the mouse TRiC-P5 subunit (also called CCT gamma) using a XbaI-DraIII fragment of the mTRiC5 cDNA. The mouse genome contains one TRiC5 gene and one TRiC5 pseudogene located on chromosomes 3F and 5B, respectively. The 2-kb transcript of TRiC5 is encoded by 14 exons distributed within 25 kb of genomic DNA. The largest exon is 312 bp and the smallest exon is 51 bp. We have used primer extension to demonstrate multiple transcription start points for the TRiC5 gene. This is consistent with the lack of any obvious TATA box upstream of the transcription start points.

The cytosolic chaperonin subunit TRiC-P5 begins to be expressed at the two-cell stage in mouse embryos.

The cytosolic chaperonin TRiC is a large protein complex involved in the folding of newly synthesized actin and tubulin. The fertilization of the mouse oocyte is followed by a remodelling of the actin and tubulin filaments. The TRiC subunit TCP1 is expressed only from the 4-cell stage on, even though actin and tubulin are synthesized in the previous stages. We investigated the onset of synthesis of another subunit, TRiC-P5, during early mouse embryogenesis. We report that TRiC-P5 is synthesized at the 2-cell stage in an alpha-amanitin sensitive manner. Thus, it is expressed before TCP1 and is one of the first proteins to be synthesized after zygotic genome activation.

Stroke-induced purpura in lesions of colloid milium.

We describe three patients with colloid milium whose lesions showed purpura upon stroking. Two of these patients had juvenile colloid milium and the third had adult colloid milium. Ultramicroscopic examination of purpuric colloid papules showed that the dermal blood vessel walls were infiltrated by colloid material. Traumatic purpura occurring in colloid milium may be analogous to that occurring in systemic amyloidosis. We suggest that the infiltrating colloid material decreases the elasticity of dermal blood vessel walls, accounting for the purpura following minor trauma.

Assignment of the human homologue of the mTRiC-P5 gene (TRIC5) to band 1q23 by fluorescence in situ hybridization.

The TCP1 ring complex (TRiC) is a molecular chaperone involved in actin and tubulin folding. Little is known about the components of this complex. The first component identified was TCP1, a protein coded by a gene in the t-complex locus on mouse chromosome 17. This locus is involved in several embryonic defects, male sterility, and the transmission ratio distortion. In humans, the t-complex genes map to chromosome 6. Other components of TRiC are thought to be TCP1-related proteins. Recently, a mouse cDNA coding for one of these proteins has been cloned and named mTRiC-P5. Here we report the cloning of a partial human cDNA clone, homologous to mTRiC-P5, and its chromosome localization by fluorescence in situ hybridization. The human TRiC-P5 gene (TRIC5) maps to human chromosome 1q23, a region known to be a preferential chromosomal breakpoint involved in leukemia. Therefore, even if TCP1 and TRiC-P5 are related proteins and are found in the same protein complex, they are not coded by syntenic genes in humans.

cDNA encoding a novel TCP1-related protein.

We report the cloning of a mouse cDNA encoding a novel protein which has significant homology with the t-complex protein 1b (TCP1b). In addition, this protein has high sequence identity with tryptic peptides from the bovine P5 subunit of the TCP1-ring complex. We named this novel protein mTRiC-P5 for mouse TCP1-Ring Complex Protein #5. Results indicate that mTRiC-P5 is a new member of the TCP1-TF55 family and is part of the TCP1-ring complex.